the dynamics of living protoplasm

THE DYNAMICS

OF LIVING

P R O T O P L A S M

L. V. HEILBRUNN University of Pennsylvania

Philadelphia, Pennsylvania

ACADEMIC PRESS INC. · PUBLISHERS NEW YORK · 1956

C O P Y R I G H T , 1956 BY

A C A D E M I C PRESS I N C .

125 East 23rd Street

N e w York 10, Ν . Y.

All Rights Reserved

N O PART OF THIS BOOK M A Y B E REPRODUCED I N

A N Y F O R M , BY PHOTOSTAT, M I C R O F I L M , OR A N Y

OTHER M E A N S , W I T H O U T W R I T T E N PERMISSION

F R O M T H E PUBLISHERS.

Library of Congress Catalog Card No. 55-12301

PRINTED IN T H E U N I T E D STATES OF A M E R I C A

DEDICATION

TO MY YOUNG COMPANIONS IN RESEARCH,

PAST, PRESENT AND FUTURE

PREFACE

On the whole, the writing of this book has been more of a pleasure than a drudgery. For as I marshalled my facts and pieced them to-gether, I felt that the story I had to tell was a good story, a story that was essentially true.

I have not tried to write an exhaustive treatise, nor have I tried to impress the reader with ponderous and abstruse data. As simply as possible, I have attempted to show that our present knowledge of the colloid chemistry of protoplasm can help to interpret and to explain some of the most puzzling and intriguing problems that physiologists have had to face.

The title of the book is perhaps redundant. But feeling as I do about the importance of studying protoplasm when it is alive, and not merely after it is dead, I have stressed this point of view in the title.

In discussing the protoplasm of living cells, I have not attempted to cover all aspects of their activity. If the living machine can be compared to an automobile engine—as it so often has been—then in terms of such an engine I have concentrated my attention on the mechanism of energy conversion within what corresponds to the cylinders. I have not been interested in the nature of the fuel, the reactions that occur when this fuel is burned, or how it finds its way into the place where it is utilized.

Because I am above all else a laboratory worker, I have tried not to neglect my research program in order to write this book. Many of the chapters were written in the laboratory while my experiments were actually in progress, during the brief intervals between measurements. At other times I have pounded my typewriter in between frequent interruptions by students who came to consult me.

I am dedicating this book to the many young investigators who have been such a help and such an inspiration to me. Needless to say, I have enjoyed working with them. Perhaps this statement of our point of view will encourage them to expand and also to correct our present store of knowledge. I have tried to train them to seek and to love the

truth. It is my ardent wish that they may continue to learn more about the dynamics of living protoplasm, no matter whether what they learn agrees or disagrees with the facts as I have presented them in this book.

Finally I should like to thank my wife, Ellen Donovan, for re-drawing several of the figures. I want also to thank the staff of Academic Press for their kind and skillful cooperation.

Philadelphia September, 1955

L. V. HEILBRUNN

1.

INTRODUCTION

Anyone who has ever looked at protoplasm under the micro-scope is almost certain to be thrilled by the very sight of it. For here is material that is endowed with the wonderful attributes of life. The protoplasm of an ameba can move, it can take in food, and it can use oxygen to burn its substance; it can grow, it can reproduce. In a muscle, the living substance can contract, and in nerve cells it can respond in intricate ways; it can even in some cases exhibit consciousness. We are alive because of the protoplasm in us, and if we are to understand the secrets of life and the mechanisms of vital action, we can find our answers only by a study of protoplasm.

One way of studying protoplasm is to look at it. But micro-scopic observation has always been somewhat disappointing, for it reveals surprisingly little. In spite of the fact that the simplest living cell is capable of much greater achievement than the most elaborate machine ever built by human hands, there is very little structure visible in such a cell, even if it is observed under the highest powers of the microscope. With an ordinary micro-scope one can perhaps distinguish a membrane around the cell and a nucleus within it; also, there are typically large numbers of granules in the protoplasm, but these appear to have rather a haphazard arrangement and, indeed, experiments show that most of the protoplasmic granules are not arranged in any definite or significant pattern. The electron microscope is more powerful than the light microscope, but the pictures obtained with it give but little additional detail and not much of any special signifi-cance.

1

2 DYNAMICS OF LIVING PROTOPLASM

Chemical study is perhaps more fruitful. We know that there are proteins and lipids and carbohydrates in cells, and modern cytochemical study has given us some information as to the distri-bution of specific proteins and other substances in the nucleus, the cytoplasm, or the granules.

But neither the morphological study, nor the morphological combined with the chemical, has told us much about the dy-namics of living matter, nor have such studies explained why an ameba moves, why a muscle contracts, or why and how a nerve cell responds. Neither pure morphology, nor chemistry, can in itself explain mechanism. Chemical reactions make an automobile run, but the exact nature of the reactions is not of primary importance. In this engine, heat is produced in one way or another and the gases which result from the combustion of liquids or their volatilization exert pressure against the pistons of the cylinders. The exact nature of the combustion and the course of the oxidative reactions do not materially affect the physical processes involved, nor the nature of the forces that produce the motion.

Similarly it is becoming increasingly clear that in any given vital process the oxidative reactions in protoplasm may vary widely. They can involve the utilization of oxygen or they can occur in the absence of it, and yet although the course of the oxidative reactions may follow one path or another, the ameba moves, the muscle contracts, the egg cell divides. Interesting as the oxidative reactions are, we need to know how the energy they produce is harnessed so as to give rise to vital mechanical activity in the living engine. And this we can not learn merely by follow-ing the oxidative reactions, no matter how patiently and carefully we study them. Over twenty years ago Meyerhof (1930) began his classic book on the chemistry of muscular contraction by saying that "The goal of muscle physiology is an answer to the question as to how the chemical processes in the organism can perform mechanical work." This is, as Meyerhof realized, a major question not only for the muscle physiologist but for all those interested in any aspect of the living engine. The question can not be answered by a purely chemical study of reactions proceed-

1. INTRODUCTION 3

ing at one rate or another. We need to know how the stuff that is protoplasm becomes capable of physical work. In order to approach this problem, we must have an understanding of the physical properties of protoplasm and how these change when the cell is thrown into activity.

It seems rather obvious that, if we are to understand the living machine from a physical standpoint, we must study the machine itself and not depend on speculation as to what the machine must be like on the basis of its chemical nature. Certainly no one would hope to interpret an automobile engine or any other com-plicated engine on the basis of the chemical compounds which could be demonstrated to be present in it as a result of a chemical analysis of the engine as a whole. From such an analysis, some things would indeed be learned. Certainly an automobile engine could not be manufactured from proteins any more than a living engine could be made of steel. Our analyses of vital material have indicated the nature of the substances of which it is com-posed; and from a knowledge of these substances, their proper-ties, and their concentrations, we can learn much. Also we know the types of fuel the living machine can utilize, and we know a great deal about the nature of the catalysts involved in the oxidation of the fuel. But this is still a long way from under-standing mechanism. In chemical studies it is usually wise to obtain substances in as pure a state as possible; and so some chemists, attacking the problem of vital mechanism, have attempted to purify as much as possible certain constituents of living tissues and to correlate the properties of these purified substances with the properties of protoplasm and cells. This, on the whole, is an unfortunate approach. For, from a physical standpoint, the living material is a colloid, and the properties and the behavior of any colloidal system are largely determined by the impurities the system contains. Hence purification is almost certain to destroy the living qualities of the protoplasmic colloid.

To call protoplasm a colloid invites a discussion of what the term colloid means. As a matter of fact, the definition has changed from time to time and, indeed, probably no science has had more difficulty in defining its concepts than the science of


colloid chemistry. The founder of the science, Graham, in 1861 defined colloids as substances which diffused slowly and were unable to pass through gelatinous or paper membranes. Some of these colloidal substances that Graham recognized were poly-mers, but by no means all of them were, as Flory ( 1953 ), in his references to Graham's work, seems to imply. Following Graham, early authorities in the field of colloid chemistry stressed the fact that many substances commonly occurring in a crystalline form could under certain conditions act as colloids, and they introduced the concept of a colloidal state which was not con-fined to certain classes of substances. The colloidal state was for a time defined as a state of dispersion in which the indivi-dually dispersed particles were larger than molecules but smaller than the smallest particles visible with an ordinary light micro-scope. The definition was rather arbitrary, nor was it strictly adhered to by colloid chemists; for they, on the one hand, were ready to investigate emulsions and suspensions in which the dispersed particles were much larger than the definition specified, and, on the other hand, they were quite ready to admit that huge single molecules might show the same behavior as groups of smaller molecules. (Indeed, at the present time in modern books on colloid chemistry, the classic definition has been largely abandoned; see Kruyt, 1949-1952; McBain, 1950.) Colloidal particles have a surface and the physical properties of the surface to a large extent condition the behavior of the colloid. However, early colloid chemists like Zsigmondy were often careful to point out that colloidal behavior might be strongly modified by chemi-cal changes occurring in the dispersed particles. These chemical changes are of particular importance in the case of huge mole-cules such as those of proteins and starches. At a time when colloid chemists were trying to explain the behavior of protein solutions and protein gels largely in terms of the physics of surfaces, protein chemists were urging the importance of classical chemistry in the interpretation of the protein colloid. Certainly much of the behavior of proteins, starches, and carbohydrate gums is indeed to be explained in terms of valence bonds and stoichiometric relationships. In relatively recent years this aspect

1. INTRODUCTION 5

of the subject has been developed by a group of excellent chemists who have introduced a new science, the science of polymer chemistry (see Meyer, 1951; Flory, 1953; Staudinger and Staudinger, 1954). The polymer chemists can no doubt explain much of the behavior of rubber, nylon, and of polymeric carbohydrates in terms of the arrangement of chemical forces within the polymer molecule, and eventually their investigations may throw a great deal of light on the colloidal behavior of proteins. But it should be remembered that a huge protein molecule can be considered as having a surface, a surface at which smaller molecules or ions can be adsorbed, and that the colloidal properties of the protein colloid are to some extent determined by the admixture of substances which are not units of the protein polymer. Indeed, the colloidal particle or micelle is typically not one pure chemical compound but an aggregate. In the study of such aggregates, the organic and polymer chemists are perhaps at a disadvantage, for they are trained to think in terms of pure substances.

There is no more complex colloid than living protoplasm. The simplest protoplasm contains a wide variety of chemical com-pounds and some of these compounds are among the most complex substances known to the chemist. Many of them are polymers of high molecular weight containing diverse types of monomers. The polymers contained in the living substance include various kinds of proteins, an assortment of carbohydrates, and some nucleic acids. The proteins can (and do) unite with any of the other polymers as well as with lipids and with both cations and anions. The living colloid is also remarkable for the various types of dispersion which may be found in it. Usually it is an emulsion containing fatty particles easily visible with an ordinary microscope; in addition, it is a suspension, for suspended in it are protoplasmic granules. These granules themselves may not be simple solid particles of undissolved solid material. Some of them at least are vacuoles, that is to say, they have an outer film or membrane inclosing a fluid interior ( Harris, 1939, 1943; Opie, 1948 ). Granules and fat droplets are suspended in the main mass of the protoplasm, which is also in a state of colloidal dispersion.


And then, in addition to the main mass of the protoplasm, there is typically a nucleus within the cell; this has its own membrane and within the nucleus is a nucleolus or nucleoli and either granular masses of chromatin or chromosomes. And, of course, there is the highly important membrane surrounding the proto-plasm. But if the protoplasmic colloid is more complicated than any known inanimate colloid, in so far as obvious visible struc-tures are concerned, it is essentially lacking in the many diverse parts that many inanimate machines possess. And, indeed, the main mass of the protoplasm can be studied from the standpoint of colloid chemistry, and so can the nucleus.

The study of the colloid chemistry of protoplasm is not new. Nearly thirty years ago a monograph was published on it ( Heil-brunn, 1928). The main purpose of that monograph was to lay the basis for what was then a new and important aspect of cell physiology. Accordingly, at that time it was necessary to review critically the early work in the field. Much of this work was not sound; some of it was based on enthusiastic but careless use of improper methods. In the early literature, various observers drew conclusions about the physical properties of protoplasm from subjective impressions rather than from genuine measurements. Work of this sort could not be repeated or confirmed; fortunately, most of this early misinformation has by now passed into the limbo of things well forgotten. In any growing science knowl-edge begins as a mixture of the true and the false. Eventuallv the truth survives, but mistaken notions often become firmly rooted in the literature and it is difficult to dislodge them. Fortunately, at the present time, students of the colloid chemistry of protoplasm have, for the most part, become aware of the necessity of making correct measurements, and the number of such measurements is constantly increasing.

The physical changes that occur in the course of the vital process are not entirely colloidal changes. Because of their osmotic properties, cells become larger or smaller as water enters or leaves them. Such changes in size are ordinarily not of any great magnitude. However, it is true that changes in the water content of cells may affect the colloidal properties of the proto-

1. INTRODUCTION 7

plasm. Our interest is in such colloidal change rather than in the osmotic behavior of the cell. The osmotic properties of cells and the changes in the permeability of the osmotic membranes sur-rounding cells have been much studied and have been reviewed in various monographs ( see, for example, Gellhorn, 1929; Brooks and Brooks, 1942; Davson and Danielli, 1952). Once it was commonly believed that permeability change was the most im-portant factor governing the vital behavior of the cell. This idea can no longer with any justice be held ( compare Heilbrunn, 1952b), and, indeed, the original advocates of such an idea eventually came to abandon it. No doubt the permeability rela-tions of a cell constitute a very important factor in determining its behavior, and probably in the differential permeability of the different cells of a higher organism one can find an explanation of why the various types of cells often react so differently to a given drug or chemical; and yet by and large the permeability of the outer membrane of the cell does not seem to play an essential role in the machinery of vital processes. There is, of course, room for a difference of opinion on this point. Be that as it may, in our discussion we shall make no effort to cover the voluminous literature on the permeability of the cell and how this permeability varies under various conditions.

However, in the biophysical study of the cell there is one aspect that can scarcely be neglected. Probably in all cells there are differences in electric potential between one part and another, and in elongated cells such as muscle or nerve fibers differences in potential along the length of the cell can readily be followed. In both nerve and muscle cells, and in elongated plant cells also, waves of potential difference can be shown to travel along the length of the cell, and such waves and the electric currents they produce are of primary importance in causing waves of activity to pass along the protoplasm. Thus a very important aspect of protoplasmic activity is dependent on electrical phenomena, and it is essential for us therefore to come to some understanding as to the nature of the potentials in living cells and how these potentials change in the course of vital activity.

Ultimately, as our knowledge of the cell and its mechanisms


increases, we will be able to relate various aspects of our knowl-edge that now are rather separate from each other. Undoubtedly, changes in the protoplasmic colloid can in themselves have an influence on bioelectric potentials, and waves of potential dif-ference passing along the surface of a living cell can induce waves of colloidal change. Moreover, beyond any doubt, the enzvmatic reactions which are involved in the metabolism of the cell depend for their intensity on the colloidal state of the proto-plasm and the enzymes in it; and it is probable also that the course and direction of these enzymatic reactions are also largely dependent on colloidal factors.

What we need is more factual knowledge. But to obtain pertinent information about the vital machine is not easy. Living cells are small, they are often so intimately bound to each other that study of the individual cells is extremely difficult; more-over the cells are fragile and can be handled only very gently or they die. But the situation is not hopeless—far from it. For, as in other branches of biology, the student of the machinery of life processes is tremendously aided by the amazing uniformity of all types of living material. No two types of cells are just alike, but almost all of them are similar in form, appearance, and content. This basic similarity has made possible the science of cytology, a science which considers all types of cells in one general treatment. So too, biochemists have more and more come to realize that, from the lowest to the highest forms of life, the chemical constitution of the living material and the chemical reactions within it are broadly and basically similar. And it is becoming increasingly probable that there is an underlying similarity in the machinery of the vital machine, just as in the material that constitutes the machine there is a similarity of form and chemical constitution. This remarkable uniformity of protoplasm has made possible the impressive advances in our understanding of the problems of heredity and development. In the fields of genetics and embryology, it is common practice to choose for investigation the simplest living systems which can be used to study a given process. The same method of approach is possible also for the physiologist, and it can serve him as well as

1. INTRODUCTION 9

it has served biologists in other fields. But in the past many physiologists have hesitated to use such an approach, for classical physiology has always been intimately related to the theory and practice of medicine, and medical men naturally prefer to work on animals close to the people on whom they practice their art. It is only since the development of general and cellular physi-ology that real progress has been made in interpreting the basic phenomena which underlie the machinery of the living processes, whether they occur in egg cells, in the cells of plants, in muscle, or in nerve.

Our knowledge is growing, and it is knowledge that may well find application, not only for various branches of theoretical biology, but for various aspects of clinical science as well. A reasonably correct understanding of the dynamics of living proto-plasm would not only make possible the development of a broad theory which would be of use in the interpretation of various types of protoplasmic activity, but it would at the same time help us to understand what happens when the living machine fails to function properly. Indeed, as has long been recognized, the basic problems of pathology are problems of the living cell. Thus, for example, if we knew what made cells divide, we would have an important clue to the nature of neoplastic growth. Also, if we understood protoplasmic dynamics, we could interpret the action of various physical and chemical agents on living cells and we would have a rational basis for the great science of pharma-cology.

2.

PROTOPLASMIC VISCOSITY

In any attempt to interpret the machinery of a living cell, it is essential to know something about the mechanical properties of the protoplasm in the cell that is being investigated. Consider a muscle cell, for example. What happens when this cell contracts? If we knew the protoplasm of the muscle cell to be essentially solid or gel-like, we might postulate one type of mechanism; but such a mechanism could hardly be applied to the muscle cell if its interior were largely fluid. The dynamics of muscle will be considered in a later chapter—for the present we are concerned only with the general problem of protoplasmic viscosity. We will want to know how it is measured and what values have been obtained for it in different cells and in different parts of the same cell.

The viscosity concept is often not clearly understood by biolo-gists and and physiologists. The term is a physical term and it should be used as the physicists use it. The common everyday concept of viscosity is clear enough. Ordinarily we think that the more readily a liquid flows, the more fluid it is and the less viscous. Actually if precise measurements of viscosity are to be attempted, there is need for a more exact definition. The unit or coefficient of viscosity of a fluid is defined as the numerical value of the tangential force on unit area of two parallel planes at unit distance apart, when the space between the planes is filled with the fluid, and one of the planes moves with unit velocity in its own plane. This unit of viscosity is the poise. Pure water at 20° C. has a viscosity of approximately 0.01 poise or 1 centi-poise.

If in the case of our postulated two planes the moving plane

10

2. PROTOPLASMIC VISCOSITY 11

were to travel at two or three times unit velocity, then the force on the stationary plane would increase two or three times; but this is true only if the fluid between the planes is a true fluid with no elastic properties. If the substance between the two planes is at all elastic, then as the moving plane travels at greater and greater speed, part of the extra force it exerts is expended in overcoming the elastic forces of the material between the two planes, and accordingly with greater and greater speed of the moving plane, there is no proportionate increase in the force exerted on the stationary plane. In the case of a true fluid, the viscosity does not vary with the speed of the moving plane; but in the case of elastic material, as the speed of the moving plane ( and its shearing force ) increases, the viscosity becomes progres-sively less. So too in the flow of a liquid through a tube. Thus a gelatin gel may not flow at all and we could consider that it had infinite viscosity; but if we exert an increased pressure on the gel, flow begins and the viscosity may become quite small. Wax will also flow if enough pressure is exerted on it. In these cases in which viscosity varies with shearing force, it is customary now to speak of an "apparent viscosity" or an "anomalous viscosity," and when a fluid shows such a variable viscosity, it is often called a "non-Newtonian" fluid (for further discussion of viscosity, see Barr, 1931; Scott Blair, 1938, 1949; Heilbrunn, 1950).

In the measurement of the viscosity of inanimate fluids, the Ostwald viscosimeter or some modification of it is commonly used. With this apparatus it is possible to measure the time it takes for a given volume of liquid to flow through a capillary of known length and bore. Then by applying Poiseuille's law for the flow of liquids through capillary tubes, it is possible to determine the viscositv.

Viscosimeters of the Ostwald type cannot be used on proto-plasm, for the protoplasmic fluid is contained within membranes or walls, and commonly if the protoplasm is released from its containing membranes, the viscosity changes sharply. Indeed it can well be said that under ordinary conditions protoplasm is never naked, for as soon as it emerges from a cell, it immediately forms a new membrane around itself (compare Chapter 5).


However, under certain conditions this formation of new films can be prevented, so that it might be possible to measure proto-plasmic viscosity by a viscosimeter of the Ostwald type, on the assumption that the treatment necessary to prevent film forma-tion has no effect on viscosity. This has almost never been done.

A common and successful method of measuring viscosity is to determine the speed with which an object of known specific gravity and of known size moves through a fluid under the influ-ence of a given force. Thus the Hoeppler viscosimeter, a stand-ard instrument, operates on this principle. In studying the viscosity of protoplasm, one can, if one pleases, introduce into cells small particles or droplets, but, in general, such techniques are only successful for large cells; and even in them, the injury attendant upon the introduction of the particle or droplet is a factor seriously to be reckoned with. Some cells take up particles from the surrounding medium and this fact can be taken ad-vantage of. Thus paramecium takes up carmine or starch particles, or indeed various types of solid or liquid particles. These are usually ingested into food vacuoles which may be heavier or lighter than the main mass of the protoplasm and can be made to move through the protoplasm by the application of centrifugal force.

However, in the case of most cells there is no uptake of parti-cles from the surrounding medium and there is no possibility of introducing such particles without rather violent injury. Fortu-nately for the student of protoplasmic viscosity, cells commonly contain granules or other inclusions and these can be made to move through the protoplasm. Occasionally the natural cell inclusions are large enough and heavy enough to move through the protoplasm under the influence of gravity, but generally centrifugal force some hundreds of times gravity, or even some thousands of times gravity, is necessary to obtain a reasonable granular speed easy to measure.

In all cases in which the movement of a spherical particle through a fluid is used to determine the viscosity of the fluid, the method depends on the application of Stokes' law. This law, derived many years ago as a mathematical exercise, has found


wide practical application in various branches of physics and chemistry. In its original form Stokes' law states that

W = 6 ιζηαυ,

where W is the force pushing the sphere through the fluid, η is the viscosity of the fluid, a the radius of the sphere, and ν its velocity. When the sphere moves under the influence of gravity, the force acting on it is its apparent weight times the gravity constant, g, and the apparent weight is the volume of the sphere times the difference between its specific gravity (σ) and the specific gravity of the fluid, which we shall call p. Accordingly,

W = 4/3 πα 3 ( σ - ρ ) g — βπηαυ. Solving for η,

_ 2 g ( a - p ) a 2

η ~ ~ ν or, solving for v,

2g ( σ - ρ ) α 2

V =

9η Obviously, therefore, if we have a sphere falling through a fluid and we know its specific gravity, its radius, its speed, and the specific gravity of the fluid through which the sphere falls, we can calculate the viscosity. If centimeter-gram-second units are used, the formula gives the viscosity in poises.

Like most laws of physics, Stokes' law holds only under certain conditions. These are discussed by various authors (see Heil-brunn, 1950 ). If the moving sphere is small and its rate of move-ment slow, and if the walls of the containing vessel are far enough distant from the sphere, the law holds very well. But it should be emphasized that the law was derived for a single sphere and not for a large number of them. If a number of parti-cles move, then it is necessary to apply a correction to the law. Several such corrections have been proposed; the only one that has been used in the study of protoplasmic viscosity is the Cunn-ingham correction (see Cunningham, 1910). If this correction is introduced into Stokes' law, then

2 g ( a - p ) a a

ν = 9qv


in which 4b ( b 5 - a 5 )

? = (b-a)2 (W-b*a-6b2a2-ba3-4a4)

In this formula, a is the radius of the sphere and b is half the distance between two adjacent spheres. But Cunningham sug-gests that it is better to substitute for b a quantity b'y which is equal to &V3/2. Ordinarily, in the measurement of protoplasmic viscosity, it is not necessary to use the Cunningham correction, for in general the biologist or biophysicist is concerned only with the changes in viscosity that occur during vital activity or follow-ing exposure to varying conditions. For such measurements of relative viscosity, the Cunningham correction is not required. It is essential only to be reasonably certain that the size of the moving particles and their specific gravity do not change markedly. In most cells there are granules that are heavier and those that are lighter than the rest of the protoplasm. Under a given condition, if the movement of both types of particles is hastened or slowed by approximately the same amount, it can safely be assumed that the change is due to a change in viscosity and not to a change in size or specific gravity of the moving particles.

Most of the quantitative measurements that have been made of protoplasmic viscosity have been made by applying Stokes' law to the movement of particles through the protoplasmic fluid under the influence of gravity or centrifugal force. Other types of measurement are also possible. As has long been known, the rate of Brownian movement in a fluid is dependent on the vis-cosity of the fluid. Thus according to Einstein's formula,

RTt D 2 , =

in which Dx is the displacement of a particle in Brownian move-ment along the X axis, R is the gas constant, Τ the absolute temperature, t the time, Ν the Avogadro number, 77 the viscosity, and a the radius of the particle. The formula contains no term for specific gravity and hence it is not necessary to know the


specific gravity either of the particle in movement or of the fluid through which it moves. One can study either the movement of large numbers of particles, so, for example, one can follow the return migration of granules previously centrifuged to one side of a cell; or one can, if he is very patient, follow the movement of a single particle. When the granules of a cell are very close to each other, as is often the case, the amplitude of the move-ments of individual granules is markedly restricted, even though the protoplasm in which the granules lie is highly fluid. So, for example, in the egg of the sea urchin Arbacia, there is little Brownian movement to be seen, but if the granules are cen-trifuged to one side of the egg, then at the boundary between granules and clear cytoplasm the granules move violently. When the concentration of particles in Brownian movement is not too high, one can measure viscosity by determining the length of time it takes for a particle to move a given distance to the right or left of its original position. This is the method employed by Pekarek (1930a, b, 1931, 1932, 1933a, b ) , and he uses a formula developed originally by Fürth ( 1930 ). Pekarek tested the method on distilled water and obtained a correct value for the viscosity of the water.

Other methods of determining the viscosity of protoplasm have also been used. Thus Heilbronn, 1922, introduced small iron rods into the protoplasm of slime molds at stages in which the proto-plasm was in the form of a large, continuous mass. By twisting the iron rods with an electromagnet and comparing the force necessary to produce an effect with the force necessary to pro-duce a similar effect when the rods were in distilled water, Heilbronn was able to make measurements of viscosity.

From various measurements of protoplasmic viscosity, it is now clear that in some cells the interior protoplasm is a fluid of no very great viscosity. This does not mean that the protoplasm of all cells is at all times fluid. Thus, for example, the protoplasm of many marine eggs is rather viscous when the egg is in the germinal vesicle stage before maturation has begun. But, quite commonly, as soon as maturation has begun, or even just before maturation division processes start, the protoplasm quickly be-


comes fluid. This can be clearly shown for the egg of the worm Nereis (Heilbrunn, 1921), and it is also true for the egg of the worm Chaetopterus (Heilbrunn and Wilson 1955b) and the egg of the clam Spisula (unpublished data)—also for various other marine eggs ( Harris, 1935 ).

It should be remembered that in most types of living cells the protoplasm contains very many granular inclusions. The vis-cosity of the fluid protoplasm in which the granules are sus-pended is one thing; that of the entire protoplasm, granules and all, is another. To some extent it is possible to measure both the viscosity of the hyaline, granule-free protoplasm and also the vis-cosity of the protoplasm plus the granules, that is to say, the viscosity of the protoplasmic suspension. Also it is possible, in some cases at least, to measure the viscosity of limited regions of the protoplasm. Thus the outer region of a cell may have much more viscous protoplasm than does the main mass of the proto-plasm in the cell interior, and the viscosity of the outer region or cortex can in a few instances be measured. But obviously it does not make good sense to try and measure the viscosity of the interior of the cell, the cortex, and the outer membrane or wall of the cell all in one. Strangely enough, some authors have described and measured by indirect procedures what they call the "viscosity" of masses of muscle cells, indeed of entire muscles. Such a measurement is largely meaningless, at least in so far as any proper understanding of viscosity is concerned. No physical chemist or physicist would attempt a measurement of the vis-cosity of a bottle of fluid, lumping together both the fluid and its container; nor would he try to determine the viscosity of an entire storage battery, sulfuric acid, lead plates, and hard rubber container.

The first quantitative measurements of protoplasmic viscosity were made by the German botanist Heilbronn in 1914. In starch-containing cells of the bean plant, Vicia faba, Heilbronn meas-ured the time it took for the starch grains to fall through the protoplasm under the influence of gravity. By comparing this speed with the speed of the same grains in water, Heilbronn was able to determine the absolute viscosity of the protoplasm of


these cells. This was on the assumption that the speed of fall was inversely proportional to the viscosity. From our discussion of Stokes' law, it is obvious that this is true, but it is also true that the viscosity is proportional to the difference in specific gravity of the falling sphere and the liquid through which it falls, and some account should have been taken of the fact that protoplasm has a higher specific gravity than water. Also there were presumably quite a number of starch grains, so that it might have been wise to introduce a Cunningham correction; moreover the space through which the grains fell in the cell was probably rather narrow (in comparison with the diameter of the grains). All these factors would tend to make the values that Heilbronn found for the protoplasmic viscosity too high. What he actually did find was that the rate of fall of the starch grains within the cell was in some cases only eight times as slow as the rate in water. In other cases the ratio was somewhat higher, and occa-sional cells were found in which the starch grains did not fall at all; here the viscosity was infinity. These cells with infinite vis-cosity were apparently in a pathological state and were probably dead. Actually what Heilbronn measured was the viscosity of the entire protoplasm, granules and all, but he gives no data as to approximately how many granules were present.

The important thing about Heilbronn's measurements is that they showed for the first time that the interior protoplasm of the cell was not the highly viscous fluid that it was commonly sup-posed to be. Early writers on protoplasm had commonly referred to it as a slime, that is to say, a fluid of very high viscosity. Actually, the viscosity of the protoplasmic fluid is, in some cases at least, about what one would expect from the fact that the protoplasm is a solution of protein in fairly high concentration. For slime molds, using the magnetic method mentioned previ-ously, Heilbronn ( 1922 ) determined the viscosity of the interior protoplasm to be 9-18 centipoises. Here too the values obtained were for the entire protoplasmic suspension.

The centrifuge method can be used to determine the viscosity of the protoplasm of the eggs of various marine invertebrates. If the eggs are centrifuged at a given centrifugal force, one can in


a series of tests determine the length of time it takes for the granules of the egg cell to be moved a given distance. This gives the velocity, υ, in the formula for Stokes' law given earlier in the chapter. In order to use the formula for determinations of absolute viscosity, it is necessary also to know the specific gravity of the moving granules, the specific gravity of the fluid through which they move, and also the size of the granules. In order to apply the Cunningham correction, we must likewise know the average distance between the granules. The specific gravity of the granules can be determined by homogenizing the eggs and then finding what strength of sugar solution has the same specific gravity. This is done by centrifuging. Knowing the specific gravity of the granules and the specific gravity of the entire egg ( and this latter value can also be determined by centrifuge tests in various strengths of sugar solution), from a knowledge of the concentration of granules within the cell, it is possible by a simple arithmetical calculation to arrive at a value for the specific gravity of the fluid through which the granules move. The values for specific gravity are not very exact, but even less exact is the value for the radius of the granules; for with very small objects it is not possible by ordinary visual observation to make accurate measurements of size. Unfortunately, moreover, the value for the radius must be squared in Stokes' law.

If the unfertilized eggs of the worm Chaetopterus or the clam Cumingia are centrifuged at forces of about 2000 times gravity, the granules in these eggs move through most of the cytoplasm in a matter of a few seconds. The protoplasm in both these cells is highly fluid. Thus for the Cumingia egg, Heilbrunn (1926a) found a value for the absolute viscosity of approximately 4 centi-poises, and this is really a maximum value, for in view of the fact that there are relatively few granules in the Cumingia egg, the Cunningham correction was neglected. In the egg of the sea urchin Arbacia, the granules which fill a large part of the egg are smaller than the granules whose movement was studied in the Cumingia egg, and accordingly their rate of movement is some-what slower. Using Stokes' law and applying the Cunningham correction, Heilbrunn found a value of 2 centipoises for the


hyaline protoplasm of the Arbacia egg. As noted previously, this value suffers from the fact that no accurate determination of the size of the tiny granules in the Arbacia egg is possible. Fortu-nately, it is possible to determine the viscosity of the Arbacia protoplasm in another way. This is done by applying the Einstein formula for Brownian movement to the return movement of the granules after they have been centrifuged. In such a determina-tion, as already noted, it is not necessary to know the specific gravity either of the granules or the fluid through which they move, for the Einstein equation does not include any term for specific gravity. However, it is necessary to know the radius of the granules. From a rough measurement of the rate of return of granules by Brownian movement, Heilbrunn (1928) obtained a value of 4 centipoises for the hyaline protoplasm of the Arbacia egg. When granules are subjected to centrifugal force, the larger they are the faster their rate of movement; on the other hand, when granules move in Brownian movement, the larger they are the slower their rate of movement. Thus any error in the determi-nation of the size of the granules would result in an opposite type of error in the two measurements. Hence it is wise to average the values obtained with the centrifuge and Brownian movement methods, and one can conclude that a fairly good value for the viscosity of the hyaline protoplasm of the Arbacia egg is about 3 centipoises.

In the egg of the worm Sabellaria, Harris (1935) was able to photograph granules in Brownian movement, and he could thus obtain an accurate measure of the speed of this movement. Harris centrifuged the eggs and then followed the return of the granules into the clear protoplasm. This is shown in Fig. 1. The return movement is very rapid and it is to some extent directed, for the granules push away from the region of higher concentration into a region of lower concentration. The reason for this is quite obvious. The granules are quite free to move toward the region of lower granular concentration, but when they attempt to move in the opposite direction, they collide with the granules already present there. It is almost as though the granules were bouncing away from a wall. However, Harris believes that the granules are


influenced by some sort of a directed streaming movement and in determining protoplasmic viscosity he makes a correction for the directiveness of the motion. This leads him to a somewhat higher value for the viscosity than he would otherwise have

FIG. 1. Return movement of granules in a centrifuged Sabellaria

eS S - ( a ) 8 min. after centrifuging; (b) 18 min. after centrifuging.

( D r a w n from Harris , photographs) .

obtained. He found a value of 20 centipoises, when in all proba-bility the true viscosity is only a small fraction of this value. A similar type of error was made by Baas-Becking, Sande Bakhuyzen, and Hotelling (1928).* They followed very care-fully the Brownian movement of tiny granules in the outer (parietal) layer of the protoplasm in the cells of the alga Spiro-gyra. This layer is very thin and is bounded by two cylindrically shaped membranes quite close to each other. The granules in Brownian movement that Baas-Becking et al. observed must have collided frequently with the limiting membranes, so that only rarely was a granule able to pursue its path for even a fraction of a second without interruption. Hence it is not surprising that Baas-Becking, et al. obtained highly variable values for the proto-plasmic viscosity. Their lowest values, presumably the correct

* For a critical review of this interesting, but rather peculiar paper, see

Heilbrunn, 1929c .


ones, indicate that the true viscosity of the protoplasm they studied was only 2 or 3 centipoises.

When a living cell is centrifuged at forces hundreds, or even thousands, of times gravity, there is always a suspicion that the cell may be seriously injured by the treatment, and also that the viscosity values obtained with these strong centrifugal forces are not the values that would be obtained if weaker forces had been used in the measurements. As to the first half of this argument, there is an abundance of evidence that when egg cells are vigor-ously centrifuged so that the granules of the interior protoplasm are made to move through the cell, the eggs are not injured; indeed the development of such centrifuged eggs is typically quite normal (see Morgan, 1927 for the older evidence on this point). Moreover, when the viscosity of these eggs with fluid protoplasm is determined at different centrifugal forces, the vis-cosity values remain the same. This was shown for the proto-plasm of the Cumingia (clam) egg by Heilbrunn (1926b). In his experiments, the centrifugal force varied from 310.5 to 4968 times gravity. A much wider range of forces was used by Howard ( 1932 ) in her study of sea urchin egg protoplasm. In her experi-ments, the forces that moved the granules varied from gravity alone to a centrifugal force approximately 4000 times gravity. Her results for temperatures of 3-7° C. are shown in Fig. 2. At this low temperature, the effect of Brownian movement is negli-gible. However, at higher temperatures, when the granules are moved to one side of the cell, their tendency to return by Brown-ian movement is a factor which must be taken into account. When this factor is corrected for, constant values for viscosity are obtained. Hence it appears that the protoplasmic fluid both in Cumingia eggs and in sea urchin eggs behaves in true New-tonian fashion. This does not necessarily mean that all proto-plasm behaves in this way, for some types of protoplasm are not in a fluid state. This aspect of the subject will be discussed more fully later.

However, it may well be concluded that in many cells the main mass of the protoplasm is quite fluid, with a viscosity only several times that of water. This is true not only for various


marine egg cells; it is true also for the interior protoplasm of the ameba. Heilbrunn ( 1929a, b ) found the viscosity of this proto-plasm to be about 2 centipoises at 18° C ; at 24° C. it was two or three times as great. These determinations were made with

6 N

2 -

1 -

0 2000 4000

F

F I G . 2. The viscosity of the protoplasm

of the eggs of the sea urchin Arbacia, de-

termined at various centrifugal forces. The

measurements were made at 3 - 7 ° C. The

abscissas give the centrifugal force in gravi-

ties. The ordinates the viscosity in arbitrary

units. ( H o w a r d ) .

the centrifuge method. Using his Brownian movement method (see above), Pekarek (1930b) found a value of 6 centipoises for the viscosity of the interior protoplasm of ameba.

These very low values for the viscosity of the protoplasm refer to the viscosity of the granule-free suspension. But, as noted previously, the viscosity of the entire protoplasm, granules and all, is not the same as the viscosity of the fluid protoplasm in which the granules are suspended. Various authors have proposed


formulas for calculating the viscosity of a suspension, that is to say, the viscosity of a composite system in which suspended material is dispersed in a fluid. The first such formulation was that of Einstein, and he deduced that the viscosity of a suspen-sion, η8, could be expressed in the following formula:

ψ — ψ(1 + 2.5c),

in which ψ is the viscosity of the dispersion medium and c is the volume concentration of the suspended particles (that is to say, the ratio of the volume of suspended particles to the total volume of the suspension).

Einstein's formula has been the basis of all further work in the field; it holds very well for dilute suspensions. But in his mathe-matical treatment, Einstein did not take into account the inter-action between the suspended particles, and this becomes very important for higher concentrations. Recent authors have de-veloped theoretical equations which do take account of the inter-action between the suspended particles. Concerning the efforts of these authors, Overbeek ( 1952 ) in his authoritative discussion of the viscosity of suspensions, writes that they "arrive at dif-ferent results and it is none too clear which of their results, if any, should be trusted." A favorite equation is that of Simha (1949, 1950). In this equation

ψ = ψ( 1 + aie + Ü2C2 + azà + . . . ) .

This equation reduces to the Einstein equation when c is small, for c2 and c 3 are then negligible. Indeed the c3 term can generally be neglected. The value of ai is taken as 2.5. For 02, on the basis of data in the literature, Overbeek prefers a value of 7.35, but other values are also possible, and some authors prefer a value of 14. ( Simha in 1952 has questioned the theoretical basis of the equation. )

If we take the viscosity of the granule-free protoplasm of the egg of the sea urchin Arbacia as 3 centipoises, then on the basis of the 7.35 value for a2, we can calculate the viscosity of the entire protoplasm of the Arbacia egg as 9.5 centipoises. In order to arrive at this value, we assume c to be approximately 0.4;


this is on the basis of Costello's estimate of the granular volume in the Arbacia egg. Costello ( 1939 ) gives the value as 0.55, but this is without any correction for the spaces between the gran-ules, and these presumably occupy a little more than a fourth of the total volume.* A direct determination of the viscosity of the protoplasmic suspension in Arbacia eggs, obtained by apply-ing Stokes' law to the movement of the relatively large pigment granules through the protoplasmic suspension, gave a value of 7 centipoises (Heilbrunn, 1926a).

Eilers (1941) in his discussion of the viscosity of emulsions emphasizes the fact that when the concentration of the sus-pended material approaches the maximum value of 74 per cent, the viscosity of an emulsion, and presumably also of a suspension, must approach infinity. This is the generally accepted view, and indeed it is borne out by experimental studies of such suspen-sions. In some types of cells the protoplasm is so tightly packed with granules that there is no room for these granules to move when the cells are centrifuged. This is the condition found in Paramecium; also in some types of sea urchin eggs (e.g., in the egg of Paracentrotus lividus). If a paramecium is allowed to ingest starch or iron particles, these and the vacuoles which contain them can be made to move through the protoplasm, and from their speed of movement the viscosity of the protoplasm can be determined. This was first done by Fetter (1926) and she obtained a value for the viscosity of the paramecium protoplasm of 8,000 centipoises. In her description of her experiments, Fetter is not very clear as to whether or not she considered the presence of a vacuole around the ingested particles, and presumably her estimate of the specific gravity of the masses that move through the protoplasm is only a rough estimate. However, the value she obtained is certainly of the right order of magnitude, for similar values have year after year been obtained by students in the course in general physiology at the University of Pennsyl-vania. These students repeat Fetter's experiment as a regular laboratory exercise, making proper estimates of the size of the

* Spheres of a given size cannot fill a volume, but only 74.05% of such a volume.


vacuole containing ingested material and its specific gravity. ( In determining the specific gravity of the vacuole, the number of particles in the vacuole is counted. )

On the other hand, Brown (1940), in what appears to be a careful study of the viscosity of the protoplasmic suspension in Paramecium, obtained a value of 50 centipoises, and this he regards as a maximum value. Perhaps some of the difference in results is due to the fact that different species of paramecium may well have been used; unfortunately, neither author men-tioned the species used in the experiments. However, another explanation of the divergence is much more interesting, and also much more plausible. Concentrated suspensions commonly ex-hibit a phenomenon known as dilatancy. Thus in dense suspen-sions of sand or quartz, or in starch pastes (all of which are dilatant), the viscosity of the suspension or paste becomes higher as the shearing force is increased. This is due to the fact that when the suspension is subjected to a shearing force, the distance between the individual particles is increased in some parts of the suspension and decreased in others, so that in some parts of the suspension flow becomes very difficult; hence the increase in viscosity. In Fetter's experiments she used a centrifugal force 709 times gravity; on the other hand, Brown used a force only 67 times gravity. This may well account for the difference in values obtained by the two investigators.

Many years ago Heilbrunn (1928) suggested that "It is very probable that the viscosity of the paramecium protoplasm varies with different rates of shear" and he thought that "It would be interesting to make determinations of absolute viscosity at dif-ferent centrifugal speeds." This has now been done by Edna Larson in some work as yet unpublished. In studies on Para-mecium caudatum she found that with centrifugal forces above 200 times gravity, with progressive increase of centrifugal force, that is to say of shearing force, there was no increase in the rate of movement of the food vacuoles through the protoplasm. This is shown in Fig. 3. When the viscosity is calculated from Stokes' law, it increases progressively with the increase in shearing force. Thus, for paramecium protoplasm, there is not one viscosity value


but an infinite number of viscosity values. (For such dilatant systems it is perhaps better to speak of "apparent viscosity," reserving the term viscosity for Newtonian fluids.) The change of apparent viscosity with shearing force is shown in Fig. 4. Mrs. Larson's work explains the difference in values obtained by Fetter and Brown.

„ 7 1 — » — ι — ι — ι — ι — ' — ' —1—

1— r

250 5 0 0 750 1000 CENTRIFUGAL FORCE IN TERMS OF GRAVITY

F I G . 3 . T h e effect of changes in shearing force

on the rate of movement of food vacuoles through

the protoplasm of Paramecium caudatum. T h e

paramecia were subjected to different intensities

of centrifugal force ( L a r s o n ) .

The protoplasm of various species of paramecium does not behave in the same manner. This might well be expected, for the work on inanimate suspensions has shown that with minor differences in concentration the behavior may change markedly. A suspension at one concentration may show dilatancy; at another concentration it may be thixotropic. Also differences in the specific gravity or the electrolyte composition of the disper-sion medium can also influence the rheological behavior.

Although the viscosity of the entire mass of the protoplasm in paramecium may be relatively high, the viscosity of the granule-free protoplasm must be very low. This is evident from the well-known fact that the protoplasm of the paramecium flows around the cell, and it is also a necessary consequence of the fact that in order for a suspension to show dilatancy, the dispersion medium


must have a very low viscosity (Freundlich and Röder, 1938; Röder, 1939 ). Hence it seems quite likely that although the vis-cosity of the entire protoplasmic suspension may vary widely from one type of cell to another, the granule-free or fibril-free

L_ , « , ι , • , ι . . . ι . ι , 1 . , • ι I

0 600 1200 1800 2400 3000 Centrifugal force in terms of gravity

F I G . 4 . Variation of the apparent viscosity of the protoplasm of Paramecium

caudatum wi th changes in shearing force ( L a r s o n ) .

protoplasm may generally be capable of existing in a highly fluid state. This does not mean that this hyaline protoplasm is always fluid, for as we shall see later, it can undergo sudden and marked changes in viscosity.

Unfortunately the protoplasmic viscosity of very few types of cells has been measured, and for most cells there are difficulties in the way of a proper measurement. In many cases the cells are very small, so that most of the protoplasm is close to the outer membrane of the cell. Often too the cell is so choked with granules that there is no room for them to move under the influ-ence of centrifugal force.


One type of cell of particular interest to the student of phys-iology or biophysics is the muscle cell. For if we are to under-stand the machinery of muscular contraction, it is essential for us to know as much as we can about the physical makeup of the machine that does the contracting. For many years those who have made theories as to the mechanism of muscular contraction have done so without any clear understanding of the physical properties of the material that contracts. Some of the older theories postulated that the muscle protoplasm was a gel which imbibed water when the muscle shortened. Other theorists have thought that when muscle contracts the protoplasm in it changes from a fluid sol to a more or less solid gel. In order to decide between these two possibilities, it is of course important to know whether the protoplasm in the resting muscle is sol or gel.

In 1863 the famous physiologist Kühne thought he had an answer to this question. He happened to see a nematode worm swimming through the protoplasm of a muscle fiber, and from this chance observation he concluded that the interior of the fiber was fluid. How fluid it was he could not tell, nor could he be sure that the presence of a rather large worm in the fiber did not produce an abnormal condition; actually it did show visible signs of abnormality. For eighty-seven years there was no essential advance in our understanding of the viscosity of muscle protoplasm, and it was not until 1949 that the first vis-cosity measurement was made. In that year Rieser (1949a) injected tiny oil drops into the interior of frog muscle fibers ( see Fig. 5), and he then observed these droplets rise through the protoplasm under the influence of gravity. Observations were made with a horizontal microscope and the speed of the moving droplets could readily be measured. Then, knowing the specific gravity of the oil and the diameter of the drop, it was a simple matter to calculate the viscosity from Stokes' law. From his measurements, Rieser concluded that the protoplasm in the interior of the frog muscle fiber had a viscosity of approximately 29 centipoises. This was on the basis of 7 out of a total of 10 measurements with various types of oil; 3 other measurements which showed a somewhat higher viscosity were discarded be-


F I G . 5. An oil drop in the interior of a

frog muscle fiber ( R i e s e r ) .

prevent the movement of the drop. Moreover, once an oil drop had moved through a fiber, if the fiber was turned end for end by a 180° revolution of the microscope stage, the oil drop was never seen to reverse its direction of movement. As a matter of fact, all the higher values that Rieser obtained may have been due to injury, and it is quite possible that the true value for the viscosity of frog muscle protoplasm is really the lowest figure that was recorded; that is to say, 14 centipoises. But whether we accept this value or the higher value of 29 centipoises, it is clear that the interior of the muscle fiber of the frog is fluid, with a viscosity that one might well expect of a suspension of granules. Presumably the dispersion medium of the muscle protoplasm contains proteins with long thread-like molecules; a solution of such protein material would tend to have a higher viscosity than a solution of proteins with globular-shaped molecules. The fact that the oil drop in the muscle fiber has an ovoid shape ( see Fig. 5 ) rather than a spherical shape, is an indication that it is subject to forces pushing on it from the sides of the muscle fiber. Such forces might result from the pressure of the membranous enve-lopes of the fiber, or they could be due to the pressure of fibrous material within the fiber.

cause it was felt that the high values were due to injury. As a matter of fact, in seriously injured fibers an oil drop is not able to move at all, and indeed it is only rarely that an oil drop can be introduced into a cell without causing an injury sufficient to


But one point is certain—the protoplasm in the interior of the fiber is definitely fluid. Indeed, this could easily have been con-cluded from the fact that when a watery solution is injected into a fiber with a fine micropipette, the injected solution swishes through the fiber. If the interior protoplasm were a gel the in-jection of a small amount of water or aqueous solution would result in the formation of a discrete droplet. Indeed, if the muscle protoplasm is converted into a gel by extracting water from it with glycerol, then when an aqueous solution is injected, it does actually form a discrete droplet.

The protoplasm of a nerve fiber may also be fluid. Rieser (1949b) injected oil drops into the protoplasm of fibers of the ventral nerve cord of the lobster and by determining their speed under the influence of gravity, he was able to measure the vis-cosity and found it to be 5.5 centipoises. These oil drops in the nerve fibers of the lobster retained a spherical shape. On the other hand, when Rieser injected oil drops into the protoplasm of the giant nerve fiber of the squid these oil drops did not move under the influence of gravity. It is possible that in the experi-ments with squid nerves, the nerve fiber was injured, for it is not an easy matter to dissect out giant nerve fibers without injury. Indeed from the very fact that removal of the giant nerve fiber involves cutting of numerous side branches, it is quite conceiv-able that in every case in which a giant nerve fiber is removed there is some gelation of the protoplasm. Cutting frog nerve fibers does produce a wave of injury ( unpublished experiments ). On the other hand, it is also possible that in the giant nerve fiber the protoplasm is much more viscous than it is in the lobster nerve fiber.

In addition to his discovery of the fluidity of the muscle proto-plasm, Rieser also made another important discovery. He tried injecting as much oil as possible into a muscle fiber and he found that no matter how much oil was injected, the entire width of the fiber could never be filled with oil. There was always a peripheral region which the oil could never displace. This, the cortex of the muscle cell, has a thickness of approximately ΙΟμ.. (The frog muscle fiber as a whole has a diameter of about 100μ. ) The


presence of a stiff cortex surrounding the fluid inner protoplasm of the muscle cell can also be demonstrated in another way. If relatively large amounts of aqueous solutions are injected into muscle fibers with cut ends, these solutions push the protoplasm out through the cut ends of the fibers; always the cortex is left behind.

Indeed there is good reason to believe that cells in general have a cortex composed of protoplasm much stiffer than that in the interior of the cell. There is, typically at least, a gel-like layer surrounding the interior fluid mass. The nature of the cortex and its physical properties will be discussed in the next chapter.

The present chapter has shown that in many cells the interior protoplasm is a fluid of no great viscosity. Later much evidence will be presented to show that this fluid is capable of sudden and rapid viscosity change which can convert it from a fluid sol to a more or less rigid gel, and then perhaps back to a sol again. These changes are closely related to the vital behavior of the protoplasmic colloid and are believed to be an important factor in the dynamics of the living system.

In addition to our knowledge of the viscosity of the cytoplasm of the cell, we have a little information about the viscosity of the fluid in the nucleus. However, changes in the viscosity of the nucleoplasm have not as yet been found to bear any relation to vital activity, and for that reason the viscosity of the nucleo-plasm, interesting as it is, will not be discussed.

3.

Protoplasm, obviously, is not all fluid. This follows from the fact that the watery cell does not disperse in the watery fluids that surround it. Of course, it might be conceivable that each cell was surrounded by a thin film of an oily fluid, but the outer boundary of a cell does not have the properties of an oil film. Thus, for example, the outer membrane of a cell is not sharply visible, as it would be if it consisted of oil with a refractive index well above that of water.

If cells were all fluid, they could scarcely be capable of ac-complishing some of their complicated functions. Muscle cells could scarcely exert the tensions they do, and an egg cell if it were merely a homogeneous fluid could scarcely be capable of forming a variety of differentiated structures. The fluid proto-plasm in the egg of a sea urchin, a clam, or a worm can be subjected to centrifugal force strong enough to move the particles suspended in it to this or that region of the cell; and yet, no matter how these particles are moved, the egg preserves its basic organization and the various parts of the embryo or larva develop normally, quite independently of whether or not they contain one type of granule or another, or are essentially free of granular inclusions. (For a discussion of the evidence in support of this point, see Morgan, 1927.) Doubtless there must be some part of the egg cell which is not moved by the centrifugal forces sufficient to move the granules in the interior of the cell, some rigid portion which can control the manner in which different parts of the protoplasm develop. The nature of this rigid layer will become evident shortly, as we proceed with our discussion.

That cells as a whole show elastic properties has been known since the time of Leeuwenhoek. He noted that red blood cells

32

PROTOPLASMIC GELS

3. PROTOPLASMIC GELS 33

were frequently distorted when they passed through narrow vessels, but that they regained their original shape following such distortion. Following Leeuwenhoek, many early students of blood cells saw them flatten when they were compressed between slide and coverslip, and then regain their original shape when the pressure on them was removed. ( For references to early observa-tions of this sort, see Heilbrunn, 1928.) Not only blood cells, but other types of isolated cells—marine egg cells and protozoa —behave in similar fashion. Cole (1932; see also Cole and Michaelis, 1932) attempted to make a quantitative study of the amount of force necessary to flatten sea urchin eggs. The force was small, indicating that the egg as a whole possessed but little elasticity. However, the force necessary to compress the egg increased as during compression the surface grew larger. This Cole believed indicated the presence of an elastic membrane around the egg, for if forces of interfacial tension alone were involved, there would be no such increase as the surface in-creased. (This follows from the fact that surface or interfacial tension does not increase appreciably as the surface is stretched. )

The elasticity of the sea urchin egg as a whole, that is to say, its ability to resist deformation, does not depend entirely on the outer membrane of the cell. For there is a cortical layer directly beneath the hyaline outer membrane that surrounds the cell, and this cortical layer is a gel. In the sea urchin egg, the cortical layer or cortex was first clearly demonstrated by Moser ( 1939 ). If the eggs of Arbacia are centrifuged with forces several thousand times gravity, light and heavy granules go to opposite ends of the cell, leaving the clear, hyaline protoplasm in the middle. But the clear region is not completely devoid of granules, for if one focuses carefully, one can see a single layer of tiny granules directly under the outer membrane of the cell. These are the granules of the cortical layer—the cortical granules; see Fig. 6. They disappear at fertilization, but very soon a new cortex forms, and this is much thicker than the cortex of the unfertilized egg. The cortical layer in the unfertilized sea urchin egg is somewhat thinner than the cortex in the egg of the worm Chaetopterus. This cortex was described by Lillie (1906); he studied living


F I G . 6. A segment of a centrifuged e g g of the sea urchin Arbacia

punctulata photographed with a television microscope. The cortical granules

appear as bright particles directly beneath the outer membrane. T h e

photograph was taken b y Dr. A. K. Parpart of Princeton University.

sea urchin egg. Lillie cites the work of older authors who de-scribed the presence of an outer layer in the eggs of various invertebrates, a layer distinguishable in one way or another from the protoplasm of the interior. These earlier authors usually referred to the cortical layer as the ectoplasm. More than anyone else, Just has emphasized the importance of the cortex or ecto-plasm. In a paper published in 1931 and in a book published in 1939, he pointed out numerous examples of a differentiated cortex in many types of cells from ameba to man, and he tried also to show that the cortex was highly important physiologically.

However, from our standpoint, the mere description of a morphologically distinct region in the periphery of a cell does not give us any real information as to the physical relationships. Much more important from our point of view are the observa-tions of Moser and Lillie on the eggs of Arbacia and Chae-topterus, as well as the work of Rieser on muscle cited in the last chapter. For work of this sort shows clearly that the fluid protoplasm of the cell is surrounded by a stiff gel-like outer layer.

centrifugea eggs and also eggs which had been sectioned and stained both before and after centrifuging. In the centrifuged egg, the cortex contains only a single layer of granules; these granules are decidedly larger than those in the cortex of the


Apparently it is this cortical gel of the Chaetopterus egg that is largely responsible for the different developmental potencies of various regions of the egg. When the egg is centrifuged with forces only strong enough to move the interior granules, then the development of the egg does not appear to be affected, ( compare Lillie, 1908 ). However, if somewhat higher centrifugal forces are used so that the granules are displaced from the cortex, then, according to Wilson and Schub (1953), normal develop-ment does not occur. This work of Wilson and Schub was reported only briefly. The subject should be of general interest to embryologists and more work needs to be done not only on Chaetopterus but also on other eggs in which the granules in the cortex can be moved by centrifugal force.

It is clear that in the cortex of some marine egg cells the granules are not readily displaced by centrifugal force and it is for that reason that we have called the cortex in these cells a gel. But perhaps at this point it might be well to inquire into the proper definition of a gel. As a matter of fact, various authors use the term in different ways. Hermans (1949) has defined gels as colloidally dispersed systems of at least two components, systems which exhibit mechanical properties characteristic of the solid state. Moreover, according to Hermans, in true gels, or at least in most true gels, the dispersed component and the dispersion medium are continuous throughout the whole system; that is to say, the dispersed material forms a ramifying network through the dispersion medium. This network may have rod-like or fibrillar units or it may have spherical particles adherent to each other in a more or less linear arrangement. Actually it is not an easy matter to discover whether a gel satisfies this criterion or not. Photographs taken with an electron microscope do show a network structure in some gels, but in order to obtain such pictures of gels in which water is the dispersion medium, it is necessary first to evaporate off all the water, and this might very well change the nature of the gel structure. If protoplasmic gels are to be studied in the living state, certainly it is not pos-sible to study them with the electron microscope. Fortunately, even in the living cell, it is possible to show that the cortex does


have the mechanical properties of a solid. Hence, in so far as it is possible to determine, and in so far as our present evidence goes, the cortex is a gel.

Let us inquire a little further into the nature of the evidence. Actually, there isn't very much, for in only two types of cells has the cortex been studied from a physical standpoint. The first studies of this sort were made on the ameba. With the centrifuge method the viscosity both of the interior protoplasm and of the cortex can be measured. In order to accomplish this, it is neces-sary to use two different species of amebas. In Amoeba dubia, the cortex is rather thin so that one can easily see through it. In this ameba then, it is possible to study the viscosity of the interior protoplasm; and one can show, as was mentioned in the last chapter, that the viscosity of this protoplasm is of the order of magnitude of several centipoises. On the other hand, in a closely related species, the common Amoeba proteus, the cortex is relatively thick; and in this ameba, although it is not practi-cable to study the viscosity of the interior, it is possible to study the viscosity of the cortical protoplasm. All that is necessary is to determine the time it takes, at a given centrifugal force, for the large granules or crystals of the cortex to move into one end of the cell. The endpoint of such a determination is shown in Fig. 7, which is a drawing of a centrifuged Amoeba proteus.

F I G . 7. A centrifuged spec imen of Amoeba proteus.


The viscosity of the cortex in Amoeba proteus is obviously high, for strong centrifugal force must be exerted for a considerable time in order to move granules through it. Moreover, the proto-plasm of the cortex is thixotropic; that is to say, it can be made to become much less viscous if it is subjected to uneven mechani-cal pressures. Angerer in 1936 found that if Amoeba proteus is placed on a shaking machine the viscosity of the cortex drops until it is only a small fraction of the original value. Eventually the cortex becomes so fluid that the protoplasm of the ameba disperses into the surrounding medium. Thus when subjected to a shearing tension, the solid cortex changes from solid to liquid; that is to say, it shows a yield value. This is an essential criterion of a gel.

The cortex of the Chaetopterus egg cell can also be shown to be a gel. For if one tries to dislodge granules from it by cen-trifugal force, this is only possible if strong centrifugal force is applied. In other words, the protoplasm of the cortex in this egg also shows a yield value. Figure 8 shows the appearance of the cortex in a Chaetopterus egg which has been subjected to a relatively weak centrifugal force; and Fig. 9 shows the effect of a stronger centrifugal force, one strong enough to dislodge the granules from the cortex.

Both in the ameba and in the Chaetopterus egg, it is possible

F I G . 8. A Chaetopterus e g g w h i c h

has b e e n subjected to a weak centrif-

ugal force. T h e cortical granules ap-

pear beneath the outer membrane .

F I G . 9. A Chaetopterus e g g w h i c h

has b e e n subjected to a strong centrif-

ugal force. T h e cortical granules

have b e e n dis lodged.


to determine the effect of various influences on the cortex. In the ameba this is done by measuring the viscosity of the cortex of Amoeba proteus by the ordinary centrifuge method. In the Chaetopterus egg, it is rather a simple matter to measure the amount of force necessary to dislodge the granules under one set of conditions or another; and in this way it is possible to discover how the rigidity of the cortex varies and how these variations are related to the vital activity of the cell. Knowing to what extent and in what fashion the rigidity of the cortex is affected by various physical and chemical agents, it is possible to draw some conclusions as to the nature of the cortical gel.

Both in the ameba and in the Chaetopterus egg, the cortex is liquefied both by heat and by cold. For the ameba, this was clearly shown to be true by Thornton (1935). His work is illustrated in Fig. 10. For the Chaetopterus egg, Wilson and Heilbrunn (1952) found that when the eggs were exposed to a temperature of 38° C , the rigidity of the cortex decreased so much that there was a 40% drop in the amount of centrifugal force required to dislodge the granules from it. And strangely enough, the rigidity of the cortex decreased almost exactly as much when the eggs were cooled to a temperature of 0° C. At this temperature the amount of centrifugal force required was 38 per cent less than that required at 21° C. The fact that both heat and cold have similar effects on the cortical protoplasm is indeed a strange phenomenon, one hard to explain in terms of colloid or polymer chemistry. And yet it is especially interesting in view of the fact that in the behavior of living systems cold and heat often act in similar fashion. Thus both heat and cold may produce the same sort of pathological effects on the proto-plasm, both heat and cold can stimulate a muscle, and both heat and cold under certain conditions can act as anesthetics. Much will be made of this later when we come to discuss the response to stimulation and the prevention of such response. At that time an attempt will be made to give an explanation of why it is that cold and heat can act in the same way.

Perhaps the most significant fact about the protoplasmic colloid that constitutes the cortex is the fact that the rigidity of this gel


seems to be dependent on the presence of calcium ion. If the calcium ion is displaced from the cortex of the ameba by immers-ing the ameba in a solution containing an excess of potassium ion, the cortex changes from a condition in which it has a high vis-

280

Temperature, eC .

F I G . 10. Viscosity temperature curves for the cortex

of Amoeba proteus. T h e ordinates represent arbitrary

values for the viscosity. T h e three curves are for amebas

in dilute KCl solution, in dilute C a C l 2 solution, and in

culture fluid ( a weak infusion of hay and w h e a t ) .

cosity to a condition of low viscosity ( Heilbrunn and Daugherty, 1932 ). Similarly if the ameba is immersed in an oxalate solution, the viscosity of the cortical gel decreases sharply (Heilbrunn and Daugherty, 1933). On the other hand, if the ameba is ex-posed to solutions rich in calcium, the viscosity of the cortex rises markedly. Exactly the same relationship can also be demon-strated for the cortex of the Chaetopterus egg cell. There, ac-cording to the work of Wilson and Heilbrunn (1952), calcium


also increases the rigidity of the cortex and oxalate markedly reduces it. Indeed, when Chaetopterus eggs are exposed to solu-tions containing potassium oxalate, the amount of force necessary to dislodge the granules from the cortex is only one-fourth that required when the cells are in sea water.

In the chapters to follow, much emphasis will be placed on this relation of the cortex to calcium, and it will be shown that much of the vital activity of the cell is dependent on the relationship. Various agents that affect the living processes apparently cause a release of calcium from the cortex. Not only does this have a marked effect on the cortex itself, but it also causes marked changes in the interior protoplasm.

As has already been indicated, the cortex is beyond doubt a highly important part of the cell. Morphologically it is seen to lie beneath the enveloping membrane of the cell. But it should be remembered that the cortical gel is a membrane in itself, and as such it may well act as a barrier to the penetration of sub-stances into the cell and the exit of substances from the cell. Thus it may well form a part of what we know, on physiological grounds, as the plasma membrane of the cell. Possibly the mem-brane that surrounds the cortex is very similar to the cortex itself both in its chemical nature and its physical behavior. The evi-dence for this idea comes from two directions. In the first place, all those agents which tend to liquefy the cortex weaken the outer membrane of the cell so that on centrifugal treatment the cell more rapidly and more readily is broken into two halves. Secondly, in view of the fact that removal of calcium from the cortex markedly weakens the rigidity of the cortex and at the same time greatly increases the permeability of the plasma mem-brane,* it seems logical to assume either that the plasma mem-brane and the cortex are part and parcel of the same thing, or that at least they have similar properties.

As we have seen, in the cells that are best known from a physio-logical or a physical standpoint, the fluid protoplasm of the in-terior is surrounded by a firm cortex. Unfortunately, however,

* For evidence on this point and references to the literature, see Heilbrunn,

1952b, pp . 165 and 166.


there are not many cells whose physical makeup is known with any certainty, and there is a strong possibility that some cells may consist very largely of material in the gel state. Thus, for a tiny cell, the cortex might occupy a large part of the whole volume of the cell, it might even constitute the entire cell. The protoplasm of the immature egg cells of various invertebrates is much more viscous than the protoplasm of the same cells after the large nucleus of these cells—the so-called germinal vesicle-breaks down. Lillie ( 1906 ) commented on this fact in his study of the Chaetopterus egg, and it is easy to show a similar behavior for the egg of the worm Nereis and the egg of the clam Spisula. In the case of Chaetopterus, the protoplasm of the immature egg is about seven or eight times as viscous as it is in the mature egg. This protoplasm appears to be slightly thixotropic, but the degree of thixotropy is not great; at least this appears to be true from the preliminary observations of Heilbrunn and Wilson ( 1955b ). In the immature Spisula egg, the protoplasm is perhaps six times as viscous as it is in the egg after maturation, but in this egg a few tests failed to give any clear evidence for thixotropy.

One thing is certain. In all types of protoplasm that have been investigated, there are, in the course of vital activity, sudden and sharp increases in viscosity, and these have in the past been interpreted as being due to gelations in the protoplasm. It is hard to know how else to interpret them. In some cases it is un-doubtedly true that one or another of the protein constituents of the protoplasm stiffens into a gel. However, the protein that forms the gel may constitute only a rather small fraction of the total protein of the cell. Hence such a gelation of merely one constituent, out of some few, would presumably not cause a very marked change in the viscosity of all of the protoplasm.

Within the fluid protoplasm of the cell interior there may be inclusions which are solid gels. Often these can be recognized because of the fact that they are biréfringent (compare Schmidt, 1937). When a cell divides, the mitotic spindle that appears in the course of the division process is definitely biréfringent, and there have been a number of studies on the birefringence of the spindle, beginning with the pioneer work of Schmidt (for the


earlier literature, see Schmidt, 1939). As a matter of fact, long before Schmidt's observations it was clearly recognized that the spindle behaved like a solid body. For if an egg cell is centri-fuged at a time when the spindle is present, the spindle can be seen to pull on the outer membrane of the cell so that the cell becomes distorted (Conklin, 1912; Heilbrunn, 1920). This is an indication not only that the spindle is solid, but also that it is attached to the periphery of the cell by solid strands that radiate out from the ends of the spindle. (If centrifuging is sufficiently vigorous, the spindle pulls away from its moorings and comes to lie in the clear region of the centrifuged cell between the lighter fat particles and the heavier granules of the cytoplasm. ) Another bit of evidence that both the spindle and the chromosomes on it are gels, is the fact that if water is extracted from a dividing cell by hypertonic solutions, the cytoplasm shrinks markedly, but the spindle and chromosomes to all intents and purposes retain their original volumes ( Belar, 1927 ). Other structures in the cell that are probably gel-like are the nuclear membrane and the membranes that surround the mitochondria.

In our discussion of the dynamics of protoplasm, we are pri-marily concerned not with inclusions of the protoplasm but with the cortex and the main mass of the cytoplasm. Our knowl-edge of the machinery of the living substance is only in its beginning stages, but the information that we do have about the colloidal properties of the cortex and the interior protoplasm can even now tell us something about the mechanical events that take place when a cell is thrown into activity. However, the mechanical changes are not the only ones that take place, for whenever a cell is aroused there are electrical effects which in some cells can be readily studied and which are undoubtedly important. Much is known about the magnitude of these elec-trical changes and the conditions which induce them, but the question as to how they can be interpreted in terms of the physi-cochemical nature of the cell is still not clear. Because of the importance of the subject, the next chapter will be devoted to a discussion of the electric properties of the cell both when it is at rest and when it is thrown into activity.

4.

THE ELECTROCHEMISTRY OF THE CELL

For over a century and a half it has been known that living systems could generate electricity; and from the first knowledge of this remarkable fact there has been a steady stream of specu-lation as to the nature of the differences of potential that are responsible for the phenomenon. In recent years large volumes have been written on the subject. Obviously in this single chapter it will scarcely be possible to review more than a small fraction of the work that has been done; and we shall be con-cerned not so much with the many interesting measurements that have been made, but rather with an attempt to understand how the presence of potential differences and the changes in potential difference can possibly be interpreted in the light of what we know about the chemistry and physics of the proto-plasmic colloid.

In recent years much of the best work in the field has been done not by general physiologists but by investigators with a special (and rather limited) interest in nerve, or perhaps in both nerve and muscle. The work of these investigators has often been a model of precision and ingenuity, but for the most part the conclusions that have been reached are not acceptable to a general physiologist whose background and training have conditioned him to believe that the behavior of protoplasm is fundamentally similar in all types of living systems. Of course, no two types of cells are exactly alike, but in any particular case until evidence is brought to the contrary, the general physiolo-gist will continue to believe that any manifestation of the vital process common to almost all types of protoplasm is funda-mentally similar in all of them.

In what follows, an attempt will be made to use as simple,

43


nontechnical language as possible. Many of those who will read this book are not well trained in the science of electrochemistry, and if the discussion is to be at all understandable to them, it must be kept simple even at the risk of offending those who would prefer a more advanced and a more impressive treatment. To them, my apologies.

Most of our knowledge about the electrochemistry and the electrophysiology of cells and protoplasm is concerned with the electric potential differences that can be shown to exist between one part of a cell or tissue and another. These potentials can be measured as voltages with electrometers, potentiometers, volt-meters, or oscillographs. They are similar to the voltages that one can measure across the poles of an electric battery. But for the colloid chemist, there is another type of potential that is of particular interest. This is the electrokinetic or zeta potential. Bubbles, drops, and large or small particles suspended in a liquid are electrically charged, either positively or negatively. And in the liquid surrounding the suspended particle there is a layer containing charges of opposite sign. Thus, just as in a condenser, a layer of positive charges is separated by a dielectric from a layer of negative charges, so too in a colloidal system of sus-pended particles, each particle is surrounded by a double layer.* The difference in potential between the two layers of opposite charge is the zeta potential. But whereas in a condenser it is possible to connect positive and negative charges with a con-ductor and so obtain an electric discharge and a flow of electric current along a wire, in the case of the double layer on colloidal particles this is not possible, and therefore the zeta potential can-not be measured in any simple or direct fashion. However, by passing an electric current through the suspension and noting the speed with which the charged particles move for a given difference of potential of the applied electric current it is pos-sible to calculate the zeta potential on the basis of theoretical

* Helmholtz thought of the double layer as consisting of a single layer of charges on the particle and a single layer of opposite charges immediate ly surrounding the particle; but more recent authors have thought of the charges in the l iquid as be ing not in a single layer, but in a somewhat dispersed region or atmosphere.

4. THE ELECTROCHEMISTRY OF THE CELL 45

equations proposed by Smoluchowski, Hiickel, and others. These equations all include a term for the dielectric constant. This constant is well known for water and for various other liquids, but it is not so well known for the solutions of electrolytes with which we are concerned, and for such solutions both the meas-urement of the constant and its calculation on the basis of theory are somewhat uncertain (compare, for example, Falkenhagen and Leist, 1952).

In the present state of our knowledge concerning the electro-chemistry of the cell, we are not so much concerned with the absolute magnitude of the zeta potential, as we are with know-ing a few elementary facts about the distribution of electric charges on the various colloids of the cell and its protoplasm.

When particles in a suspension are exposed to an electric cur-rent, if they are positively charged, they move toward the cath-ode; and if they are negatively charged, they move toward the anode. This migration is called electrophoresis. There have been some few studies on the electrophoresis of blood cells, sperma-tozoa, and bacteria. These small cells are relatively easy to study, for because of their small size they stay suspended long enough for measurements to be made. With relatively large cells measurement is more difficult, but by watching the fall of marine eggs through sea water across which an electric current was flow-ing, Dan (1933, 1934) was able to obtain values for the zeta potential of the eggs (on the assumption that the dielectric con-stant of sea water is the same as that of distilled water, which it is not).

In general, whether cells are large or small, almost all of them have been found to migrate toward the anode. The speed of this migration and the electrokinetic potential as calculated from the speed vary somewhat for different types of cells. But for the same type of cell under different conditions of rest and activity, in so far as known, the zeta potential does not seem to vary very much, and those who looked for changes in the zeta potential as an index of vital activity have on the whole been disappointed. In interpreting the values obtained for the speed of migration or for zeta potential, it should be remembered that


the theory of electrophoresis has been developed for small, homo-geneous particles, in which the electric charge is all at the sur-face. Thus for a quartz particle or for the metallic particle of a colloidal metal the problem of electrophoresis is simpler than it is for a cell which contains within it various colloids with their own electric charges. If we could assume that, when cells are placed in the path of an electric current, none of the current passes into the cell, we could then conclude that the values obtained for the zeta potential are values that pertain only to the outer surface of the cell. But in the light of modern knowl-edge as to the permeability of the plasma membrane for ions no such assumption can properly be made; although in solutions of high conductivity, a relatively small amount of the current penetrates deep into the cell interior. If ions enter the cortex readily, as they apparently do, the colloids of the cortex might to some extent influence the electrophoretic behavior of the cell as a whole.

The fact that, in general, the surfaces of cells are negatively charged indicates that the proteins which are present at the surface are on the alkaline side of their isoelectric point. On the alkaline side of the isoelectric point, proteins are combined with cations, and the protein part of the molecule behaves as an anion. Thus the proteins at the surface (and presumably also in the cortex) are probably largely in the form of calcium and magne-sium proteinate.

It would be interesting if we could determine the isoelectric point of the proteins at the cell surface. If we place cells in solu-tions of lower and lower pH, there should be some pH at which the cells migrate to the cathode rather than to the anode. How-ever, generally speaking, it is only at a pH well below 4 that such a reversal in the direction of migration occurs, and at such a low pH the cell is certainly dead.

In this brief discussion of the electrophoretic behavior of cells no attempt has been made to cite references to the literature. Many of these may be found in Heilbrunn, 1952b. The main point that has been clearly established is that the outer surface or outer region of the cell contains proteins (and perhaps other


substances) at the alkaline side of their isoelectric point, so that presumably the proteins are capable of combining with cations. As will be shown later, this is a fact of some importance for the understanding of the potentials responsible for the electric cur-rents generated by living cells.

The outside of a cell is negatively charged—but what about the colloids inside? We must admit that there is very little informa-tion, and this is partly because the information is rather hard to come by and partly also because the data are rather hard to interpret. As far back as 1864 Kühne let an electric current flow through the protoplasm of a slime mold and he saw the proto-plasmic granules move toward the cathode. There are other early observations of the same sort. Then, in a short paper published in 1913, Hardy presented what appeared to be unequivocal evidence that the protoplasm in the cells of an onion root tip migrated toward the cathode. Hardy illustrated what he saw by a drawing that looks decisive. However, Hardy's experiment is not an easy one to repeat. As Hardy himself found, if too much electricity is allowed to flow through the root, the protoplasm reverses its charge and migrates toward the anode. Thus in any repetition of the experiment, it is essential that the current be not too strong; nor can it be too weak, for then the protoplasm will not move at all. Moreover, there is another difficulty with the experiment. Just as soon as the current ceases to flow through the plant cells, there is undoubtedly a polarization current that starts to flow in the opposite direction. But the cells in the onion root can not be studied until sections have been made. Hence as soon as the current ceases to flow it is essential to kill and fix the root as rapidly as possible so that the protoplasm which has moved to the cathode will not have time to move back again. In my laboratory, attempts to repeat Hardy's observations have at best been only partially successful. The experiment is not as simple as one might conclude from reading Hardy's paper.

Two facts are certain. In protoplasm that has been killed the charge on the protoplasmic particles is negative, just as it is on most particles suspended in water. And secondly, the chromatin in the nucleus is negatively charged. Such a negative charge


on chromatin and chromosomes has been reported not only by Hardy, but also by various other observers (Pentimalli, 1909, 1912;' McClendon, 1910; Botta, 1931; Lehotzky, 1935; Yamaha, 1937; Churney and Klein, 1937). In Hardy's figure he shows no movement of the nucleus either to the anode or to the cathode. When an electric current is sent through the salivary gland cells of fly larvae, Churney and Klein found that the nucleus as a whole moves toward the cathode, an indication perhaps that the cytoplasm is a positively charged colloid, for the nuclear mem-

F I G . 11. T h e effect of an electric current

on t w o brain cells of Torpedo ocellata. The

arrow shows the direction of the current.

The nuclei have pushed over toward the

cathode. Also the chromatin has migrated

toward the anode, not only in the brain

cells, but also in the connect ive tissue cells

which appear at the right of the picture.

brane would have adsorbed on it the protein of the cytoplasm. Churney and Klein's observation about the movement of the nucleus is not the only one in the literature, for many years ago Dahlgren (1915) showed a movement of the nucleus toward the cathode in brain cells of the electric ray. One of Dahlgren's figures is reproduced as Fig. 11. In the electric ray, the animal is accustomed to electric currents, so that presumably the cells were not injured by the passage of the current.

In any attempt to determine the sign of the charge on the colloidal particles of the main mass of the protoplasm, it is essen-tial that the protoplasm remain alive during the course of the tests. Accordingly, instead of working with cells like those of


the onion root tip, cells which can not be seen until the material is killed and sectioned, it is advisable to study cells which can be observed in the living condition and in which it is possible to determine when the cell is seriously injured. Heilbrunn and Daugherty (1939) passed electric currents through the trans-parent leaves of the common water plant, Elodea. In the living cells of this plant, the protoplasm constantly rotates around the cell carrying with it easily observable green chloroplasts; such rotation ceases when the cell dies. Using currents which were not strong enough to kill or seriously injure the cells, Heilbrunn and Daugherty determined the effect of the current on the rate of travel of the chloroplasts with the electric current and their rate of travel against the current. The results were at first thought to be disappointing, for in some cases the chloroplasts moved more rapidly toward the cathode, in other cases their speed was more rapid toward the anode. Of 40 cells tested, only 16 showed a greater speed of chloroplasts toward the cath-ode, 24 showed a greater speed toward the anode. Thus it would appear that the charge on the protoplasmic colloid is a matter of no great significance, for sometimes it appears to be positive and sometimes negative, more often negative. But let us consider for a moment what determines the sign of the charge. Un-doubtedly the hydrogen ion concentration of the protoplasm is a factor of primary importance. All living protoplasm is con-stantly producing large amounts of an acid, carbonic acid, and this would tend to give the colloidal particles of the protoplasm a positive charge, especially in the immediate neighborhood of inclusions which are centers of carbon dioxide production. But in cells which are actively engaged in the process of photosyn-thesis the carbon dioxide which is produced is immediately utilized. Hence in such cells the protoplasm may be decidedly less acid and this would tend to permit a negative charge on the colloidal particles of the protoplasm. The cells which Heilbrunn and Daugherty studied in their first series of experiments were cells exposed to rather strong light, and undoubtedly photosyn-thesis to a greater or less extent was occurring in them.

Obviously it would be interesting to know about the sign of


the charge in Elodea cells which were not undergoing photo-synthesis. This is not so easy to discover. In the dark, of course, photosynthesis stops, but in the dark it is not possible to observe the cells under the microscope. What Heilbrunn and Daugherty did was to keep the Elodea plants in the dark for 6 to 9 days; then after they were removed from the dark, there was a short time interval during which photosynthesis did not occur. Measurements made during this time interval gave consistent results. In 24 of the 26 experiments the chloroplasts showed a very significant increase in speed when they were moving toward the cathode. In 2 experiments there was no significant difference in speed towards anode and cathode. Thus it seems clear that in the case of Elodea cells not undergoing photosynthesis the charge on the particles of the protoplasmic colloid is positive. Also, apparently, this charge is dependent on the production of carbon dioxide in the cell. When cells are killed so that no carbon dioxide is produced the charge on the particles of the dead protoplasm becomes negative. Thus when Elodea cells are exposed to strong electric currents, currents strong enough to stop the streaming of the protoplasm and presumably to kill or at least to seriously injure the cells, the charge on the chloroplasts of the protoplasm is negative (Tobias and Solomon, 1950).

When an electric current is passed through an ameba, there is a vigorous movement of the cytoplasmic inclusions toward the cathode. This has frequently been noted by observers in the past (see, for example, Verworn, 1896; Mast, 1931; Hahnert, 1932) and it can easily be demonstrated by beginning students in the laboratory. Indeed it is a pretty sight to watch. Through the body of the ameba the protoplasmic granules rush toward the cathode quickly reversing their direction as the current is reversed. The ameba as a whole moves in the same direction as the granules. Most of those who have studied the phenomenon have suggested that the movement of the granules is a conse-quence of the movement of the ameba and that this is due to some change at the surface of the cell. Thus it might be sup-posed that the cortex liquefies on the cathodal side and that this then causes ameboid (and granular) movement toward the


cathode.* It seems wiser to assume that the cytoplasmic granules and inclusions of the ameba are positively charged and that it is their electrophoretic movement that is responsible for the move-ment of the ameba toward the cathode. Particles moving electro-phoretically can exert considerable pressure, pressure sufficient to cause the liquefaction of a thixotropic gel such as is present in the ameba cortex. If it is true that the movement of the ameba in an electric field is due to movement of positively charged particles, then if it were possible to reverse the charge on these particles, it should be possible to make the ameba move toward the anode. Actually this can be done. For if the interior proto-plasm of the ameba is made alkaline, then the ameba, if it does not die, usually moves toward the anode (Heilbrunn and Daugherty, 1939).

When an ameba is moving in a relatively strong electric field, it often happens that the anodal end of the cell breaks, and the protoplasmic inclusions are set free in the surrounding medium. When this occurs, the protoplasmic inclusions which were mov-ing vigorously toward the cathode immediately move in the opposite direction. Probably this is due to the fact that once released from the cell the granules are no longer in a medium rich in carbon dioxide, and thus as the pH of their immediate environment becomes higher, they become negatively charged.

In the discussion thus far, we have tried to show that although the exterior region of the cell has negatively charged colloids, the colloids of the main mass of the protoplasm are positively charged, or at any rate they are positively charged in cells in which carbon dioxide is being rapidly evolved. We can thus assume that the proteins or macromolecular compounds in the interior cytoplasm of the cell are on the acid side of their iso-electric point. This is rather hard to believe, for the isoelectric points of most proteins are lower than what is generally believed to be the pH of the protoplasm. However, we do not really know

* As a matter of fact, if the electric current is strong enough, it causes a

l iquefaction not at the cathodal end but at the anodal end, a l iquefaction doubt-

less to be associated wi th the migration of calc ium from the cortex at the anodal

end.


the isoelectric points of proteins in living cells where conditions are quite different from what they are in a test tube. Another possibility is that the hydrogen ion concentration at the surface of, or in the immediate vicinity of a granule which is actively and vigorously producing carbon dioxide may be decidedly greater than the hydrogen ion concentration either of the protoplasm at a little distance from the granule or of the material within the granule, and this may account for the electrophoretic behavior of the granule. Actually there is evidence that at least some of the proteins in the cell interior do indeed act chemically as though they were on the acid side of their isoelectric point. This will be discussed later.

Our concept of a cell as having an outer region with colloids negatively charged, an interior with colloids positively charged, and within that a nucleus whose inclusions bear a negative charge, might seem to open up various possibilities of potential differences between different parts of the cell. But it must be remembered that the charges and the electrokinetic potentials we are discussing are balanced by opposite charges in their im-mediate vicinity, so that they can not be responsible for any flow of electrons which would produce an electric current. Never-theless, if we assume proteins on the alkaline side of their iso-electric point in the outer rim of the cell and proteins on the acid side of their isoelectric point in the cell interior, we do indeed have a possible mechanism for interpreting bioelectric potentials and bioelectric currents. But before we enter into this discussion, it will be necessary to present a very brief resume of some of the facts known about bioelectric potentials. Nothing like a com-plete statement is possible. More details and some references to the literature will be found in Crane (1950) and Heilbrunn (1952b).

Broadly considered, the bioelectric potentials of living cells have the following characteristics:

1. The potential differences actually can produce a flow of current which may continue for a considerable time period.

2. Any injured region is negative to an uninjured region, that is to say, outside the cell, current will flow from an uninjured region to an injured region.


3. Any region which is actively responding to a stimulus is negative to nonactive regions.

4. In large cells, the interior of the cell is negative to the ex-terior. ( This does not necessarily apply to cells capable of photo-synthesis. )

5. Agents which decrease the rate of respiration decrease bio-electric potentials, but there is no close correlation between the lowering of the respiration rate and the change in potential.

6. Various substances, but especially the potassium ion and fat solvent anesthetics, depress bioelectric potentials. (On the basis of an old theory, the Bernstein theory, the substances which depress bioelectric potentials are often called depolarizing sub-stances. )

There is a question as to whether any general theory of bio-electric potentials is possible, whether the potential differences which arise in the cells of the tissues of one organism are essen-tially similar to those which arise in the cells of other tissues of the same or widely different organisms; whether, moreover, similar explanations can be used both for injury potentials and for action potentials. Modern biology and modern biochemistry have shown the amazing similarity of all sorts of living material; and until it is necessary to assume one type of theory for one cell and another for a different cell, it seems logical to assume that one explanation, with minor modifications, will serve for all. The injury and the action potentials of cells of the algae Chara and Nitella have similar characteristics to the injury and action potentials of human nerves. Moreover, whereas relatively mild stimulation causes excitation or response in protoplasm, ex-cessive stimulation results in injury. Hence it is logical to sup-pose that action potentials and injury potentials depend on the same changes in the cells from which they both originate.

The Bernstein theory, a favorite theory for so many years, did indeed postulate a similar mechanism for both action and injury potentials. This theory assumed that the cell was surrounded by a membrane permeable to cations but not to anions. The potas-sium ion commonly present in large amounts in living cells was supposed to pass through the membrane, but to be held im-


mediately outside the cell by a layer of anions unable to escape through the membrane. The membrane thus separated a layer of positive charges on the outside of the cell from a layer of negative charges on the inside. Injury broke through this double layer and discharged the condenser-like system of opposite charges. Excitation had a similar effect, for it was thought to increase permeability of the membrane and to permit a passage of ions across it at the point of excitation. The theory had a great appeal, especially to the many general physiologists who believed in the permeability theory of excitation, now generally aban-doned.

The Bernstein theory is no longer tenable for three reasons. In the first place, bioelectric currents in many cases do not resemble the discharge of a condenser, for the currents may persist for hours. Thus, when a muscle is cut, the injury current continues to flow for a long time, and after it begins to fade, it can always be renewed by making a fresh cut. Secondly, recent studies on permeability give no support to the idea that cell mem-branes are generally impermeable to anions. And thirdly, in the case of both nerve and muscle, various observers have found that on excitation the potential between the inside and outside of a cell does not merely drop to zero, as it would have to do on the basis of the Bernstein theory, but is actually reversed, so that the inside of the cell, originally negative to the outside, becomes strongly positive to it. Because of this last difficulty originally discovered by Hodgkin and Huxley in 1939 and then confirmed by Curtis and Cole ( 1942 ) and many others, Hodgkin and Katz (1949a) proposed a modification of the Bernstein theory. They suggested that although in the resting nerve fiber the membrane surrounding the fiber is more permeable to potas-sium than it is to sodium ion, at the moment of excitation, a great increase in permeability to the sodium ion occurs, so that the membrane becomes much more permeable to sodium than to potassium. Hodgkin and his various collaborators devised many experiments to support this hypothesis, and the hypothesis was accepted immediately and enthusiastically not only in England but also in various other parts of the world. In the short space of


a few years many papers on the subject have appeared and most of them support the Hodgkin hypothesis. It would be tak-ing us too far afield to attempt a survey of this voluminous literature; useful reviews have been published by Hodgkin (1951) and Eccles (1953). Hodgkin believes that at the instant the action potential is increasing in magnitude, there is an inrush of sodium ion into the cell, then when the potential is falling off, there is a rapid exit of potassium ion, and that these ionic changes are the cause of the changes in potential. Actually there is no doubt that when a nerve fiber is aroused sodium does indeed enter the fiber and potassium does leave, but whether the sequence of sodium gain and potassium loss is as Hodgkin believes it must be, would be rather hard to determine, for the rise and fall of the action potential occur in very tiny intervals of time and this fact seems to preclude the exact measurements necessary for a complete justification of the theory. If the Hodgkin theory is correct, then in the complete absence of sodium, no action potential should arise. For the squid nerve, Hodgkin and Katz have been able to show that when the sodium of the environment is replaced by sugar or by choline, the action potential does not appear. This experiment is perhaps open to question, for one of the difficulties of understanding the action of sodium is to find substitutes for it; this has long been recog-nized by general physiologists. A very large proportion of the osmotic pressure of sea water and of body fluids is due to sodium salts, and it is practically impossible to replace this sodium with other substances that are without effect. Also when sodium is ?*emoved, the potassium ion is no longer properly antagonized, so that the result may not merely be a loss of sodium but also an increase in the potency of the potassium ion that remains. In the squid nerve, action potentials do not appear except in the presence of sodium or lithium ions. However, in the case of crab muscle, the sodium ion may be replaced by tetraethylammonium ions and under these conditions the action potentials are even larger than they are in the presence of sodium ions (Fatt and Katz, 1953). Concerning the frog nerve, there is much contro-versy. Lorente de No (1949) placed frog nerves in solutions in


which the sodium ions had been replaced by various quaternary ammonium ions and found that action potentials could still be obtained, but his work has been attacked on the ground that the sheath surrounding the frog nerve may prevent the sodium ions surrounding the nerve fibers from diffusing away. At present, the subject of frog nerve is controversial. But even if the action potential did occur in the presence of ions other than sodium, it might be possible for supporters of the Hodgkin theory to maintain that the various substitutes for the sodium ion can act like it and can rush into the cell and thus be responsible for the action potential.

But there is one type of action potential which can certainly be produced in the complete absence of sodium (or any other ion) in the environment. Years ago, Osterhout and Hill (1933) placed Nitella cells in distilled water and found that the action potentials of these cells, which are very much like the action potentials of nerve and muscle cells, persisted for at least two days. And, finally, after the cells in the distilled water were no longer able to give an action potential, the ability to produce an action potential could be restored not by sodium ion, but by the addition of a trace of calcium ion. Thus, in the case of Nitella cells, the ion which is important for the genesis of the action potential is calcium. In frog eggs also, according to Umrath (1954), action potentials appear in the absence of sodium.

We are now in a position to correlate our knowledge of bio-electric potentials with the information we already have about the nature of the living cell. Instead of postulating, as Bernstein did, a hypothetical membrane which can now clearly be shown to be nonexistent; or imagining, as Hodgkin does, a membrane with extremely peculiar properties which can neither be proven to exist or be absent, we should perhaps fall back upon what we actually know about the membrane or membranes that sur-round the cell. The cortex of the cell from a physical standpoint is a gel which owes its stiffness primarily to the presence of calcium in it. For, as has been shown, the outer region of the cell contains proteins on the alkaline side of their isoelectric point, and these proteins bind cations and especially calcium.


This calcium can be delivered to the cell interior. Thus very definitely the cortex is a calcium electrode. Also, as will be shown in a later chapter (Chapter 13), every known type of stimulating agent releases calcium from the cortex. Such a release of calcium could be, in part at least, responsible for the action potential. On the basis of known facts, this must be true. Ordinarily in a resting cell, the calcium electrode of the cortex holds its calcium tightly, and thus the solution pressure of the calcium of the electrode is low. However, within the cell interior, the proteins or at least some of them are, as we have shown, on the acid side of their isoelectric point, and hence they have very little affinity for calcium. We can conceive of a system like this:

<

Ca proteinate of cortex j cytoplasm | Ca proteinate of the cell interior

In such a system, calcium ions would tend to leave the interior protoplasm and migrate toward the cortex. And if the two elec-trodes were connected by a wire or other conductor, a current would flow through the cell from the interior to the exterior, as the arrow indicates, and outside the cell from the exterior to the interior. In other words, in the usual terminology, the exterior is positive to the interior. But this positivity would decline or disappear during excitation, if at that time the cortex lost its power to bind calcium—as it actually does. For a very small interval of time, the exterior might even become negative to the interior.

This, of course, is only part of the story. The action potential —and bioelectric potentials in general—are algebraic sums of various potential differences, and these may be of various sorts, some tending to increase the potential at the surface of the cell, others tending to decrease it. For one thing, the cortex of the cell binds not only calcium, but other cations as well, so that when calcium is released as a result of stimulation, doubtless magnesium and potassium are released also. Indeed there have been many papers published to show that calcium, magnesium, and potassium ions are all released from muscle when it is stimu-lated, and there is some evidence that this is not merely due


to a change in permeability, but involves a release of ions from a bound state. The literature concerning this release of cations will be considered in a later chapter (Chapter 13). The im-portant point for this part of our story is that we really have to deal not only with a calcium electrode, but also with a magne-sium electrode, and a potassium electrode as well.

Nor is this all. In the early part of this chapter, much emphasis was placed on what evidence there is to show that in the interior of the cell the colloids of the cytoplasm bear a positive charge. Thus we would expect that at least some of the cytoplasmic proteins are on the acid side of their isoelectric point. These proteins would bind not cations but anions. Accordingly, in the cell interior there would be protein chloride and protein com-bined with any other anions that might be present. Actually there is chloride in cells, although for a while many biochemists felt that there was none (see Heilbrunn and Hamilton, 1942; Shenk, 1950, 1954). Moreover, according to Hamilton, much of this chloride is bound. (Unfortunately, Hamilton's work has not yet been published, although it was done some years ago.) She found that when she attempted to demonstrate chloride in muscle cells histochemically with the aid of a modified Mac-Callum method, she could only obtain positive tests when the cells were injured. Two of Hamilton's figures are reproduced in Fig. 12. Apparently injury causes a release of the chloride that is normally bound in the cell interior.*

If chloride is bound to protein in the cell interior and if this bound chloride can be released, it is obvious that in addition to the calcium electrode, or, perhaps more properly speaking, the cation electrode, we have a chloride electrode (or anion elec-trode) within the cell. Such an electrode would make a definite

* The suggestion that chloride in the cell is bound to proteins in the cell

interior may seem rather strange. However , there is support for the concept

in the fact that replacement of chloride b y bromide in l iving systems takes a

long t ime. A cation such as magnes ium has an almost immediate effect on a

l iving cell or tissue, robbing it of its irritability. Irritability is also lost in the

presence of bromide, but this action may take m a n y hours. For further discussion,

see Heilbrunn 1952b, Chapter 3 3 .


contribution to the resting or the injury potential of the cell. This is illustrated in the following scheme:

Protein chloride in cortex |cytoplasm] Protein chloride in interior

Because chloride ( and other anions ) are bound little or not at all in the cortex, there would be a greater solution pressure of

(a)

(b)

F I G . 12. A positive test for chloride in two muscle fibers. T h e reagent

used was a mixture of equal parts of 60 per cent silver nitrate and 6 0 per

cent nitric acid. T h e fiber above was injured b y exposure to ultraviolet

radiation; the upper part of the fiber b e l o w was injured by be ing com-

pressed with a glass rod w h i c h was rolled over it up to the point marked

by the arrow. The lower part of this fiber, not exposed to pressure, did

not give a positive test for chloride.


chloride ion in the chloride electrode of the cortex than there would be in the chloride electrode of the cell interior. Hence chloride ion would tend to move through the cell from the out-side toward the inside and this would mean that if the electrodes were connected by a wire or other conductor, there would be a flow of current in a direction opposite to that taken by the negative chloride ions. Thus the chloride electrodes of the cell, like the calcium electrodes, make the outside of the cell positive to the inside. The arrow in the above scheme shows the direction of the current through the cell. Some types of stimulation might have little or no influence on the chloride binding in the cell interior, but on strong electrical stimulation, entrance of the electric current into the cell interior would perhaps cause a release of chloride, just as injury does.

If the chloride electrode (or the anion electrode) in the cell interior is partly responsible for the fact that the interior of the cell is negative to the exterior, then obviously anything that would increase the binding of chloride within the cell would cause an increase in the resting or injury potential, and anything that would cause decrease in chloride binding would have the opposite effect. Thus an increased acidity in the cell interior would cause an increase in the magnitude of the resting or action potential. Here then we have an explanation of why it is that agents which depress respiration and carbon dioxide production also depress bioelectric potentials. Moreover, we can understand why it is that when carbon dioxide is removed from a nerve, the injury potential drops; this fact was reported by Lorente de No ( 1947 ). It is only when cells are rich in carbon dioxide that the chloride electrode of the cell interior would function as it does. In cells undergoing active photosynthesis, in the absence of much carbon dioxide, there would be less tendency for the interior of the cell to be negative. This may be why in the alga Valonia the interior of the cell is slightly positive to the exterior (for a discussion of the potentials in Valonia and Halicystis, see Blinks, 1949).

The theory that has been outlined will also explain the so-called depolarizing effect of the potassium ion and of fat solvents.


For these substances can clearly be shown to release calcium from the cortex (see Chapter 13) and they would therefore act like stimulating agents in cutting down the magnitude of the injury potential.

But, best of all, the theory offers, in terms of known facts, a rational explanation of why all sorts of stimulating agents do actually produce an action potential. For, as will be shown later, all the various types of stimulating agents do indeed cause a release of calcium from the cortex of the cell, and such a release can also occur in vitro from the proteins and other compounds that go to make up the protoplasm. Thus the theory has a great advantage over the Hodgkin theory, which would have a hard time explaining how cold and heat, ultraviolet light, pres-sure, and the potassium ion, among other things, could all cause any sort of known or postulated membrane suddenly to change its permeability in such a way as to open its pores to sodium ions and not to potassium ions.

5.

The living machine is a cellular machine, that is to say, the work it does is done within the confines of a single, small proto-plasmic cell. In the chapters that have preceded this one, an attempt has been made to tell, from a colloidal standpoint, what is now known of the physical makeup of the living cell and the protoplasm in it. But as yet, little has been said about how this cellular engine performs, and what changes occur in it when it is active. What indeed does happen to the protoplasmic colloid during activity? This is the question that will occupy us in the chapters to follow.

In an earlier monograph (Heilbrunn, 1928), one of the central themes was the demonstration that the living colloid is not like an ordinary protein or a mixture of protein and lipid. This is not surprising, although it is a fact never clearly recognized by the chemists who have so often speculated and written about living cells without ever having seen them, and without ever having bothered to read the rather voluminous literature about them. It is true that in some respects the behavior of protoplasm is like that of an ordinary protein. Expose a cell to a protein coagulant like mercuric chloride, and the proteins of the proto-plasm will coagulate in much the same way that ordinary proteins do. But over and above this behavior due to its constituents, there is a colloidal behavior characteristic of living colloids, a behavior which is for the most part not shared by nonliving colloids which may to some extent have a similar chemical com-position. To cite only one or two examples, in some types of protoplasm, both increase and decrease in temperature cause a drop in protoplasmic viscosity; and to add to the complexity, a still further rise or a fall in temperature can both cause great

62

THE SURFACE PRECIPITATION REACTION

5. THE SURFACE PRECIPITATION REACTION 63

increases in protoplasmic viscosity. Also, commonly, when proto-plasm is exposed to very tiny electric currents, it can change from sol to gel and it can undergo this change very rapidly. Ether and other fat solvents also have strange effects on different parts of the protoplasm—complex effects. Clearly, if we are to under-stand the colloidal behavior of protoplasm, we must study the protoplasm itself, not some combination of proteins or proteins plus lipids, even though such a combination, for one reason or another, is believed to be more or less of a replica of the living colloid. And, certainly, no purified substances obtained from living cells can possibly duplicate the behavior of the living protoplasm they came from.

In the monograph written in 1928, early studies of the colloid chemistry of protoplasm were discussed, and an effort was made to separate the wheat of correct factual knowledge from the chaff of speculation and subjective impressions. There is no need to go over this ground again. Nor will any effort be made to present an exhaustive survey of all the early and late work that has been done on the colloidal behavior of protoplasm. We are interested primarily only in what happens to the protoplasmic colloid when the machine which it constitutes engages in vital activity. Hence we will need to discuss only that aspect of the colloidal behavior of protoplasm which has to do with the func-tioning of the vital machine.

Protoplasm is a unique colloid. Its uniqueness depends prima-rily on the fact that a basic colloidal reaction occurs in it, a reac-tion not found in colloids which have never had the spark of life in them. Because this basic reaction is in large measure responsible for the action of the living machine, we shall discuss it at some length and in some detail.

The basic reaction of protoplasm is a reaction that is very readily observed. It can be seen without recourse to electron microscopes or to other elaborate apparatus now so common in biological laboratories. Indeed the reaction was observed by the very first pioneer in the study of protoplasm, Dujardin in 1835 and 1838, and even long before that by the early micro-scopists who looked at living protozoa with their primitive micro-


scopes. For they could see that when the protozoa were broken by the pressure of a coverslip, droplets emerged which did not diffuse through the surrounding medium.

When a cell is torn or broken, the emerging protoplasm typically does not continue to flow out. Instead a membrane is soon formed about the droplet of protoplasm that has escaped from the cell, and the flow of protoplasm is halted. The fact that this can and does occur is of fundamental importance for the cell, for if every slight injury to the cell surface were to cause a loss of the protoplasmic substance, cells with weak outer membranes might not be able to endure long. The ability of a cell to heal itself, and to protect itself against injury is an illustration of the fact that all living systems tend to preserve their balance and are capable of resisting untoward factors of their environment ( compare Chapter 14 ). Moreover, the reaction of a cell to injury may really be the fundamental basis for the irritability of proto-plasm. For all the stimuli that act on a living cell are essentially alterations of the environment, alterations not quite strong enough to cause what we can recognize as injury. As a matter of fact, there is no sharp line between the response to strong stimulation and injury; and, as we have seen, there is a similarity between the electrical effects when a cell or tissue responds to stimulation or when it is injured. Thus it might perhaps be said that it is of the nature of life itself to take advantage of injury, and that the injury reaction is a fundamental reaction of proto-plasm. As will be seen later, this does appear to be the case.

Over the years many authors have described what happens when, as a result of a break or a tear, the protoplasm emerges from a cell and forms a discrete droplet which does not diffuse through the outer medium. This type of behavior has been shown to occur in the cells of both lower and higher plants, in protozoa, and in various types of marine egg cells. There is also a similar reaction at the cut surface of muscle fibers of crabs, insects, frogs, snakes, and rats. Because of the universality of the reaction, it deserves a name and in 1927 Heilbrunn decided to call it the surface precipitation reaction or s.p.r.

In spite of the fact that the reaction is so easy to see and has


so often been observed, for many years there was scarcely any attempt to do anything more than to draw pictures of it. This failure to study the reaction was probably due, in part at least, to the belief that the explanation of it was simple. Thus it was usually stated that the membrane was of the same sort as the membrane that forms at the air-water surface of solutions of surface active substances. If a solution of peptone is allowed to stand for some time, the peptone molecules, because they lower surface tension, accumulate at the surface film and their con-centration in the film becomes so great that they are thrown out of solution. Hence a more or less solid film forms, a so-called haptogen membrane. This is the explanation suggested by Bayliss and it is to be found in all four editions of his classic book on general physiology (on page 128 of the first edition and page 129 of the fourth edition).

Now the idea of a haptogen membrane forming on the surface of droplets squeezed out of cells was perhaps a proper enough idea when Bayliss wrote, for at that time there was no clear knowledge as to whether or not the protoplasm of a cell was sol or gel, and most of the students of protoplasm who poked at cells with microdissection needles did indeed think that protoplasm was a gel. But if the protoplasm of a cell is a watery sol, as we know now that it is in many cells (see Chapter 2), then when a cell is broken, there is no surface or interfacial film at which substances could accumulate to form a membrane.

In 1926(c), and more especially in 1927, Heilbrunn was able to show that when a sea urchin egg is broken, the emerging protoplasm does not seal off to form a droplet unless calcium ion is present. Thus the s.p.r. in the sea urchin egg depends on the presence of calcium. Figure 13 shows the s.p.r. in the egg of the sea urchin Arbacia as it occurs in sea water, and Fig. 14 shows a similar egg crushed in sea water from which the calcium has been removed. The same dependence on calcium ion can also be demonstrated for the s.p.r. in other types of marine eggs, and it can likewise be shown very clearly for some protozoan cells. Figure 15 shows the beautiful s.p.r. that occurs when the ciliate protozoan Stentor is broken by compressing it between


F I G . 1 3 . An Arbacia e g g crushed F I G . 1 4 . An Arbacia e g g crushed

in sea water. in sea water from w h i c h calcium has

been removed.

F I G . 1 5 . Normal surface précipita- F I G . 1 6 . Absence of the reaction

tion reaction in Stentor. w h e n Stentor is crushed in dilute

sodium oxalate solution.

a slide and coverslip; but when this is done in the presence of a small amount of oxalate, no reaction occurs and the protoplasm flows out of the cell without hindrance; this is shown in Fig. 16.

The surface precipitation reactions of various types of cells


show individual differences. In some cells the reaction is very rapid and sharp. This is true of the sea urchin egg, the egg of the starfish, and the egg of the worm Cerebratulus; there is also a very rapid reaction in Stentor. In these cells, it is only when

F I G . 1 7 . Surface precipitation reactions of various e g g cells, (a) The

annelid Chaetopterus. (b) T h e starfish Asterias. ( c ) T h e sand dollar

Echinarachnius. (d) The annelid Hydroides . (e) T h e clam Cumingia.

(After Cos te l lo ) .

the cell is crushed very rapidly and harshly that the reaction does not occur. But in other cells, especially those provided with a thick outer membrane, the reaction can not be seen so easily. Thus in Amoeba proteus, the cell must be broken gently if a reaction is to be seen. However, by adding a little calcium to the medium, the reaction can be hastened.

Details of the reaction also vary. Figure 17 shows the s.p.r. in some types of marine eggs studied by Costello (1932), and Fig. 18 shows photographs of starfish eggs broken in sea water,


and giving an s.p.r., and broken also in sea water from which calcium has been removed so that no s.p.r. occurs. In some marine eggs when an s.p.r. occurs there is a breakdown of many of the cytoplasmic granules. This happens in the starfish egg,

(a) (b)

F I G . 1 8 . (a) Eggs of the starfish Asterias forbesii broken in sea water,

( b ) and in sea water from which calcium has b e e n removed. In the absence

of calcium, there is no surface precipitation reaction, and the protoplasm

flows out of the eggs in a steady stream.

the sea urchin egg, and the egg of the worm Cerebratulus. It is especially evident in the egg of the sea urchin Arbacia; the red pigment granules of this egg disappear in the course of the reac-tion and their pigment diffuses through the part of the protoplasm that has emerged from the cell, as well as into the outer sea water. However, in many eggs, although various types of granules, including pigment granules, may be present, there is no breakdown of granules during the course of the s.p.r.

The breakdown of pigment granules, so striking in the Arbacia egg, is an interesting reaction to follow. Like the s.p.r., it de-pends on the presence of calcium. For if the eggs are broken in sea water from which the calcium has been removed by oxalate, the pigment granules remain intact. The breakdown of


the granules may indeed be related to the s.p.r. For, if Arbacia eggs are vigorously centrifuged so that all the pigment granules are thrown down to one end of the cell, that end of the cell if it is broken gives a vigorous s.p.r. On the other hand, if the egg is broken at the light end, where pigment granules and other granules as well are lacking, the s.p.r. if it occurs at all is very weak.

In the frog egg also, there is an s.p.r., and this has been studied by Terry ( 1950 ). Figure 19 shows the surface of an ovarian frog

F I G . 1 9 . Surface of the exu-

date from a frog e g g cell

broken in a dilute solution of

calcium chloride ( T e r r y ) .

F I G . 2 0 . Surface of the exu-

date from a frog e g g cell

broken in a dilute solution of

sodium citrate ( T e r r y ) .

egg broken in the presence of calcium chloride—the border is smooth. But if the egg is broken in the presence of sodium citrate, the granular material of the protoplasm diffuses out through the medium surrounding the egg; this is shown in Fig. 20. In the s.p.r. of the frog egg, granules or inclusions are involved. The frog egg contains large numbers of yolk platelets. These are of various sizes and some of them are very much larger than ordinary cytoplasmic granules. But like the granules in starfish and sea urchin eggs they break down in the presence of calcium. This is illustrated in Fig. 21.


Essner (1954) and Gross (1954) have studied the kinetics of the reaction involved in the lysis of the yolk platelets. Essner has determined the effect of varying concentrations of calcium

F I G . 2 1 . Breakdown of cytoplasmic in-

clusions (yolk platelets) of the frog e g g as

a result of the addition of calcium ion. Cal-

c ium is moving into the preparation from

the lower right ( T e r r y ) .

on the rate of the lytic reaction. His results are shown in Fig. 22. A very slight increase in the concentration of calcium causes a very marked increase in the rate of lysis. Essner also plotted the effect of temperature on the rate of the reaction. This is shown in Fig. 23. According to Gross, if the yolk platelets are heated to a temperature of 65° C., they are no longer subject to break-down in the presence of calcium. This is an indication that an enzymatic reaction is involved. Additional support for this idea is the fact that the lytic reaction has a sharp pH optimum (at about pH 6). Also Gross was able to show clearly that the yolk platelets contain a substance that catalyzes their own break-down. However, as yet, no enzyme has been isolated.

In the sea urchin egg, the breakdown of pigment granules also appears to be dependent on enzyme action. Hultin (1950) found that if the pigment granules were isolated and washed in a potassium chloride solution, they no longer broke down when exposed to calcium.


Although in some respects the lysis of the yolk platelets of the frog egg is similar to the lysis of the pigment granules of the sea urchin egg, the resemblance is not complete. For whereas the yolk platelets are dissolved by strong salt solutions, in the

100, 1

• 0.025MCaCI 2

Time, minutes F I G . 2 2 . Rate of lysis of yolk platelets of the

frog e g g at various concentrations of calc ium

( Essner ) .

case of the granules of the sea urchin egg, their breakdown is prevented if the salt concentration of the medium is high. Not only do sodium and potassium chloride in high concentration prevent the breakdown of the granules in the presence of calci-um, but calcium itself will prevent the reaction if its concentra-tion is strong enough. Heilbrunn (1930) showed that in the presence of a molar solution of calcium chloride, the pigment granules of the Arbacia egg did not break down. This is similar to a phenomenon observed by Delezenne (1905) in a study of the proteolytic action of pancreatic juice. Only when calcium


is added to the pancreatic juice, does the trypsin become active, but in the presence of somewhat higher concentrations of calci-um, the proteolytic activity is lost. With rather keen insight, Delezenne compares this strange behavior of the calcium ion

100

J* ?

0.02MCaCI 2 30°C.

î^a02 M CaCI2 26°C.

16 24 32 40

Time, minutes

F I G . 2 3 . The action of temperature on the rate of

lysis of yolk platelets of the frog e g g in the presence of

calcium ( E s s n e r ) .

with a similar action in blood coagulation. A little calcium is necessary for the clotting of vertebrate blood; larger amounts of calcium inhibit the clotting. When Delezenne wrote, he did not know what is now well known that the action of the enzyme thrombin involves not only clotting but also proteolysis, nor could he have known that trypsin and other proteolytic enzymes can induce the clotting of the blood of higher animals. Accord-ing to Delezenne, calcium ion is rather specific for the activa-tion of trypsin; but it is not specific for the breakdown of cyto-plasmic inclusions. In the lysis of sea urchin granules, strontium


can take the place of calcium; and both strontium and barium can act like calcium in causing lysis of yolk platelets. Magnesium also can cause the breakdown of the granules of the sea urchin egg, but it is far less potent than calcium and it must be present

F I G . 24 . Absence of a surface precipitation re-

action in Stentor w h e n the cell is crushed in the

presence of 1 per cent ether. T h e normal reaction

is shown in Fig . 15.

in much higher concentration in order to be effective. In a later chapter, in discussing the anesthetic effect of the magnesium ion, it will be shown that this relative weakness of the magnesium ion in relation to the s.p.r. can serve to interpret the anesthetic action of the ion.

The surface precipitation reaction can be prevented by cold; at temperatures slightly above the freezing point, no visible re-action occurs in the sea urchin egg. In the protozoan Stentor, the reaction can also be prevented by dilute solutions of fat solvents (Heilbrunn, 1934a, b) , see Fig. 24. This figure shows the absence of the reaction in the presence of 1% ether. A Stentor lives in fresh water, in which there is at best only a very low concentration of calcium. In sea water there is much more calcium. This apparently is the reason why dilute solutions of


ether do not prevent the s.p.r. in sea urchin eggs, for the calcium in the sea water overrides the effect of the fat solvent. If, how-ever, the calcium concentration of the sea water is reduced to the point where the s.p.r. barely occurs, then dilute solutions of ether do indeed prevent the s.p.r. in sea urchin eggs. Dilute solutions of quinine prevent the s.p.r. in Stentor, and they also prevent granule breakdown in the sea urchin egg. This action of quinine only occurs after the cells have been immersed in solu-tions of the drug for some few minutes. Why quinine acts as it does is not yet clear.

When a sea urchin egg is broken, not only is a membrane formed at the surface of the exuding droplet, but typically a wave of destruction passes across the egg from the point of injury. This is indicated in Fig. 13. In the wave of destruction, not only do the pigment granules break down, but there is a vacuolization of the protoplasm. Typically this wave of granule breakdown and vacuolization does not proceed completely across the egg, but stops when a fraction of the egg has been traversed. The magni-tude of this fraction is dependent on the force with which the egg has been broken; the greater the injury, the farther the death wave proceeds. One wonders why the wave does not go all across the cell. If the egg is broken in isotonic calcium chloride solution instead of in sea water, the death wave is never stopped but passes across the entire cell, and this it does in a matter of about four seconds. If the egg is broken in a solution of isotonic magnesium chloride, the death wave also goes com-pletely across the cell, but the time it takes to complete its path is about fifteen seconds. Why is it that the wave is stopped in sea water? Apparently the sodium ion or the potassium ion, or both, are involved, but this may not be the whole story.

The surface precipitation reaction has often been studied in plant cells (for discussion and references to literature, see Heil-brunn, 1928). When the long, filamentous cells of algae or the root hairs of higher plants are crushed, the protoplasm emerges in balls or droplets, each of which is surrounded by its own newly formed membrane. Within the droplets, numerous vacuoles may appear. Crustacean nerve fibers behave in somewhat similar


fashion. The protoplasm in these fibers is highly fluid. If a fiber is cut, the protoplasm flows out readily. But according to Yung ( 1878 ), if this protoplasm is squeezed out, it does not mix with the surrounding medium, but forms balls or droplets, which rapidly become "granular."* Actually this granular appearance seems to be due to vacuoles, as is evident from Yung's drawing of the droplets. This is shown in Fig. 25. The protoplasm of the giant nerve fibers of cephalopods also shows a surface precipita-tion reaction; this is described in Chapter 8.

F I G . 25 . Droplets of protoplasm squeezed out

from crab nerve (redrawn after Y u n g ) .

The vacuolization reaction that travels across a cell as a result of injury is a reaction that has been observed ever since the concept of protoplasm was first postulated. The originator of the protoplasm concept, Dujardin, in his first paper on what he called sarcode, and later came to be called protoplasm, wrote (in 1835), "Mais la propriété la plus étrange du Sarcode c'est la production spontanée, dans sa masse, de vacuoles ou petites cavités sphériques." This property of being able to form vacuoles, Dujardin was inclined to think was the property that most clearly distinguished it from inanimate substances such as gelatin, mucus, or albumin (Dujardin, 1841).

Following Dujardin, there have been numerous descriptions

* Haeckel in 1857 also described a surface precipitation reaction in the proto-

plasm of crustacean nerve. W h e n he pressed the fluid protoplasm out of a

crayfish nerve, it c lotted in the form of "droplets, threads, granules, e tc ." From

one of Haeckel's figures, it is obvious that h e uses the term granules to designate

what w e today w o u l d call vacuoles .


of vacuolization phenomena in protoplasm that has been exposed to injury of one sort or another. But this information, voluminous as it is and important as it is, has never found its way into the books on cytology. Cytologists for the most part have not bothered to look at living cells, and in the past as morphologists they have never been very much interested in what happens to the cytoplasm when it is treated in one way or another. Mostly they have been concerned with problems of heredity and re-production, and although they have often thought and written about what the basic structure of protoplasm might be, they have done little to show how the structure changes as a result of one influence or another.

To attempt to gather together the scattered literature on vacuolization phenomena in protoplasm would be a major task, far outside the scope of this book. Before cytologists became enamored of the art of fixing and staining cells, the older ob-servers reported many instances of vacuolization in protoplasm; and even after the science of cytology became very largely a study of dead material, observers continued to report instances in which vacuoles appeared in the protoplasm of many types of cells.* Some of the older literature has been cited by Heilbrunn (1928). In general, heat and cold, hypotonic and hypertonic solutions, mechanical injury, radiation of various kinds, electric shocks, and many chemical stimulating agents; can cause vacu-olization. A notion of how much is written is given by the fact that Heilbrunn and Mazia in their review of the literature on the effects of radiation on protoplasm published in 1936, were able to cite 37 papers in which the authors described vacuoliza-tion of protoplasm after exposure to radiation. Such vacuolization

* It should be remembered also that Biitschli, a great student of l iving

protoplasm in protozoa, thought that all protoplasm h a d w h a t he cal led an

"alveolar" structure. Biitschli ( s e e his book publ i shed in 1 8 9 4 ) often saw in

the protoplasm of the cells he studied masses of vacuoles that gave the proto-

plasm a foam-like appearance. Biitschli be l i eved that all protoplasm was like a

foam, except that instead of the cavities in the foam b e i n g filled wi th a gas,

they were filled wi th a fluid. T h e Biitschli idea, though it had a great influence

for a t ime, has not b e e n fruitful. But what Biitschli did see was the c o m m o n

occurrence of vacuoles .


occurred in yeast cells, in the cells of Elodea, onion, and various other higher plants, in protozoan cells, marine egg cells, cancer cells, nerve cells, gland cells, etc. Descriptions of vacuolization go back to the early days of cell study. Figure 26 is a copy of two drawings published by Haeckel in 1857. In the normal state, the protoplasm in the nerve fibers of the ventral nerve cord of the crayfish is clear and fluid, but when death processes begin, the protoplasm coagulates in the form of droplets or vacuoles.

(a) (b)

F I G . 2 6 . Parts of two fibers from the ventral nerve cord of

the crayfish, ( a ) A smaller fiber wi th relatively small vacuoles .

(b) A larger fiber with huge vacuoles .

The literature of pathology is full of descriptions of vacuoles which have formed in protoplasm as a result of degenerative processes. Often such vacuolization is referred to as an example of what the pathologists call "cloudy swelling." This term has been variously used and the process of cloudy swelling has been interpreted in many ways. Cameron (1952) writes of cloudy swelling that "no pathological condition has given rise to so much uncertainty as this degeneration." Textbooks have varied in their definition of the process. Thus in 1921 Davidman and Dolley remarked, "As a pathological phenomenon, excessive water imbibition with the consequent vacuolation of the cell . . . is in the majority of text-books connected as a phase of cloudy swelling . . . . Others, though noting the connection, list it separately as hydropic degeneration, some with a separate sub-division for what is called vacuolar degeneration." In tissue culture preparations, in which cells can easily be studied in the


living condition, that is to say, without fixation and staining, vacuolization has often been observed. Thus according to Lewis (1919): "This vacuolization of the cytoplasm is one of the com-mon modes of cell degeneration and death in tissue-cultures."

The main point to be emphasized is that vacuolization follows injury of all sorts in many different kinds of cells. And more specifically, it is injury caused by excessive stimulation that produces this effect. For agents like heat and cold, mechanical impact, and ultraviolet radiation are stimulating agents. Perhaps the best illustration of the fact that excessive stimulation leads to a vacuolization reaction is the behavior of marine eggs. When these eggs are stimulated, they divide. But if the stimulating agent is too strong, or exposure to the stimulating agent is too prolonged, the egg becomes filled with a mass of vacuoles. This was observed many times by Loeb, who rather unwisely referred to the reaction that occurred as cytolysis (compare Loeb, 1913). Loeb was not much interested in the morphological details of cytolysis, and he went to no great lengths to describe the ap-pearance of the cytolyzed protoplasm. Sometimes the vacuoles that appear in the protoplasm are larger and more evident than they are in other instances. Figure 27 shows an example of a cytolyzed sea urchin egg with unusually large vacuoles. In the ganglion cells of cats and sparrows, overstimulation also results in the formation of numerous vacuoles in the cytoplasm ( Hodge 1892).

The vacuolization reaction that occurs so often in so many kinds of protoplasm is essentially a form of the s.p.r. (It might even be called an internal s.p.r.). As noted previously, a violent s.p.r. causes vacuoles to appear in the protoplasm that exudes from the sea urchin egg cell—this is also true for the s.p.r. as it occurs in other types of egg cells and as it occurs in the protozoan Stentor. If a cell is broken in the absence of calcium, there is no s.p.r. and, moreover, there is no vacuolization. Hence vacuole formation also depends on the presence of calcium. Now, when vacuolization occurs in an intact cell as a result of excessive stimulation, does this mean that calcium has been released into the cell interior? Actually, as will be shown in a later chapter,


stimulation does indeed release calcium into the cell interior, and thus it is easy to understand why excessive stimulation or injury so often causes vacuolization.

In his early work, Heilbrunn (1927) assumed that vacuole formation, involving as it seemed to do, the appearance of new films throughout the cell, was essentially the same sort of film formation as that which occurs at the boundary of the droplet of protoplasm that emerges from a broken cell. But, according to

F I G . 27 . A sea urchin e g g w h i c h

has b e e n complete ly cyto lyzed (Loeb) .

Harris (1943), in the sea urchin egg pigment granules can coalesce to form large vacuoles and small colorless granules can do the same thing. Whether all vacuoles form in this way is not certain. Also it seems clear that although some granules may enlarge to form vacuoles, others disappear in the course of the s.p.r.

Often the vacuoles produced as a result of injury are small and tend to escape observation. In the death wave that passes along a muscle fiber when one end is injured, vacuolization is not very obvious, although apparently it does occur (Woodward, personal communication ). Let us now consider the death wave in muscle fibers, and the s.p.r. that precedes it.

If an isolated skeletal muscle fiber of a frog is injured with a microneedle, the fiber immediately seals itself. The reaction is very rapid. It can be studied very simply by isolating fibers, cutting them across, and watching the reaction at the cut surface.


Immediately a "plug" forms which seals the cut end. Such plug formation has long been known to pathologists, who have seen it in cut or injured muscle and have referred to it as "waxy degeneration" ( see, for example, Thoma, 1906 ). The plug forma-tion which occurs whenever a muscle fiber is cut can be seen not only in the muscle fibers of the frog but also in the fibers of turtles, snakes, rats, crabs, and insects. However, if the reaction

F I G . 2 8 . Surface precipitation re-

action in a frog muscle fiber.

is to be properly observed, care must be taken to isolate the fibers with a minimum of injury. Dead fibers do not show the reaction. Figure 28 shows the reaction in the muscle fiber of a frog. The plug formation is apparently a surface precipitation reaction, but when the fiber is cut, the protoplasm does not exude. This is perhaps due in part to the relatively high viscosity of the muscle protoplasm, in part also to the fact that the s.p.r. in muscle proto-plasm is extremely rapid. The similarity of the plug formation in muscle fibers and the s.p.r. in marine eggs is indicated by the fact that both reactions are typically dependent on the calcium ion. Proctor ( 1952 ) found that in crab muscle, the reaction does


(a) (b)

F I G . 2 9 . Surface precipitation reaction in a crab muscle fiber; (a) shows

the reaction in sea water; (b) shows absence of the reaction in calc ium-

free sea water.

although the reaction is weak if fibers are cut in a mixture of sodium and potassium salts, ordinarily the reaction still occurs. Moreover, in frog fibers the reaction is not prevented by oxalate or citrate. This apparently is due to the fact that oxalate and citrate induce a secondary reaction. If oxalate or citrate is in-jected into the interior of the frog muscle fiber, in that part of the fiber where the injection is made, the muscle protoplasm preserves its normal appearance, but at a little distance from the site of injection, the muscle undergoes a reaction similar to that which occurs at the cut ends of the fibers, and two waves of

not occur at all in calcium-free sea water. This is shown in Fig 29. Also in the muscle fibers of grasshoppers no plug forms if the fibers are placed in isotonic salt solutions containing a small amount of sodium oxalate. However, in frog muscle fibers,


destruction then proceed from these points in the vicinity of the injection toward the ends of the fiber. *

When a frog muscle fiber is cut in an isosmotic calcium chloride solution, not only does a plug form at the cut end, but just as in the case of the s.p.r. in the sea urchin egg, a wave of death passes along the length of the cell. And again, as in the sea urchin egg, this wave is faster in the presence of high con-centrations of calcium than it is when little calcium is present in the surrounding medium (Heilbrunn, 1940; Woodward, 1948). According to Woodward, whereas in Ringer's solution the wave moves at the rate of only 0.23-0.30 mm./min., in isosmotic calcium chloride solutions, the wave travels at the rate of 1.09-1.89 mm./min. Thus in isosmotic calcium chloride solutions, the speed is at least 50 times as great as it is when the concentra-tion of calcium is low. Figure 30 illustrates the passage of a wave along a frog muscle fiber immersed in isosmotic calcium chloride solution. As the wave passes, the muscle fiber shortens until it is only a fraction of its original length.

There are certain obvious resemblances between the s.p.r. and the reaction, or reactions that occur when the blood of higher animals clots. The rupture of a cell is to some extent like the rupture of a blood vessel, for just as the flow of blood from the vessel is prevented by a precipitation or clotting process that requires the presence of calcium ion, so too the flow of proto-plasm from the broken cell is stopped by a precipitation reaction requiring calcium. There are other resemblances between the s.p.r. and blood clotting. Both s.p.r. and blood clotting are pre-vented by cold; in both cases also, although a small amount of calcium is necessary for the reaction, too much calcium impedes it. But there is an additional and even more striking resemblance between blood clotting and the s.p.r. The literature on blood

* Oxalate and citrate may have strange effects on vertebrate animals. Thus

if oxalate or citrate is injected into the blood stream, the blood instead of

clotting more s lowly w h e n it is drawn from the body, clots more rapidly. It

should be noted also that the described difference in the behavior of frog and

grasshopper muscle fibers may be due to the fact that Proctor in his experiments

with grasshopper muscle fibers used dilute solutions of oxalate, whereas in the

studies on frog muscle fibers, somewhat more concentrated solutions were used.


F I G . 3 0 . Shortening of an isolated frog muscle fiber in isosmotic cal-

c ium chloride solution. T h e first photograph was taken in Ringer's fluid;

the second 1 0 sec. after the addit ion of calc ium chloride. Subsequent

pictures were taken at 1 0 - s e c . intervals.


clotting is vast, and there have been many differences in opinion as to various aspects of the process. But one fact is rather universally agreed upon. There are two major stages in the blood clotting process. The first of these stages requires calcium; the second does not. Once blood has clotted, the blood contains a substance, thrombin, which can clot a fresh sample of blood in the complete absence of calcium. The protoplasmic colloid be-haves in somewhat similar fashion. This was shown by Heilbrunn (1927) in the following way. Sea urchin eggs were broken up by shaking them with splintered glass coverslips. This prepara-tion of broken eggs then contained a substance which would cause an s.p.r. in eggs even when calcium had been completely removed from the solution surrounding the eggs by the addition of oxalate. In this s.p.r. in the absence of calcium, the red pigment granules of the sea urchin egg protoplasm do not break down. The substance which causes the s.p.r. in sea urchin eggs in the absence of calcium is not a specific substance present only in sea urchin eggs, for it can be obtained from various types of injured cells. Thus Heilbrunn et al. (1946) were able to obtain effective extracts from the heat-killed tissues of sea anemones, worms, clams, squids, lobsters, fish, and frogs, and all these extracts acted on sea urchin eggs in the same way as did the preparation of smashed eggs.

In view of the many resemblances between blood clotting and the s.p.r. it seems proper to use the term protoplasmic clotting for the reactions in the protoplasm that are so similar to the re-actions involved in the coagulation of vertebrate blood. In the next chapter additional justification will be given for this point of view, and additional details will be presented concerning the process of protoplasmic clotting. The concept of protoplasmic clotting can help to interpret many mysterious aspects of cell physiology and is basic to our understanding of the dynamics of living protoplasm.

6.

Let us suppose that some rash biologist were interested in making a living cell from inanimate materials, or at least a cell endowed with some of the attributes of life. If he were at all aware of the nature of living substance, he would not follow the pattern of those who believe that electronic devices act like brain cells, but he would try to construct a cell out of some colloidal material. And because living cells are extraordinarily sensitive he would try to choose for his colloidal material some-thing that would react to sudden physical and chemical changes in the environment, and would react in such a way as to do work. It is not easy to imagine such a colloidal material; obviously it could not be a simple chemical compound or compounds. Moreover, if a colloid is to be alive, it must be able not merely to respond to some change in the environment, it must also be able to revert to its original resting state, the state it existed in before it responded. A primary characteristic of living material is the fact that it tends to preserve its balance; in general, influ-ences that induce change in one direction immediately set into play forces which tend to reverse such a change. How this can possibly occur will be considered in the chapter on Cellular Homeostasis (Chapter 14), but for the present we are concerned only with the properties a colloid must have if it is to be alive.

If the living colloid is in a state of balance between factors or influences which tend to transform it in one direction and those which act in exactly opposite fashion, it must be in a state of equilibrium between the two sets of factors. What are these factors and how do they operate?

In the last chapter it was shown that protoplasm of all sorts (plant protoplasm, protoplasm of protozoan cells, and protoplasm

85

PROTOPLASMIC CLOTTING


of mammalian cells) can undergo a basic reaction, a clotting reaction dependent on the presence of calcium, a reaction similar in many respects to blood clotting. In our discussion of the protoplasmic colloid, we may therefore with some propriety take vertebrate blood as a model.

The blood of mammals—our blood—is in a constant state of equilibrium between the factors which tend to cause clotting and the factors which tend to inhibit or reverse such clotting. As the blood flows through the arteries and veins, there may frequently be small incipient clots, especially in the neighbor-hood of local tissue injury. This is well shown by the work of Knisely and his associates; they have developed methods for looking at blood as it flows inside the body. If a tissue near a blood vessel is injured, some portion of the blood within the vessel may form a thick, pasty sludge in which some few blood corpuscles are imbedded; this sludge moves with difficulty through the smaller vessels (Knisely, Eliot, and Bloch, 1946). Extensive injury leads not merely to a sludge but to a thrombosis. Apparently what is happening is that the injured cells send into the blood substances which favor clotting.

The important point is that in blood there are various sub-stances which favor clotting and, as we shall see later, other substances which inhibit or reverse clotting. In protoplasm a similar situation holds, and some of the substances which act on the clotting of blood are similar to those which act on proto-plasm. In this chapter, and to some extent in chapters to follow, information will be presented concerning the clotting of proto-plasm and the factors concerned in this clotting. The information we have is rather scant. This is due to the fact that there have been very few workers in the field. For every student of proto-plasmic clotting, there are hundreds of students of blood clotting. Moreover, whereas the student of blood coagulation can have buckets of blood at his disposal, the student of protoplasmic clotting, if he wishes to work on the living cell, must be content to examine his material in minute containers with which he can not do much tampering, if he is to preserve the vital properties of the material he is interested in. Nevertheless, in spite of these

6. PROTOPLASMIC CLOTTING 87

difficulties, we do have some interesting information about proto-plasmic clotting and how it is related to vital processes.

If, as we have postulated, the basic colloidal reaction of living material is a clotting reaction in which calcium plays a major role and which exists in two stages, the second of which involves a substance that is similar to thrombin, then it is not surprising that in vertebrates this colloidal reaction or series of reactions should have developed to prevent loss of the precious blood fluid from arteries and veins, just as the s.p.r. prevents loss of proto-plasm from cells. In support of this idea is the fact that all of the substances involved in the complicated reactions of blood clotting come from cells. If we want blood to clot rapidly, we add thromboplastin, a substance which hastens the formation of thrombin from its precursor, prothrombin. The ordinary prepara-tion of thromboplastin that is sold in drug stores is just dried rabbit brain that has been ground up. In other words, the cells of the brain contain thromboplastin. And so do the cells of muscle, lung, and various other organs and tissues. In a lifetime of research, Alexander Schmidt, the discoverer of thrombin and no doubt the leading pioneer in the science of blood coagula-tion, showed that extracts of various types of cells and tissues hastened the clotting of horse blood. Schmidt's work and that of his many students is reviewed in his two books on blood clotting, published in 1892 and 1895. In his studies, Schmidt used horse blood because it is slow to clot. Modern workers in testing for the presence of thrombin generally use oxalated or citrated blood. But the discovery that calcium is important for blood clotting was made late in Schmidt's life and he did not take kindly to the use of oxalate. Thus in his tests he used horse blood without removing the calcium from it, and the fact that the extracts of various tissues hastened clotting of this blood might well be interpreted as meaning that these extracts con-tained what we now know as thromboplastin. Be that as it may, Schmidt found that all sorts of cells contained substances which had a marked influence in speeding up blood clotting. And this is true not only for the cells of vertebrates, but also for the cells of various lower organisms, even the cells of yeast. Ob-


viously the cells of lower organisms do not contain thrombin or thromboplastin merely for the purpose of hastening the clotting of vertebrate blood, and the clot-promoting substances of cells like those of yeast doubtless serve some function in the cells that contain them.

Now if it is true that cells—all cells—contain substances which induce or favor the clotting of blood, and presumably the clotting of protoplasm, then if there is to be action and counteraction, there should also be substances in cells which prevent clotting, and perhaps other substances which reverse clotting. In blood the most important anticoagulant is a polysaccharide called heparin. The chemistry of heparin is not as well understood as it should be; actually there are doubtless many kinds of heparin, or more properly speaking, many heparins. Heparins are muco-polysaccharides; that is to say, they are polymers of certain acid derivatives of sugars, the uronic acids. (If a sugar like glucose has its alcohol group in the number 6 carbon position oxidized to an acid, the result is a uronic acid.) Also heparins contain amino derivatives of sugars (hexosamine or glucosamine) and in addition they also contain a rather large amount of sulfuric acid. At any rate, ordinary heparin, the heparin that is sold and used as a drug, apparently has such a composition. But whether all heparins have exactly this composition does not appear to be certain. At any rate there are heparin-like substances which do not have such a composition, and at present in the uncertainties of the knowledge now available it is hard to decide whether a substance is a heparin or a heparin-like compound.

Heparins and heparin-like substances are widely distributed in organisms—from bacteria to man. However, in higher animals it has been claimed, and it is commonly believed, that heparin is not present in all cells, but occurs only in the mast cells (see Jorpes, 1946). These mast cells are connective tissue cells con-taining an abundance of large granules which give a histo-chemical test for heparin. There is no question at all but that the mast cells are rich in heparin, but this does not necessarily mean that all other cells are wholly devoid of heparin. The histochemical detection of heparin depends on what is called a


metachromatic reaction; that is to say, a reaction in which a dye changes color. The dye commonly employed in tests for heparin is toluidine blue; this dye is blue in solution, but in the presence of heparin it becomes reddish. Now heparin, which because of its chemical composition is one of the most acid substances found in living material, combines readily with proteins, especially with basic proteins. In such a combination, it is often no longer metachromatic. Thus a cell which contains an abundance of heparin might have the substance in a combined form and so it might not give a positive histochemical test.

Charles and Scott (1933) determined the amount of heparin present in various tissues and organs of the ox. Their values are shown in Table I.

T A B L E I

Distribution of Heparin in Various Tissues of the Ox

Purified heparin per kilo of tissue

mg . Units of activity

Liver 190 1900

Spleen 2 3 0 7 0 0

Heart 54 3 8 0

Blood 6 6 6 0

Thymus 3 1 0 3 5

L u n g 2 3 0 2 2 0 0

Muscle 6 0 0 1900

Table I is interesting for two reasons. In the first place, it is clear that muscle is rich in heparin, and it would be hard to maintain that muscle is rich in mast cells. Secondly, the table shows a surprising lack of correspondence between the amounts of heparin obtained by the authors and the activity of their heparin preparations as measured by the effect on blood clotting. The suspicion is strong that not all the preparations contained the same chemical entities. In a later discussion on cell division (Chapter 11), we shall have occasion to note that there may be various types of heparin-like substances, and that some of them act more strongly on blood, but that others have less of an action on blood and more of an action on protoplasm. The table offers some support for this idea.


That cells other than mast cells do indeed contain heparin or heparin-like substances is indicated by the fact that many cells show metachromatic staining. Thus, for example, the egg cells of various animals do ( Kelly, 1950, 1954 ). The jelly of sea urchin eggs can be shown to be either a heparin or heparin-like sub-stance (Immers and Vasseur, 1949); it prevents the clotting of vertebrate blood. Even when cells fail to show a metachromatic reaction, such a reaction can sometimes be induced by first sub-jecting the protoplasm to tryptic digestion. This at any rate is true for the muscle fibers of the frog ( Couillard, personal com-munication). Certainly muscle fibers contain a heparin-like sub-stance, for such a substance is liberated from fatigued muscle (see Chapter 7). At the University of Pennsylvania, Thomas (1951, 1954) showed that various tissues of the surf clam con-tained a substance that had a powerful anticoagulant action. This work was later taken up by the Lederle Company ( Fromm-hagen, et al, 1953; Love and Frommhagen, 1953). The presence of heparin-like substances in clams is not due to mast cells; indeed even the eggs of the clam contain these substances.

Although much more work needs to be done, it seems likely that heparin and heparin-like substances are a common constit-uent of animal and perhaps also of plant cells. Certainly they are not confined to the mast cells. Hence we have commonly present in protoplasm, substances which promote clotting and also those which tend to prevent it.

The evidence presented so far indicates that substances from living cells can hasten blood clotting, and that other substances from living cells can also inhibit blood clotting. But what about the protoplasm? Do we know that these types of substances also have an effect on the protoplasmic colloid? The evidence is not very abundant, but it does exist. In the last chapter it was shown that when living tissues are injured so that presuma-bly their protoplasm is clotted, they produce substances which induce the s.p.r. in the absence of calcium. These same sub-stances, when they enter cells, also cause a sharp increase in the viscosity of the protoplasm of the cell interior. This was shown to be true for various extracts of heat-killed tissues from a number


of animals (Heilbrunn et al.> 1946; compare also Harding, 1951). (When protoplasm is killed by heat at relatively low tempera-tures, this is due not to an ordinary heat coagulation of proto-plasm, but to a clotting reaction; see Heilbrunn, 1924; Heilbrunn, 1952b.)

But do heparin and heparin-like substances also act on proto-plasm as they do on blood? Ordinary heparin has a rather large molecule and does not enter cells readily. However, in studies on the egg of the worm Chaetopterus, Heilbrunn and Wilson (1949) were able to show that dilute solutions of heparin pre-vented the protoplasmic clotting or gelation that precedes the appearance of the mitotic spindle. This aspect of the subject will be discussed more fully in the chapter on cell division. Suffice it here to say that not only heparin, but various heparin-like substances as well, have an anticlotting effect on protoplasm. Such substances can be obtained from various tissues, especially from ovaries (Heilbrunn, Wilson, and Harding, 1951; Heilbrunn et ah, 1954). Heparin and heparin-like substances also have an effect on muscle. If, as we shall try to show in the next chapter, the contraction of muscle is due to a gelation or clotting of the muscle protoplasm, then we might expect that heparin, if it could get into the muscle fibers, would prevent contraction and keep the muscle in a relaxed state. Striated muscle fibers do not appear to be very permeable to heparin and, indeed, the osmotic membrane of these fibers seems to be a relatively impermeable structure. However, there are two types of muscle fibers which seem to have membranes relatively permeable to dissolved sub-stances. The smooth muscle fibers in the walls of blood vessels are sensitive to calcium ion and appear to be permeable to it; the muscle cells of the frog heart show a similar behavior. Both these types of muscle are also sensitive to heparin. Blood vessels are dilated by heparin, and the frog heart exposed to heparin stops beating in a relaxed state.

Up to this point no attempt has been made to discuss details of the clotting of protoplasm. As a matter of fact, not many details are known. It has already been shown that the s.p.r. occurs in two stages, the first of which requires the presence of


calcium, the second does not. As a result of the action of calcium, a thrombin-like substance is formed, a substance which can cause a precipitation at the surface of the emerging protoplasm of a crushed cell. The same type of substance doubtless is important for clotting in the cell interior. What are the properties of the thrombin-like substance?

In recent years students of blood clotting have shown more and more conclusively that thrombin, in addition to being a clotting enzyme, is also a proteolytic enzyme. This is scarcely surprising, for various proteases are known to clot blood; also the rennin which acts on milk is both a proteolytic and a clotting enzyme. Thrombin is now known partially to digest fibrin, for it splits off a peptide from it (Lorand and Middlebrook, 1952; Lorand 1952 ). Thus thrombin is both a proteolytic enzyme and at the same time a clotting enzyme activated by calcium. There is an increasing body of evidence to indicate that in protoplasm there are proteolytic enzyme systems activated by calcium ions ( Gross, 1952; Lundblad, 1952, 1954; Lundblad and Lundblad, 1954; compare also Goldstein, 1953), and there is every reason to believe that these enzymes are the ones concerned with the clotting reaction.

No doubt it can safely be concluded that protoplasmic clotting is, in the main, similar to blood clotting. The information we have, although certainly not voluminous, is consistent in so far as it goes. In the chapters that follow, some additional facts will be presented, especially concerning the effect of stimulating and anesthetic agents on the protoplasmic viscosity. Obviously we should like to know more about the course of the reactions that occur when protoplasm clots. Fortunately clotting can be studied in homogenates made from cells, and in this way much can be learned. The advantage of such techniques is that the reaction or reactions can be studied in test tubes, under controllable con-ditions, and with relatively large masses of material. In other words, in homogenate study the protoplasmic colloid is no longer confined to individual cells. Of course in making the homo-genates, there is always of necessity a dilution of the protoplasm; and also there is undoubtedlv the possibility, indeed the proba-


bility, that the colloid changes as it emerges from the cell. How-ever, by removing calcium and preventing the s.p.r. these changes can be minimized.

The pioneer in the study of colloidal change in cell ho-mogenates is Hultin (1949, 1950a, b ) . More recently, the work

200L

IOOL

Ü5B

x v C a free homogenate

ο ο Qoo5 M CaCij added

025 Q5 575 To F I G . 3 1 . The effect of 0 . 0 0 5 M

C a C l 2 on the viscosity of sea

urchin e g g homogenates . The ab-

scissa shows the concentration of

homogenate expressed as a fraction

of the most concentrated homoge-

nate ( H u l t i n ) .

F I G . 3 2 . Clott ing of homoge-

nates from sea urchin eggs . Both

tubes shown in the figure were

similarly treated. The tube con-

taining calc ium shows particles of

c lotted protoplasm sticking to the

walls of the tube; in the absence of

calc ium there is no clot ( G r o s s ) .

has been taken up by Gross ( 1955 ). If sea urchin eggs are broken in the absence of calcium, and then calcium is added later, there is a sharp increase in viscosity. This is shown in Fig. 31 taken from Hultin. The reaction of calcium-free homogenate when calcium is added is strikingly like that of calcium-free blood after the addition of calcium. This is shown in Fig. 32.


Just as in blood the addition of calcium to blood previously oxalated or citrated causes a separation out of a solid phase (fibrin), so in sea urchin eggs, the addition of calcium to the

F I G . 3 3 . An electron microscope photograph of the protoplasm of sea urchin e g g homogenates , clotted as a result of the addition of calcium ( G r o s s ) .

calcium-free homogenates also causes a separation out of a solid phase. This is clearly shown by the work of Gross. In the cell homogenates, the solid phase does not separate out as fibrillar material, but as an aggregation of small globular particles. These


are about 350-400 A. in diameter; they represent either macro-molecules or groups of smaller molecules. Figure 33 is a photo-graph taken with an electron microscope of the aggregated material produced as a result of the addition of calcium to a homogenate of sea urchin eggs. Electrophoretic studies indicated that the solid material separated out from the homogenates con-sisted of a single specific component. This component apparently contains nucleoprotein, for after it has been precipitated out, spectrophotometric study indicated a loss of nucleic acid.

Hultin in his study of homogenates showed not only that calcium caused a lysis of granules and an increase in viscosity, but also that following the addition of calcium there was an increase in the acidity of the homogenate and also a sharp rise in the amount of oxygen taken up per unit of time. Both these phenomena are important. When protoplasm is injured, it be-comes acid (Chambers and Pollack, 1927); such an increase in acidity also follows the activation of the egg at fertilization ( Runnström, 1930 ). Hence the addition of calcium to protoplasm causes not only clotting but also changes which are to be as-sociated with injury and stimulation. Perhaps in cells generally, protoplasmic clotting can induce the increase in respiration com-monly associated with protoplasmic activity. We shall return to this point in our discussion of excitation ( see Chapter 13 ).

In this chapter and the chapters that have gone before it, an attempt has been made to show the nature of the protoplasmic colloid and the way it behaves and reacts. In the chapters to follow, various vital processes will be interpreted in the light of this knowledge.

7.

For many, many years muscle has been a favorite object of study in physiological laboratories and the amount of work done on it has been enormous. As a result of all this work a vast amount of information has been gathered. We know the time sequence of events when a muscle contracts, we know how much force it exerts and how much work it can do, the electrical changes that occur, the heat that is given off, and we also know many details concerning the complicated oxidative reactions that go on in a contracting muscle. Moreover, many workers have studied the effect of this, that, or the other physical or chemical agent on muscular contraction. And yet, in spite of our vast store of detailed information, there is still no certainty as to why it is that a muscle shortens. In other words, we do not really understand the mechanics of muscular contraction.

There have been no lack of theories, no lack of attempts at interpretation. For a time a favorite idea was that the proteins of the muscle underwent a change in molecular shape. Direct evidence for this point of view was hard to obtain. Astbury in his X-ray diffraction studies thought that in living muscle he could on contraction detect a molecular superfolding of protein molecules (see, for example, Astbury, 1939). But this evidence unfortunately failed to materialize, for in living muscle it is not possible with present methods to detect such superfolding even if it does occur. This was shown by Spiegel-Adolf, Henny, and Ashkenaz (1944), and it has also been recognized by Astbury himself (1947).

Because of the fact that a muscle is an internal combustion engine, or at any rate oxidizes organic materials just as an internal combustion engine does, biochemists have been greatly con-

96

MUSCULAR CONTRACTION

7. MUSCULAR CONTRACTION 97

cerned with the various reactions involved in such oxidative processes, and most physiologists have been deeply impressed with the work and the ideas of their chemically-minded col-leagues. In this chemical work the emphasis has from time to time shifted. In the years before 1930 it was generally thought that the oxidation of carbohydrate to lactic acid was the reaction of primary importance, and that in some way or another the lactic acid made the muscle shorten. But in 1930 Lundsgaard found that when a muscle was poisoned with iodoacetic acid, it no longer was capable of producing lactic acid and nevertheless was able to contract. This inspired what the distinguished English physiologist Α. V. Hill called the "revolution in muscle physiology/' After the revolution attention became centered on compounds containing phosphate, and for a short time the sub-stances that were thought most significant for contraction were the so-called phosphagens (combinations of phosphate either with the base creatine or with the basic amino acid arginine), and it was believed that it was the breakdown of phosphagen into base and phosphoric acid that was the reaction that made the muscle shorten. But this belief was short-lived and ever since Lohmann in 1934 showed that phosphagen breakdown as it occurred in extracts of muscle depended on the presence of adenosine triphosphate, the latter substance has been thought by many to be primarily responsible for muscular contraction. Adenosine triphosphate, or ATP, is indeed a remarkable sub-stance; it can give up one, two, or three phosphate groups, and in giving up the first two it yields a great amount of energy. Perhaps it is this energy that is responsible for the contraction of muscle; such indeed is the opinion commonly held at the present time, although there is little or no direct evidence to prove it. This will become apparent later.

In the attempt to correlate the metabolic changes of contract-ing muscle with the machinery of contraction, various workers in the past have attempted to use models of one sort or another. Thus in 1893 the famous Dutch physiologist Engelmann sus-pended a violin Ε string (made of catgut) in water and attached it to a recording apparatus or kymograph. When the catgut


string was heated by a coil of wire wound around it, it con-tracted, and then it relaxed again on cooling. Here then was an imitation muscle. Not only did heat cause a shortening, but also acid and indeed the acid produced by muscle—lactic acid. This Engelmann model made such a deep impression on physiologists at the turn of the century that it was widely referred to in various texts of physiology and it continued to be referred to for many years. Engelmann thought that in muscle, as in catgut, the swelling of gels induced shortening. But in 1915 Bernstein showed that the catgut in violin strings is composed of groups of threads wound spirally around each other; if these threads are unwound, no shortening occurs when swelling is induced by heat or acid. It is the thickening of the individual threads as they wind about each other that causes the entire string to shorten. But muscle is, of course, not composed of spirally wound threads and as a result of Bernstein's work the argument in favor of the Engelmann theory completely collapsed.

Various other authors have used models in an attempt to interpret the contraction of muscle. Szent-Györgyi in 1940 used a variation of the old Engelmann model. Instead of catgut, for one model he used a group of parallel wool threads with gelatin gel in between the individual threads, and for another model he used artificial silk threads with agar gel interspersed. As the gels took up water, the threads became bowed out so that the distance between their ends decreased. This shortening of the group of threads was taken to be similar to the contraction of muscle.

On the basis of their model experiments, both Engelmann in 1893 and Szent-Györgyi in 1940 thought that the contraction of muscle was due to a taking up of water by gels. But by 1947 (and before that, probably as early as 1941), on the basis of another model experiment, Szent-Györgyi changed his mind completely and thought that the contraction of muscle was due not to an addition of water to a gel or gels but to a loss of water from the gels of the muscle. The new model that Szent-Györgyi proposed was chemically much more like muscle than threads of catgut, wool, or artificial silk. Instead of these imitation


materials, Szent-Györgyi used a thread of actomyosin. Now actomyosin is a protein, or a combination of proteins, that occurs in muscles or at least is supposed to exist in muscles, for it can be extracted from them. When ATP is added to a thread of actomyosin, the thread shortens violently and undergoes a very marked decrease in volume. This is shown in Fig. 34. Concern-ing this shortening, Szent-Györgyi wrote (in 1951): "Contrac-

F I G . 3 4 . (Above) Actomyos in thread. (Below)

The same a f ew minutes after the addition of ATP.

Magnification 1 : 3 0 ( S z e n t - G y ö r g y i ) .

tion of actomyosin seems to be a simple colloid-chemical process, synaeresis, taking place in a specific structure, built of two specific colloids. In muscle, the steric orientation, called struc-ture, may still add to the specificity of the reaction, but there can be little doubt that in essence both contraction of muscle and contraction of actomyosin are identical phenomena." One difficulty with the actomyosin model is that when it shortens, it also incurs a great decrease in volume, but in muscular con-traction there is no such decrease in volume. But if the actomyo-sin threads are allowed to dry with their ends fixed, then when the dried threads are treated with ATP, shortening does not appear to be accompanied by any decrease in volume. Figure 35 shows the behavior of such a dried thread after treatment with ATP.

The actomyosin model was an advance over the earlier models, for instead of being silk or wool, it consisted of material extracted from the muscle itself. In his later work Szent-Györgyi has used models even closer to the living muscle, for he has used


dehydrated that is to say, glycerinated, muscle fibers. These later model experiments have had a great influence on the think-ing of muscle physiologists throughout the world, and they have been the inspiration for much interesting research. In his studies on glycerinated muscle fibers Szent-Györgyi (1949) uses the psoas muscle of the rabbit. This is a long, slender muscle which has parallel fibers running from one end to the other. Small bundles of fibers are removed; usually these are attached to sticks in order to keep them stretched, then they are plunged

F I G . 35 . Contraction of a dried

actomyosin thread after treatment

with A T P (Szent -György i ) .

into 50 per cent glycerol. In the glycerol they can be kept at a temperature of -20° C. for many months. Then when they are brought back to room temperature, if a few drops of 0.25 per cent ATP are placed on the fibers, there is a very decided shortening.

It would be difficult to survey and more difficult properly to evaluate the large amount of research done by Szent-Györgyi and his group of enthusiastic workers. Fortunately Szent-Györgyi has published, mostly in book form, various accounts of his work (1945, 1947, 1948, 1951, 1953), and these should be consulted by anyone who is interested in what is generally regarded as the most exciting development in modern muscle physiology. Like so many other workers in the field, Szent-Györgyi is impressed with the importance of ATP; in his colorful way of writing, he calls it "the master substance of muscle" (1951, p. 27).

One thing is certain, adenosine triphosphate, or ATP, is a powerful word in all our modern thinking about muscle, just as deoxyribosenucleoprotein is a powerful word for those who are concerned with the process of cell division. These words seem to have taken the place of the incantations used by the magicians


in the days before modern science. Whereas the old magicians were content with their abracadabra, we now have much more impressive words like adenosine triphosphate. Adenosine tri-phosphate—and the muscle contracts; adenosine triphosphate— and it relaxes again.

Let us look a little further into the importance of ATP for muscular contraction. In the first place, it should be remembered that there has never been any direct evidence to show that in living muscle ATP breaks down in the course of muscular con-traction. Lohmann, the discoverer of ATP breakdown in muscle extracts, was never sure of the importance of the reaction for the contraction process. And in 1947 that great authority on muscle metabolism, Otto Meyerhof, wrote: "We should not . . . treat this hypothesis . . . that ATP breakdown initiates the chain of events in activity as an established fact. . . . To my knowledge, nothing besides the earlier arguments of Lohmann was dis-covered which adds to the evidence of such a series of reactions, and we should treat the problem, accordingly, with caution." And in 1949, Α. V. Hill (1949a) wrote: "In truth, the break-down of ATP has never been observed in an intact muscle except in extreme fatigue verging on rigor."*

But let us return to the model experiments. All of them involve a fundamental error. For in every single model that has been chosen, or at least in every model that has been discussed in these pages, the inanimate material believed to simulate the living muscle is a gel. But, as shown in an earlier chapter, the interior protoplasm of muscle fibers is a fluid sol. If one squirts water from a micropipette into the interior of a muscle fiber, the water quickly mixes with the interior protoplasm, but if one injects water into a glycerinated muscle fiber prepared accord-ing to Szent-Györgyi's recipe, the injected fluid remains as a discrete droplet, a fluid droplet in a solid gel. To take a dried gel, to find that some substances cause it to shorten—others to

* Since the above was written it has b e e n shown rather clearly that ATP

does not break d o w n w h e n a muscle contracts, and it has been suggested that

this m a y we l l inspire "a second revolution in muscle physio logy" ( s e e F lecken-

stein et al., 1954; Mommaerts , 1 9 5 4 ) .


lengthen—all this gives no real information concerning the proto-plasmic colloids of the muscle fiber. What we need is informa-tion about the living colloid itself; not guesses and hypotheses based on highly insecure analogies.

As to the role of ATP on models of muscle fibers or on dead muscle fibers, years ago, long before the magic of ATP came to be recognized, Hiirthle (1909) immersed glycerinated muscle fibers of insects in water and saw them shorten just as the fibers in Szent-Györgyi's model do. No ATP—no source of phosphate bond energy—just water. Similarly, Hopkins (1955) found that frozen dried muscle fibers of the frog on being returned to dis-tilled water or salt solutions at room temperature shorten very markedly. As to whether or not ATP makes a living muscle fiber contract is a debatable question. The early observations, for example, those of Buchthal, Deutsch, and Knappeis ( 1944, 1946 ) indicated that ATP caused a contraction of isolated fibers. How-ever, in this work barium was used in the preparation of the ATP and it is hard to get rid of the last traces of this element. Barium exerts a very powerful contracting effect on muscle protoplasm; thus for the muscle of the frog heart, it is eight times as potent as calcium (Dreyer, 1925), and as has already been indicated and will be shown more fully later, calcium is very potent in causing muscle protoplasm to contract.* When ATP is injected into the isolated muscle fibers of the frog, according to the un-published observations of F. J. Wiercinski, little or no effect is produced, certainly nothing to compare wich the effect of various other substances which can cause violent shortening. In smooth muscle ATP causes relaxation (see, for example, Folkow, 1949) and it makes the frog heart stop in diastole (Chaet, personal communication). Obviously the living muscle does not behave like glycerinated fibers do, and it is the living muscle rather than the dead muscle that we are primarily concerned with.

Indeed this set out to be a book on living protoplasm, and it is high time we returned to our subject. What can our knowledge

0 In our experience some commercial preparations of ATP, supposedly free

of barium, do not cause isolated frog muscle fibers to contract w h e n the fibers

are immersed in them.


of the physical nature of muscle protoplasm and of protoplasm in general tell us about the mechanism of muscular contraction?

Before we can go on with this subject, it will be necessary to review, ever so briefly, the main facts about the contraction of muscle. In the first place, a living muscle can be made to con-tract or shorten by the application of any one of various stimulat-ing agents, and this is one of the essential ways in which it differs from a dead muscle. The stimulating agents which cause muscle to contract are the same agents which act on protoplasm gener-ally. Sudden heat, sudden cold, sharp uneven pressure such as is given by a blow, ultraviolet radiation, an electric shock; all these agents can cause contraction of muscle just as they can cause the excitation of a nerve or the stoppage of movement in the rotating protoplasm of plant cells. Because the subject of excitation is a general one, because so many types of proto-plasm respond to the same types of stimuli, it will be necessary to devote a special chapter to the understanding of excitation. The chapter on excitation will come later, but at this point it is scarcely proper to discuss the nature of muscular contraction without mentioning, as we have just done, the agents which initiate the contraction. Also, in considering electrical stimula-tion, it should be pointed out that when an electric current is sent through a muscle, the muscle always begins its contraction at the cathode. This long-known fact is of importance for the theory that will be developed later.

Secondly, we must keep in mind what happens when a muscle is exposed to a single stimulus. The muscle shortens and speedily it relaxes again. This relaxation is not a passive process, that is to say, it can occur even when the muscle is not loaded down by a weight. The time relations of the contraction are important. These vary somewhat, for some muscles are much slower than others. Mostlv we know about the course of muscular contrac-tion as it occurs in one or another of the muscles of the frog leg. Almost every student of biology or physiology has seen a frog muscle contract, and has either himself made, or has observed, records of such contractions on a kymograph. Exposed to a single stimulus, a frog muscle shortens and then relaxes to its original


length again in the short space of time of a tenth of a second. The time consumed in contraction is a little shorter than the time required for relaxation. But before the muscle begins to shorten, there is a very brief period in which it lengthens slightly. This slight relaxation, which occurs before contraction begins, was for many years called RamYs nose, because of the fact that it was discovered by Rauh (1922) and the dip in the record of contraction resembles more or less the shape of a nose. Now, in what is perhaps more dignified terminology, the relaxation before contraction is called "latency relaxation" (see Sandow, 1947).*

F I G . 36 . Fat igue in a frog sartorius

muscle , st imulated once per second. (F le t -

cher, after Starl ing) .

A third important body of facts is concerned with the fatigue of a muscle. When a muscle is repeatedly stimulated at frequent intervals, after a time the contractions become weaker; then they cease completely and the muscle remains in the relaxed state, even though it is subjected to electric shocks. The muscle is then said to be fatigued. Figure 36 is a typical record of a muscle subjected to a series of stimulating shocks. Note that the height of contraction increases with the first few stimulations. This phenomenon has been called by the German word "Treppe" or by its English equivalent "staircase." The point to be em-phasized here is that after a number of stimulations the muscle stays in the relaxed state. The type of record shown in Fig. 36 is obtained if an isolated muscle is stimulated in a small volume of fluid. Under such conditions, if the muscle is stimulated once a second, it typically becomes fatigued in a few minutes.

* Rauh's kymograph records indicate that the latency relaxation might be

more pronounced with weaker stimulation. In the l ight of colloidal theories to

be deve loped later, this might have theoretical significance.


However, if the muscle is bathed in a fluid that flows over it so that the fluid is constantly being replaced, the muscle may not show any signs of fatigue for hours. This was clearly shown in Asher's laboratory at the University of Bern by a number of workers (for example, Labhardt, 1927; Witschi, 1927). Thus there is an indication that the muscle as it is being stimulated, gives off to the surrounding medium substances which hasten fatigue or enhance it. This is an old idea, one that goes back to Ranke (1863). He stimulated the leg of a frog to complete fatigue, then perfused it with salt solution and obtained recovery; also he found that the water extract of a fatigued muscle caused fatigue in a second muscle. A more striking experiment was performed by Mosso in 1890. By making a dog run, he got it into a thoroughly fatigued state, then he took the blood from this animal and injected it into the blood stream of a second animal. The second animal promptly showed signs of fatigue. Ranke (1863, 1864) introduced the idea of fatigue substances, and he thought that one of the most essential of these was lactic acid. This idea has often been suggested, but it is certainly not correct (Abelous, 1894; Simonson, 1935). The essential point is that when a muscle contracts it produces a substance or substances which tend to prevent contraction; thus the very act of contrac-tion creates an inhibitor of contraction. If we knew the true nature of the inhibitor, this might well help to explain some of the mysteries of muscular contraction.

When a muscle is stimulated, at the point of excitation the surface of the muscle becomes negative electrically to other parts of the muscle. Thus there is an action potential and an action current. Action potentials have already been discussed in Chapter 4. A complete theory of muscular contraction will have to take them into account.

Finally, there is an enormous amount of scientific informa-tion about the chemical changes that take place in muscle and in extracts of muscle. Certainly it is true that when a muscle con-tracts there is an increase in the oxidative processes going on in the muscle protoplasm. Biochemists and physiologists with a leaning toward chemical interpretation (and this would include


most present-day physiologists ) tend to believe that the oxidative processes going on in muscle, processes which take place at a more rapid rate during contraction, are in one way or another responsible for the contraction of the muscle. But neither the biochemists nor the physiologists have ever developed any con-vincing theory of why it is that oxidative reactions, whether they involve phosphorylations or not, can so affect the proteins of the muscle as to make the entire muscle contract. From a chemi-cal standpoint, muscle is primarily a protein machine; from a physical standpoint, it is a colloidal machine. How can any of the substances built up or broken down in the muscle, as a result of the processes of oxidation or reduction, so change the proto-plasmic colloid as to make it first shorten and then lengthen? Moreover, the fact that oxidative processes increase in rate during the contraction of a muscle does not necessarily mean that the increased rate is a cause of the reaction, for it is just as logical and perhaps more logical to assume that it is a result. Indeed there appears to be a growing belief that the oxidative and phos-phorylation reactions are more concerned with relaxation than with contraction. Lohmann himself, the discoverer of the ATP reaction in muscle, liked to think of muscle as a spring poised to shorten, and he believed that perhaps the phosphorylation reac-tions in muscle might serve the purpose of stretching the spring again ( Lohmann, 1937 ). But as to how the stretching might be accomplished in terms of phosphorylations has remained a mystery, concerning which there has been little or no direct evidence and not even much speculation.

The last few paragraphs have been little more than a list of some of the changes that occur when a muscle contracts. Cer-tainly it is far from being a complete list, but it gives a rough idea of some of the facts that a theory of muscular contraction should attempt to interpret. Certain other aspects of muscular contraction scarcely require interpretation. For if there are oxi-dative reactions, there must be heat produced, and the total amount of heat given off by the muscle is an algebraic sum of the heat given off and the heat taken up by all of the reactions involved.


Obviously at the present time no complete theory of muscular contraction is possible, but in the pages that follow an effort will be made to show how known facts can be used to interpret the contraction of muscle in terms of the known properties of the protoplasmic colloid. The discussion will be simple, for the facts are simple, these things are known:

1. A muscle fiber cannot contract in the absence of calcium. Unfortunately the usual opinion is that the ion most important for the response of muscle is sodium. But this opinion is wrong. It is based on a famous and oft-quoted paper by Overton written in 1902. What Overton did was to immerse a frog muscle in a sugar solution; after a time the muscle lost its irritability and would no longer contract when subjected to an electric shock. According to Heilbrunn and Ashkenaz (1941), what actually happens in the sugar solution is that the muscle as a whole soon fails to contract, but that the muscle fibers in the immediate neighborhood of the stimulating electric current continue to show a localized contraction for a long time. In the absence of electro-lyte, the electric current does not travel up and down and through the muscle; moreover, the local electric currents, which, as we shall see in the next chapter, are responsible for conduc-tion, are impeded or inhibited by the absence of a medium cap-able of conducting electricity. After a muscle has been in a sugar solution for a considerable time, if the individual muscle fibers are dissected out from the interior of the muscle, they are still capable of response to electrical stimulation. If now the fibers in the sugar solution are transferred to an isotonic sodium chloride solution, within a minute or two they lose their ability to respond to electrical stimulation. This is due to the fact that the sodium ion replaces the calcium of the cortex, presLimably by a process of ion exchange. If the muscle fibers which have lost their irritability are then transferred from the sodium chloride solution to a solution of sodium chloride plus calcium chloride, the fibers regain their irritability and respond readily to electrical stimulation. Also if the fibers are placed in a solution containing calcium and no appreciable amounts of any other cation, they respond readily to electric shocks, although in


such solutions they live only for a short time. Clearly it is calcium and not sodium that is necessary for the contraction of the fiber to occur.

2. In heart muscle the magnitude of the contraction is pro-portional to the amount of calcium in the surrounding medium In the study of the action of chemicals on protoplasm there is always a basic difficulty in that it is hard to know whether a chemical really enters a cell or not. Thus a substance might have a very drastic effect on protoplasm, but if it were prevented from entering the cell interior by its inability to penetrate the plasma membrane and the cortex with any rapidity, it might appear to be without effect. Fortunately, cells differ widely in their permeability and there are some types of cells that are much more permeable than others. Although some cells appear to offer considerable resistance to the entrance of cations (es-pecially calcium), the cells of heart muscle and the smooth muscle cells in the walls of blood vessels apparently allow calci-um ions to enter rather rapidly. In this way they differ from skeletal muscle fibers, which act as though they are impermeable to calcium ion (compare Heilbrunn, 1940).

The sensitivity of the protoplasm of the frog heart to calcium has been long known. If the heart is perfused with a solution containing no calcium, it very quickly stops in a relaxed state; then if calcium is added again, it rapidly resumes its contractions (De, 1928). Back in 1912 Straub suggested that the sensitivity of the frog heart could be used as a means of titrating the con-centration of calcium, and in 1921 this was actually done by Trendelenburg and Goebel. According to Clark, Percival, and Stewart ( 1928 ), the mechanical response of the frog heart varies directly as the concentration of calcium ions—the more calcium in the medium, the greater the contraction. The use of the frog heart preparation as a means of titrating for calcium ions was perfected by McLean and Hastings (1934). Thus everyone is agreed that with increased calcium in the outer medium, there is increased contraction, the amount of contraction being propor-tional to the amount of calcium (for the lower ranges of calcium concentration). It might be assumed, as Clark, Percival, and


Stewart actually do assume, that the effect of calcium is on the surface of the cell. However, there is a mass of evidence, both for protoplasm in general and for muscle protoplasm in par-ticular, that calcium primarily affects the interior protoplasm, and in the case of muscle, causes it to contract. This will become clearer in the course of the discussion.

Calcium also causes a contraction of the smooth muscle cells in the blood vessels; this is evidenced by a constriction in the caliber of the vessels (Hooker, 1911; Alday-Redonnet, 1920). Likewise various other types of smooth muscle are made to contract by calcium, but this is not true for all smooth muscle. Apparently the differences are largely the result of differences in permeability. In general, there is good reason to believe that whenever calcium enters a muscle cell in any appreciable con-centration, it causes a contraction of the muscle protoplasm.

3. All the agents that cause muscle to contract cause a release of calcium. Actually, it is not only calcium that is released, but magnesium and potassium as well. What happens is that the proteins of the cell and, presumably, the proteins of the cortex, which, as we have seen in Chapter 4, bind cations, to some ex-tent lose their affinity for cations. This, as we shall see later, actually happens. But for the present, we are concerned only with the evidence that muscular contraction is actually accom-panied by a release of calcium. Following muscular exercise, there is an increase in the concentration of calcium in the blood. This may or may not be significant, for during exercise, the blood loses water. However, if muscles contract very violently, the increase in the calcium of the blood is so great that there can be no question but that calcium has been released into the blood. Strychnine causes violent muscular contractions. Miko and Pala ( 1927 ) injected strychnine into rabbits, and found that the con-vulsions that resulted were accompanied by a 25-30 per cent in-crease in the calcium concentration of the blood. In similar experiments on dogs Beznâk (1931) found a 15-46 per cent increase in blood calcium. Lissâk (1934) injected both rabbits and dogs with the toxin that produces tetanus or lockjaw; the muscular spasms that resulted were accompanied by a significant


increase in the calcium of the blood. Coombs, Searle, and Pike (1934) produced convulsions in cats by operations on their brains; these convulsions also caused a sharp increase in blood calcium. Perhaps the most interesting work is that of Wacker (1929). He stimulated muscles all over a rabbit's body elec-trically and found that the concentration of calcium in the blood increased as much as 50 per cent.

Heart muscle also gives off calcium, and the amount of calcium given off increases as the frequency of the beat increases. Yasutake (1925) perfused a turtle heart with Ringer's fluid and determined the amount of calcium that was given off to the perfusing fluid. When the heart was made to beat more rapidly by stimulation of the sympathetic nerve, there was approximately a 20 per cent increase in the amount of calcium released. When isolated skeletal muscle fibers of the frog, or small bundles of such fibers, are made to undergo contracture by stimulation with ultraviolet radiation, they lose approximately 5 per cent of the calcium they contain to the surrounding medium ( Ashkenaz, 1938a). In some careful experiments, unfortunately never pub-lished in detailed form, Woodward (1949) studied the release of radioactive calcium from sartorius muscles of the frog which had been made to take up the element. Upon stimulation these muscles gave up 30-200 per cent more radioactive material than control unstimulated muscles.

Clearly, when a muscle contracts, calcium is freed and given off to the surrounding medium. But does any of this liberated calcium stay in the muscle or enter the interior of the muscle fibers? There isn't much evidence on this point, but what evidence there is indicates that it does. In 1934 Weise studied the free and bound calcium of rat muscle. In the resting muscle of the rat she found 4.3-7 mg. per cent of calcium, but none of this calcium was free, for none of it was able to pass through a collodion ultrafilter. On the other hand, after the rats were fatigued by making them run for 5 or 6 hr., Weise found that their muscles contained 1-3.7 mg. per cent of diffusible calcium.

If it is true that when a muscle is stimulated to contract, calci-um is released from a bound state, then we should perhaps in-


quire as to why it is that the various diverse types of stimulat-ing agents are able to cause such a release of calcium. This is a question that will be discussed more fully in a later chapter, for the stimulating agents that act on muscle also act on various other types of protoplasmic systems. The problem is a general one and can best be interpreted on the basis of information obtained from all sorts of cells, some of them with protoplasm much more amenable to study than is the protoplasm of muscle. Suffice it to say at this point that such stimulating agents as heat, cold, and potassium chloride, which at first glance have little in common and could scarcely be expected to produce the same effect on protoplasm, actually do, all of them, cause a release of calcium from homogenized muscle fibers of the frog. This has been shown by Weimar ( 1953 ) ; she homogenized the muscle fibers in a Waring Blendor and tested the binding of calcium by the muscle brei.

4. Calcium release can cause an action potential. This was shown to be true in Chapter 4. For if the cortex of the cell is a calcium electrode, then any decrease in its affinity for calcium would produce a potential drop in the outer region of the cell. Muscle behaves in the same way as do other types of living material. The only reason for mentioning action potentials at this point is to make it clear that a reasonably complete theory of muscular contraction should offer an explanation of the electric changes that precede (or accompany) the shortening of the muscle (ATP enthusiasts, please note!).

5. When muscle is stimulated by an electric current passing through it, contraction begins at the cathode and may be limited to the region of the cathode. This indicates that some cation is responsible for the initiation of the contraction. The fact that muscle responds at the cathode is old knowledge, which has been pushed very much into the background by newer and, at the moment, more impressive investigations. It is a phenomenon of genuine importance, not to be neglected by those interested in the understanding of muscular contraction. Nearly a century ago, in 1860, Bezold reported to the Berlin Academy of Sciences that if a frog muscle was placed in an electric circuit, as soon as


the circuit was closed contraction began at the cathode. Bezold ( 1861 ) used the sartorius muscle, which has long, parallel fibers, and he treated the muscle in order to prevent any effects due to nerve. Subsequent to Bezold's report, various other workers became interested in the problem. Notable among them was Engelmann (1867, 1868, 1870). Engelmann tried various types of experiments; among other things, he suspended a long, slender, frog sartorius muscle and then showed that only at the cathode did it contract. This is illustrated in Fig. 37. (Presumably the

υ

(α) (6)

FIG. 37. (a) A. suspended sartorius

muscle of a frog connected with wires

from a battery; the circuit has not yet

been closed, (b) Contraction at the

cathode after closing of the circuit.

passage of the electric current through the muscle prevents the passage of a wave of contraction ).

Even more convincing proof that contraction occurs at the cathode when an electric current is sent through a muscle, was given by Biedermann (1879). He used a technique that had been proposed by his teacher, Hering (1879). A curarized sartorius muscle of a frog was suspended in a horizontal posi-tion and held tightly in the center. With the aid of nonpolariz-able electrodes an electric current was sent through the length of the muscle, and then contractions were recorded from both ends of it on a double kymograph. Only the cathodal end of the muscle showed a contraction. In this experiment relatively weak


currents had to be used; with stronger currents, contraction spread through the muscle. Of course, if an electric current is passing through a muscle and the circuit is broken, a polariza-tion current then flows in the opposite direction; as a result contraction now takes place at the new cathode ( the old anode ). This also was observed in 1860 by Bezold.

If some substance that migrates toward the cathode, that is to say, a cation, causes the contraction of muscle, which cation is it? There are only four cations present in any considerable quantity in muscle. These are sodium, potassium, magnesium, and calcium. Which of these is the one that is responsible, or is there only one that is involved? The answer is clear, as will be shown immediately.

6. Of all the cations present in muscle, calcium is the only one that has a shortening effect on the interior protoplasm. This follows from the work of Heilbrunn and Wiercinski ( 1947 ). They studied the effect of injecting with a micropipette various salt solutions into the interior of isolated muscle fibers of the frog. When a frog Ringer solution, containing a small amount of calci-um, was injected into the fibers, they contracted immediately until they were only a little over half as long as they were origi-nally. If in the Ringer solution, magnesium was substituted for calcium, injection of this magnesium Ringer caused first a slight lengthening of the fiber, soon to be followed by a return to the original length. There was very little if any effect when sodium or potassium solutions were injected into the fibers; in these in-jections, an isotonic solution (0.123 molar) was used. On the other hand, if a very much more dilute solution of calcium chloride was injected, the muscle shortened violently. Thus a 0.002 molar calcium chloride solution made the muscle shorten to about half of its original length. This shortening occurred whether the solution was slightly acid (pH 5.65) or slightly alka-line (pH 8.0).

Calcium exerts an effect on the muscle protoplasm, even in very high dilution. In some earlv experiments Heilbrunn and Wiercinski cut isolated muscle fibers (of the frog) into pieces of equal length; then injected into one of the pieces various con-


centrât ions of calcium chloride. A noticeable shortening could be observed even with concentrations as low as 0.0002 molar. The experiment is illustrated by Fig. 38. In interpreting experi-ments of this sort, it should be remembered that the solution of calcium chloride that is injected into the muscle fiber is injected in the form of a tiny droplet, which is immediately diluted by the protoplasm of the muscle. Thus, apparently, the concentra-tion of calcium chloride that is effective is something less than one-five-thousandth molar.

F I G . 3 8 . T h e t w o pieces of a frog muscle fiber

shown in the figure were originally of equal length.

Into one of them a dilute solution of calcium

chloride was injected with a micropipette; this

p iece then shortened greatly ( Wiercinski ) .

We have seen that stimulating agents of various types cause a release of calcium in muscle and that this calcium is capable of causing muscle protoplasm to shorten. But why? Ordinarily, calcium in dilute solutions has no great effect on protein systems. However, in vertebrate blood, calcium is the ion essential for blood clotting and in earlier chapters of this book it has also been shown to be essential for protoplasmic clotting. The clotting of muscle protoplasm may well be like the clotting of blood, as discussed below.

7. Muscle contains substances known to favor blood clotting and other substances known to inhibit blood clotting. This is a difficult subject to discuss, for some of the information concern-


ing it is rather old and is perhaps in need of reinterpretation in the light of modern knowledge in the field of blood coagulation. It has long been known to surgeons, both those who practice their art on human beings and those who experiment on animals, that it is often possible to stop a hemorrhage by placing a freshly cut piece of muscle in contact with the flowing blood. Early students of blood clotting emphasized the fact that extracts of muscle and other tissues could markedly hasten the clotting process ( see Schmidt, 1892 ). This is especially noticeable in bird blood. Thus Delezenne (1897) found that if the blood of a duck was drawn carefully (so as not to come in contact with tissues), it did not begin to clot until 3 days had elapsed and the clot was not complete until the 4th day. However, if blood from the duck was allowed to come into contact with a fragment of muscle, clotting commenced in 20 sec. and was complete in 1 min.

In a study of rabbit muscle Kraus and Fuchs ( 1929 ) found that if they froze the muscle rapidly in liquid air, they could demon-strate the presence of considerable amounts of thromboplastin and prothrombin in it, but no thrombin. However, if the muscle was subjected to mechanical injury so as to cause what the authors call a disturbance of cell structure, then thrombin was formed in the muscle. This is rather interesting, for it indicates that in muscle, as in blood, thrombin is formed as a result of a clotting reaction. When muscle is subjected to lack of oxygen, the amount of thromboplastin that can be extracted from it in-creases (Stoner and Green, 1947). According to Fischer (1934), chicken muscle and horse muscle contain thrombin as well as prothrombin, for extracts made from these muscles were able to clot oxalated blood and heparinized blood; they could also clot solutions of pure fibrinogen. Thus muscles contain prothrombin, thromboplastin, and some muscles at least contain thrombin.

Clotting substances are also found in the muscles of inverte-brates. This was shown by Leo Loeb in a long series of papers (see especially Loeb, 1905, 1906). Loeb found that the muscles of the lobster contained a substance which hastened the clotting of lobster blood, as well as a substance which prevented this clotting.


In the last chapter evidence was presented to show that cells in general, and muscle cells in particular, contain heparin or heparin-like substances. These substances can prevent proto-plasmic clotting, at any rate this is true for the heparin-like substance which is found in lobster muscle (Heilbrunn and Wilson, 1955c).

If it is true that the action of calcium in causing shortening of muscle is due to a clotting reaction similar to the clotting reaction in blood, then since thrombin is now known to be not only a clotting enzyme, but also a proteolytic enzyme, and since pro-teolytic enzymes like trypsin cause blood clotting, it seems logical to assume that proteolytic enzymes should cause a clotting and a shortening of the protoplasm of muscle cells. This is true, as will be shown immediately.

8. Proteolytic enzymes clot and shorten the protoplasm of muscle fibers. Woodward ( 1948 ) immersed isolated frog muscle fibers with cut ends in a dilute (0.1 per cent) solution of crystal-line trypsin in Ringer's fluid. The trypsin greatly hastened the clotting of the protoplasm of the muscle fibers; indeed the rate of clotting was 38 times as fast as in the control without trypsin. The clotting was, as always, accompanied by a shortening. Chymotrypsin and papain acted in similar fashion, although they did not act nearly as powerfully as the trypsin.

An even more striking experiment was performed by Wiercin-ski and Cookson ( 1949 ). They injected a 0.05 per cent solution of crystalline trypsin into frog muscle fibers. Immediately the protoplasm contracted so violently that it broke into pieces. Wiercinski took a motion picture of this phenomenon. Figure 39 shows two of the frames taken from this motion picture. More dilute solutions of trypsin, 0.01 per cent or even 0.001 per cent also caused a contraction of the muscle protoplasm, but with these solutions, the contraction was less violent.

9. When blood clots, substances are produced which cause muscles to contract. The clotted defibrinated blood of a cat will make the heart of the same cat contract much more violently than it does normally; although the unclotted blood is totally without effect (Heuking and Szent-Györgyi, 1923). There is

117 7. MUSCULAR CONTRACTION

much literature to show that clotted blood or blood serum will cause various types of smooth muscle to contract; this literature goes back as far as 1869. Unfortunately it is still in an unsatis-

F I G . 39 . T h e action of a dilute solution of trypsin

on the protoplasm of frog muscle fibers ( Wiercinski ) .

factory state. Useful papers include those of Janeway, Richard-son, and Park (1918), Reid and Bick (1942), Zucker (1944), and Moussatché and Cruz (1952), and these give references to the older literature. The prevailing opinion is that the substance in clotted blood which causes muscular contraction comes from the blood platelets; probably it is some sort of a precursor or co-factor of thrombin; but it is not thrombin itself, for it is heat


stable and dialyzable. The fact that the potent substance of clotted blood is dialyzable probably explains why it can enter muscle cells, but apparently it can enter only those muscle cells which are more readily permeable than the cells of striated muscle.

10. When a muscle is stimulated to fatigue, heparin or a heparin-like substance is given off to the surrounding fluid. This follows from the work of Most ( 1950). If frog muscles in Ringer's fluid are stimulated repeatedly so that they are no longer able to contract, the fluid comes to contain a substance which will prevent the clotting of (human) blood. Action of this anticlot-ting substance is antagonized both by protamine and by toluidine blue; both of these substances are well known antagonists of heparin. According to Nekrassow and Nekrassowa (1936), if a few drops of blood serum are placed on a fatigued frog muscle, the muscle recovers its power of contraction; blood serum might well counteract the effect of heparin.

11. Heparin can cause relaxation of muscle. In the clinical literature there are various indications that heparin (and other anticoagulants) can cause small blood vessels to dilate so that more blood can flow through them (Gilbert and Nalefski, 1949; Ahlquist, 1950; Abrahams and Howarth, 1950; Engelberg and Massell, 1953; Matis and Scheele, 1953; Storti, Vaccari, and Scardovi, 1954). The dilatation of the blood vessels is due to a relaxation of the smooth muscle cells in the walls of the vessels. These cells seem to be especially permeable and that is probably why heparin can act on their protoplasm.

As noted previously, the muscle fibers of the vertebrate heart seem to have membranes which are relatively permeable to dis-solved substances. Thus it is not surprising that the frog heart is affected by solutions of heparin. These solutions cause the heart to stop in diastole; in other words, they inhibit the contraction of the heart muscle. The effect of heparin on the heart was described first by Kraus, Fuchs, and Merländer in 1931. They showed that not only heparin, but other blood anticoagulants as well, all stopped the heart in diastole. In a recent, careful study, Chaet (1955) has repeated the earlier work of Kraus,


Fuchs, and Merländer. One of Chaet's kymograph records is shown in Fig. 40. According to Chaet, the effect of heparin solutions on frog heart muscle is not due to the large heparin molecule itself, but to some smaller molecule, one capable of passing through a dialysis sac. This smaller molecule may perhaps be a breakdown product of the heparin.

F I G . 4 0 . Effect of heparin on frog heart ( C h a e t ) .

When a muscle contracts, there is a great increase in the rate of the oxidative processes going on in the protoplasm. What is responsible for this increase in oxidation? Certainly the various types of stimulating agents, for example, sudden cold or the electric current, could scarcely be thought of as directly produc-ing an increase in the activity of oxidizing enzyme systems. Presumably the oxidative changes are related to the colloidal changes. But how? This we shall now consider.

12. The clotting of muscle protoplasm could logically cause an increase in oxidative processes. For, if protoplasmic clotting is, as there is so much evidence to indicate, fundamentally similar to blood clotting, then the fact that the clotting of blood is to some extent an oxidative reaction, could perhaps explain the relation between colloidal change and oxidations in muscle. There is plenty of evidence to indicate that blood clotting does involve oxidation. As far back as 1936 Mathews wrote "there is no doubt


that an oxidation and reduction system of some kind plays a role in clotting, for hydrocyanic acid and cyanides and H 2S and hyposulfites inhibit thrombin clotting." More recently various authors have suggested that the change of fibrinogen to fibrin involves an oxidation of the —SH groups of fibrinogen to S-S groups (see, for example, Baumberger, 1941; Chargaff and Bendich, 1943; Gerber and Blanchard, 1945; Lyons, 1945; Jeener, 1947). If such an oxidative reaction occurs when muscle proto-plasm clots, it could be the spark that starts electrons flowing rapidly when a muscle is thrown into activity.

Something of this sort may perhaps happen in blood, although in view of a rather vigorous controversy,* the evidence is some-what hard to evaluate. If blood is allowed to clot, some of the sugar it contains is broken down to lactic acid—this is the same sort of process, glycolysis, that occurs in contracting muscle. But if the blood is oxalated so that clotting does not occur, the sugar content remains constant. Thus there seems to be a relation between clotting and glycolysis, and Stuber and Lang in a long series of papers urged that the glycolysis was the primary cause of the clotting (see Stuber and Lang, 1926, 1930; and references cited in the 1930 book). This theory of Stuber and Lang was strongly criticized (Hartmann and Kühnau, 1930; Partos, 1932) and even the factual basis of the theory was questioned. To these criticisms Stuber and Lang reacted strongly (1931, 1932). The truth of the matter is that the theory of Stuber and Lang will not hold, for there are many well known facts in contradic-tion to it. However, it does seem to be true that when blood clots there is glycolysis and when clotting is prevented glycolysis fails to occur (see, for example, Fuchs and Falkenhausen, 1931).

* Some recent authors have presented serious objections to the v iew that

w h e n fibrinogen clots to form fibrin, there is an oxidation of - S H groups to S -S

groups ( s e e especial ly Mihâlyi and Lorand, 1948; Edsall and Lever, 1951; also

papers cited b y Edsall and L e v e r ) . It is possible that who le b lood behaves

differently from pure fibrinogen. In Jeener's studies he used chicken blood. Al-

though it may wel l be true that the transformation of fibrinogen to fibrin is not

an oxidative reaction, it n o w seems certain that the presence of S-S groups is

important for the clotting process ( Carter and Warner, 1954 ) .


Hence clotting of blood can initiate reactions like those which occur in contracting muscle.

Although there is perhaps some doubt as to whether blood clotting can cause an increase in the rate of processes associated with metabolism, there does not seem to be any doubt but that when protoplasm clots, there is an increase in the rate of oxidative processes. At any rate this is true of sea urchin egg protoplasm, for when calcium is added to homogenates of these eggs pre-pared in the absence of calcium, as the protoplasm clots there is a sharp increase in the uptake of oxygen (Hultin, 1950a).

13. Muscle can be prevented from contracting by all those agents (anesthetics or narcotics) which prevent protoplasmic activity. If muscular contraction is initiated by one or another type of oxidation or phosphorylation reaction, then how can one explain the inhibiting effect of ether and other fat solvents? This is a broad, general question, one that will be discussed in a later chapter. There it will be shown that ether and other fat solvents prevent protoplasmic clotting. Our knowledge of the action of these substances fits in very well with what is known about the colloidal machinery of muscular contraction.

The facts as we have presented them not only offer firm sup-port for a theory of muscular contraction, they essentially con-stitute such a theory. When stimulating agents act on muscle, they cause a release of calcium and this calcium clots the proto-plasm in the interior of the muscle fibers. This causes the muscle to shorten. No other theory that has been proposed can explain the peculiar dependence of muscular contraction on calcium, the strange fact that both sudden heat and sudden cold can cause contraction, that contraction occurs first at the cathode, and that contraction is preceded by an action potential. The theory also provides an interpretation of muscular fatigue, and it likewise explains why it is that heparin can cause relaxation; it can even offer an interpretation of why the metabolism of the muscle in-creases as soon as contraction begins. The theory has the ad-vantage, moreover, that it is a theory that has its basis in an understanding of protoplasmic behavior. In later chapters it will be shown that the general nature of the response of muscle is


similar to the response of other types of protoplasmic systems. If the living material in all organisms and all tissues is as uniform as the morphologists and chemists have led us to believe, then we should not have one kind of response in muscle protoplasm and a totally different kind of response in the protoplasm of other cells.

Although the thirteen points that we have brought forward offer rather strong evidence that the contraction of a muscle fiber is due to some sort of a coagulative or clotting process in its protoplasm, there is no conclusive evidence that in normal con-traction the protoplasm does actually show such a change. We do know from Rieser's measurements (see Chapter 2) that the protoplasm in the interior of a resting frog muscle fiber is a fluid; and if, as seems certain, muscular contraction is dependent on some colloidal change, the only pronounced colloidal change that could occur in the interior protoplasm would be a change from sol to gel. That such a change did occur was a favorite belief for many years. In the past many authors thought that contraction was the same sort of process that occurs in rigor, an idea that goes back to Nysten, who wrote in 1811, and probably goes back to even earlier authors. (Nysten believed that rigor mortis was the last contractile effort of the vital forces in muscle struggling to combat death). Some have pointed out that when a muscle is thrown into rigor either by heat or by chemical agents known to be coagulative, the muscle shortens and thickens, just as it does in a contraction. Certainly this is a point worth con-sidering; it has been discussed pro and con by various physi-ologists. If a muscle is essentially fluid, and if it undergoes a clot-ting or coagulative process during contraction, we might expect that it would show changes in transparency, like those which occur when the white of an egg is heated or when blood plasma clots. If such a process were to occur, it would only be evident in muscles which were highly transparent. The ordinary muscles of a frog or a mammal are relatively opaque. In these muscles changes in transparency do occur, but they are relatively minor; and in order to be sure of them, one has to use rather accurate methods of study. In such muscles there does appear to be an


increase in opacity during contraction, and this has been related to colloidal change (Schaefer and Göpfert, 1937; compare also Buchthal, Knappeis, and Sjöstrand, 1939 ). However, the changes in opacity are not great and there is plenty of room for differences in opinion about them, for the subject is complicated by the fact that in striated muscle the striations can serve as a diffrac-tion grating.

But there are some muscles that in the living relaxed state are beautifully transparent, and in these muscles colloidal change should be relatively easy to observe. According to Biedermann ( 1909), the retractor muscles of the worm Sipunculus are remark-ably clear—a transparent blue. In these muscles even the slightest contraction causes a whitening and an increase in opacity. This is easily visible to the naked eye. Similarly in the foot of a snail, the longitudinal fibers, according to Biedermann, are glassy clear. Waves of contraction along these muscles show very sharply as cloudy whitish bands. In sea cucumbers also, there are muscles that become visibly opaque when they contract. According to Young (1938), the muscles of the squid Loligo, transparent and clear when relaxed, become opaque on contraction. This is shown in a drawing of Young's reproduced in Fig. 41. If these observa-tions of Biedermann and of Young had been made with elaborate apparatus, with photoelectric cells contained in spectrophotom-eters or colorimeters, they would have had far more influence on physiological thought than they have had. But the fact that the changes in opacity are so pronounced that they can be seen with the naked eye has, strangely enough, made them much less impressive to the scientific community. In describing what he saw, Biedermann wrote that he could see no possibility that the obvious opacity of every contracting region in comparison with the complete transparency of the relaxed region could be interpreted in any other way than as an indication of colloidal change. I should be inclined to agree with Biedermann, but not all writers have felt this way (compare Pauli, 1912), especially if they thought that the protoplasm of a relaxed muscle was a gel.

A theory, if it is to be successful, should invite criticism. Only in this way can a decision be reached as to how much truth there


F I G . 4 1 . Diagram of the mantle of

Lol igo to show areas which have be-

come opaque after local stimulation.

Electrodes were placed at A and B,

and after the application of a few

stimuli the whole of the units in which

the electrodes lie become opaque.

Fenn (1945) in discussing the idea that muscular contraction depends on calcium release says that "the theory is based largely on the contraction which results when a muscle is immersed in isotonic solutions of CaCh" and that such a concentration is far beyond physiological limits. Work, for the most part pub-lished after Fenn's criticism, shows that very dilute concentra-tions of calcium are effective if they enter the muscle protoplasm. Also Fenn refers to earlier work of his own in which stimulated gastrocnemius muscles of the cat showed no loss of calcium as compared with normal, unstimulated muscles ( Fenn et al., 1937 ). In this work Fenn and his collaborators estimated the total calci-um (bound and free) in whole muscles. When muscle fibers are stimulated, calcium is liberated, but most of this calcium prob-

is in it. What arguments have been presented against the point of view outlined in the preceding pages? Of published criticism there has been very little, and the theory suffers more from being disregarded than from any actual attacks on it.


ably stays in the fibers; some enters the intercellular spaces and some enters the blood capillaries. An analysis of a whole muscle —blood and all—might well not reveal any changes in total calcium.*

A favorite criticism of the calcium release theory, one that perhaps often comes to the minds of those who learn about the theory, is that in the tetany which follows deprivation or lack of function of the parathyroid glands, there is a drop in the calci-um of the blood, and nevertheless a great contraction of the muscles. Parathyroid tetany is doubtless a complicated process in which perhaps increases in the concentration of clotting enzymes may occur, but the point to be brought out here is that following removal of the parathyroid glands, although there is indeed a decrease in blood calcium, there is no decrease in the calcium content of the muscles. In parathyroidectomized cats Burns (1933) found a slight increase in the calcium of the muscles, an increase probably not significant, and in dogs, Dixon, Davenport, and Ranson (1929) obtained similar results. How-ever, in view of the fact that after parathyroidectomy the calcium content of blood and body fluids is low and in view of the fact also that muscle contains both fibers and intercellular fluid, Burns' data indicate a calcium content of the fibers somewhat above the normal. In rats Bülbring (1931) found a marked increase in the calcium content of the muscles of parathyroidectomized animals; when the rats were on a diet rich in calcium, the calcium content of the muscles doubled after parathyroidectomy.

A more interesting criticism of the theory was made by a lead-ing physiologist in an unpublished discussion. He expressed doubt as to whether in the small amount of time required for a muscle to shorten in a muscle twitch, there would be time enough for the calcium released from the cortex to penetrate into the cell interior. This is a serious question. Fortunately Hill ( 1948 ) has calculated the amount of substance that would enter a muscle

* It should perhaps be noted that F e n n and his collaborators found m u c h

more calc ium in cat muscle than did earlier workers. T h e y found seven times

as m u c h as Burns ( 1 9 3 3 ) and several t imes as m u c h as Heubner and Rona

( 1 9 2 3 ) .


fiber in the time it takes for it to contract. He assumed a frog muscle fiber 100 μ in diameter; at 30° such a fiber contracts in about a twentieth of a second. Because in a muscle twitch a muscle contracts only about half as much as it does when the contraction is complete (in a tetanus), only half of the muscle substance would need to be affected by the entering calcium. Hill found that in the allotted time (1/20 sec) , 40 per cent of a substance released at the surface of a fiber would penetrate half the volume of the fiber, and he concluded that there would be ample time for a substance arriving by diffusion from the surface to "trigger" the process of contraction. In Hill's calculation he assumed a diffusion coefficient of 5 χ 10° (for a hypothetical substance). Actually at 30° the diffusion coefficient of calcium is 14.8 χ 10"6 (Stiles, 1923), so that on the basis of Hill's calcula-tions, 40 per cent of the calcium set free from the cortex would penetrate into half of the muscle fiber in about one-third of the time required for the contraction in a muscle twitch. As a matter of fact, because of the extreme sensitivity of the muscle protoplasm to calcium, as noted in an earlier part of this chapter, it is doubtful if as much as 40 per cent of the calcium set free would be needed. Another factor (of minor significance) is the thickness of the cortex, for the entering calcium would diffuse from the inner border of the cortex and the cortex of a frog muscle fiber is 10 μ thick. This factor would still further shorten the time necessary for sufficient calcium to diffuse into the in-terior protoplasm.

In the final paragraph of his 1948 paper Hill writes: "The results of this calculation are suggestive and permissive only. They merely allow, by experiment and calculation, to examine further the hypothesis that propagation of contraction inwards is due to the diffusion of a substance liberated at the surface of a fibre during excitation. That hypothesis may be found untenable for other reasons. It is not ruled out, however, by the length of time necessary for diffusion."

However, in 1949, on the basis of experiments in which muscles were stretched immediately after they were stimulated, Hill com-pletely reversed his stand and argued that diffusion processes


could not possibly account for the speed with which a muscle changes from a state of rest to a state of maximum activity. In this later discussion Hill is impressed by the fact that in the muscles stretched immediately after stimulation, a maximal ten-sion is developed in about 40 msec, that is to say l/25th of a second (at 0 ° C ) . The time is not so much faster than that required for contraction in a normal twitch, but Hill emphasizes the fact that it is a maximum tension that is developed, a ten-sion as great as that which the muscle is capable of exerting in a tetanus. Hill's argument applies only to the stretched muscles and not to muscles contracting normally. Why are stretched muscles different? On the basis of the information presented in Chapter 2 we conceive of a muscle fiber as consisting of a tube of solid material, the cortex, surrounding a fluid interior, some-what like an ordinary piece of laboratory rubber tubing with a liquid of no great viscosity inside of it. If we rapidly stretch such a tube, the agitation of the stretching might well have an effect in stirring up the fluid contents. Under conditions like these, ordinary diffusion constants mean little; one would not measure diffusion constants in the presence of a stirring ap-paratus. The corrections to Hill's calculations noted in the pre-ceding paragraph would permit a four- or five-fold decrease in the time required for sufficient calcium to diffuse from the cortex to the protoplasm that contracts in a muscle twitch, but even this decrease might not be enough to account for the speed with which the maximum tension is reached in stretched muscle. However, these corrections, in combination with the added effect caused by agitation, would probably be sufficient to account for the abnormal speed with which tension is developed in the stretched muscle. And how else could one account for it? For what other type of process would be so hastened by a mere stretching of 15 per cent? Hill believes that some unidentified, mvsterious process is involved.

Over the years muscle physiology has suffered from the invoca-tion of mysterious processes and magic words. The theory out-lined in this chapter is based on known forces and known facts. Calcium is released when muscles are stimulated in any one of


a variety of ways, calcium does cause the protoplasm in the interior of a muscle fiber to shorten. The calcium release in it-self can cause an action potential. Thus excitation would involve calcium release and this released calcium would then act on the interior protoplasm and cause it to shorten or contract. Both calcium release and shortening are logical sequences of the known chemical and colloidal properties of the cortex and the protoplasm in the interior. Much still remains to be explained— the theory will no doubt have to be extended and modified— but even in its present status, the theory does far more to in-terpret the physical behavior of muscle than does any other theory.

8.

There is an enormous literature on the physiology of nerve fibers, a literature that is constantly growing. In the past this literature has, for the most part, been concerned with the prob-lem of the conduction of an impulse along a nerve, and it is easy to see why this should be so, for the primary function of nerve is to conduct. But in order to conduct an impulse, a nerve fiber must first be excited. In this chapter we shall be concerned only with the problem of excitation; conduction will be con-sidered briefly in the chapter to follow.

Before discussing the mechanism of excitation in nerve, it will be necessary to review briefly what is known about the physico-chemical nature of the nerve fiber and its protoplasm. It is assumed that the reader is acquainted with the ordinary facts about the microscopic anatomy of nerves and nerve fibers. The essential part of the nerve, in so far as its function is concerned, is the protoplasmic part of the fiber. This is ordinarily a very narrow strand of protoplasm, although fortunately for the nerve physiologists, there are exceptional cases of nerve fibers with a relatively huge volume of nerve protoplasm. Thus, for example, there are the famous giant nerve fibers of the squid, fibers which may reach a diameter of nearly a millimeter.

What is the nature of nerve protoplasm? For years cytologists have looked at it under the microscope and, as is usual in such studies, the microscope shows little that is of any help in in-terpreting why it is that a nerve can act as it does. Perhaps nerve protoplasm has fibrils—perhaps it doesn't. In living nerve fibers the fibrils appear more and more clearly as the protoplasm dies (Young, 1936; Bear, Schmitt, and Young, 1937). They show most clearly after the protoplasm has been thoroughly killed and

129

THE EXCITATION OF NERVE


coagulated by the harsh treatments used for so many years in classical cytology. In 1899, Fischer showed that cytological fixing agents could produce granules or fibrils from solutions of inani-mate proteins, and that these artifacts closely resembled some of the structures that cytologists had so carefully drawn in their pictures of fixed cells. This work of Fischer's and other work of the same sort made cytologists more cautious, and especially in recent years they have often attempted to check their studies of fixed cells by comparison with what can be seen in the living. But with the advent of the electron microscope there has been a new wave of enthusiasm for morphological study, and there have been various new attempts to discover in the protoplasm of nerve and other cells some structure or structures which would help to explain the mysteries of vital processes. Actually these studies have not been very successful. For the techniques neces-sary in the preparation of material for examination with an elec-tron microscope can do even more in the way of producing artifacts than the older methods of the cytologists. Caution is necessary in interpreting the pictures of nerve fibers and nerve fiber protoplasm that the electron microscope reveals. This has been shown by Wohlfarth-Bottermann ( 1954 ), who has been able to duplicate some of the structures observed with the electron microscope in fixed and sectioned cells by preparing solutions of pure proteins with the same techniques as those used by the electron microscopists on the cells. And the electron microscope has the additional disadvantage that, by the very nature of the apparatus, it is not possible to make any studies on protoplasm maintained in a living condition. Thus we still do not know whether the nerve protoplasm does or does not contain fibrils. Nor does it matter much, for even if fibrils of one size or another did exist, it would be difficult to correlate this information with any of the facts that are known about the vital processes that go on in nerve protoplasm.

So once again we are driven to the realization that morphologi-cal study of protoplasm, whether it be in nerve or in other cells, has not contributed very much to an understanding of vital machinery, and once again we must attempt to discover what we

8. THE EXCITATION OF NERVE 131

can about the physicochemical properties of the colloidal material that constitutes the axoplasm.

The information that we have is unfortunately slight. In a few measurements never reported in detail Rieser ( 1949b ) found that the viscosity of the protoplasm of the large nerve fibers in the ventral nerve cord of the lobster was that of a fluid sol. He injected small oil drops into the protoplasm and determined their rate of movement under the influence of gravity. In this way he found that the protoplasm of the lobster nerve had a viscosity of only 3 centipoises, that is to say, it was only three times as viscous as water.* However, most of those who have studied the squid nerve fiber are agreed that the protoplasm of this fiber is much more viscous. This may be due in part to the fact that observations are generally made on isolated nerve fibers. In isolating the fibers various branches have to be cut and the injury due to cutting may induce a change in the viscosity of the proto-plasm as a whole. For, as shown in Chapter 5, when a cell is torn or injured, a wave of coagulative change may pass through the protoplasm from the site of the injury. In experiments in which they studied squid nerve fibers in situ, Chambers and Kao ( 1951 ) found that when they injected solutions of acid dyes into the interior of the fiber, there was "definite evidence of a fluid core. When microinjected, the solution takes a preferred route of running along the central core through the nerve. The dye then diffuses radially . . .," presumably more slowly. This work might indicate a fluid central part of the axoplasm and a more solid cortex. According to Young ( 1936), any injury to the sheath of the giant nerve fiber of Sepia causes fibrillation and granula-

* This measurement of Rieser's is in accord with earlier observations and

opinions about the protoplasm of crustacean and other nerve fibers. Many

workers have urged that nerve protoplasm is fluid. These ideas go back to the

doctoral dissertation that the y o u n g Helmhol tz wrote in 1842. Haecke l ( 1 8 5 7 ) ,

Yung ( 1 8 7 8 ) , and Krieger ( 1 8 8 0 ) saw the protoplasm flow out of the cut nerve

fibers of Crustacea, and both Krieger and Yung saw Brownian m o v e m e n t in the

protoplasm. Hardy ( 1 8 9 5 ) was m u c h impressed wi th the extreme fluidity of

the protoplasm of crayfish nerves. According to Hardy, air bubbles are often

present in the protoplasm of crayfish nerves, and these could b e m a d e to m o v e

about.


tion. The effect of such an injury is shown in Fig. 42. These changes due to injury or to exposure to the outer medium seem to be similar to the coagulative changes that occur in cells generally in the surface precipitation reaction or in the proto-plasmic clotting that follows this reaction ( see Chapter 5 and 6 ). That a surface precipitation reaction does occur in nerve proto-

F I G . 42 . Giant axon of Sepia officianalis w h i c h has

b e e n punctured b y a fine needle . T h e fibrils were not

visible before the injury ( Y o u n g ) .

plasm has been clearly shown for crustacean nerve by Haeckel ( 1857 ). He found that the protoplasm which flowed out from a cut fiber "clotted in the form of droplets, threads, and granules." Similar observations were made by Yung (1878); he figures vacuolated droplets forming from the merging protoplasm. More-over, in the squid axon, there is also evidence of a surface precipi-tation reaction. Thus Flaig (1947) writes that: "When a giant axon was punctured fibrils about 5 microns in width formed rapidly at the point of contact between sea water and axoplasm and extended backward into the axoplasm." Sometimes the coagulative changes at the cut surface were sufficient to form a plug which prevented further exit of protoplasm.

It is not known whether coagulative changes are responsible for the gelled condition of the protoplasm of the isolated squid


axon, but it is certain that in the usual preparation of the axon, the protoplasm is rigid. This follows from the work of Hodgkin and Katz (1949b). They pulled out pieces of the nerve proto-plasm with a pipette and found that then the walls of the proto-plasmic gel did not close in to fill the space left by the pipette. If these pieces of gelled protoplasm are placed in contact with solutions containing calcium, then the gel is rapidly liquefied. Chambers and Kao (1950, 1952) confirmed the work and the conclusions of Hodgkin and Katz, and there does not seem to be any doubt that calcium does actually cause a liquefaction of the gelled protoplasm cut out from the squid axon.

This seems rather suprising, for in earlier chapters it was shown that in the presence of appreciable amounts of calcium ion, proto-plasm soon changes from sol to gel. Is it possible that calcium can cause both a clotting and a liquefaction of protoplasm; and if so, how can this be explained? Fortunately the explanation is quite simple. For, as noted previously, calcium activates a proteolytic enzyme that is at the same time a clotting enzyme. This enzyme can cause both clotting and liquefaction. Thrombin seems to behave in similar fashion ( Lorand, 1954 ). Also, as will be shown later (see Chapter 10), in the immature eggs of Chaetopterus, release of calcium causes a transitory clotting followed by a pronounced liquefaction.

Obviously we know very little about the colloidal properties of nerve protoplasm, and nothing at all about the cortex. More work is needed. We should like to know if watery solutions of various types diffuse through the protoplasm when injected with a micropipette or whether they form discrete droplets, as they would do if the protoplasm were wholly a gel. Also it would be of interest to know if a calcium-activated proteolytic system is present in nerve protoplasm and this should be rather easy to determine.

In studying the physiological behavior of nerve in the attempt to relate this behavior to the physical nature of the substances of which it is composed, there are great difficulties. The fact that a nerve is excited or responds can be detected only by its effect on muscle or by its electrical behavior. In their attempts


to interpret nerve excitation, physiologists have, in general, thought only in terms of membranes. Is the interior protoplasm without any function in relation to nerve excitation; does it merely act as a support for a membrane that is a functional part of the nerve? One is reminded of the speculation about the thyroid gland before some of its functions became known. Some physiologists thought its only purpose was to fill out and beautify the neck. Perhaps the protoplasm in the interior of a nerve fiber supplies energy for the excitation and the propagation of the impulse. There is no good evidence either for or against this point of view. In conduction the interior of the nerve may well serve as a pathway for the local action currents that are responsi-ble for the passage of the impulse along the nerve, but is this its only function?

Let us come now to the main point of this chapter. What we want to understand is how to explain the excitation that occurs in nerve. In the past there has been no adequate, general theory. We need to know why and how it is that not only the electric current, but also heat, cold, mechanical impact, chemical stimuli, and solutions of high osmotic pressure can all arouse a nerve. That they do this is obvious to anyone who has ever had an exposed nerve in a tooth or who has ever been worked on by a dentist.* In the past there has been very little attempt at ex-planation, except in so far as electrical stimulation is concerned. And even for electrical stimulation there is no adequate theory. In his excellent book on "Electric Excitation of Nerve," Katz, after mentioning briefly some of the hypotheses that have been proposed writes:

"In the absence of any critical evidence there seems to be no profit in discussing the relative merits of each of these hypotheses. It is a considerable relief, however, to find that Lapicque ( 1934 ) and Hill (1936) attribute now very little importance to their own earlier attempts of interpreting the physical nature of the excitation process . . . ."

"We are forced, with Lapicque and Hill, to admit our igno-

* For a discussion of the various agents which stimulate nerve, see Cremer,

1909; Fröhlich, 1929.


ranee, at this stage, of the intimate nature of the excitatory disturbance."

Since 1939, when the above sentences were written, there has been no marked improvement in our understanding of the "in-timate nature of the excitatory process" in nerve. It is true that various authors, notably Hodgkin, have advanced theories as to the origin of the action potential, and have shown that after excitation there is an increased rate of diffusion of various ions into and out of the nerve fiber, but these authors have not tried to explain why such changes in diffusion rates occur.

From the standpoint of general physiology, and more especially of cell physiology, it seems clear that the problem of excitation in nerve is essentially the same as the problem of excitation in other types of living material. If the various diverse types of stimulating agents excite muscle protoplasm, the protoplasm of marine eggs, and also the streaming protoplasm of plant cells, if in these other types of protoplasm the manifestations of the excitatory process are the same as they are in nerve, then it would indeed be strange if nerve excitation were a totally dif-ferent sort of phenomenon. In other branches of the broad fields of biology and biochemistry workers have learned to appreciate the uniformity of living material and to profit from it. Perhaps it is time that the nerve physiologists gave less thought to the exercise of their mathematical talents and less attention to the complexities of their material and their techniques, and con-sidered to some extent what application they could make of the facts discovered by students of excitation in types of material better suited for study from a protoplasmic standpoint.

One point cannot be too strongly emphasized—a complete theory of excitation, either for nerve or for any other living system, must take account of the fact that anesthetic or narcotic agents prevent excitation and response. By and large, the agents which prevent excitation in many diverse kinds of cells are effec-tive in preventing excitation in nerve.

In the chapter on muscle, evidence was presented to show that stimulating agents—cold, heat, ultraviolet radiation, etc., all cause a release of calcium and that by their very nature and


the nature of the colloids in the protoplasm, they would be ex-pected to do so. Is there any evidence that when nerve is stimulated there is also a release of calcium? The evidence that has been published is not very extensive and perhaps not very impressive, but what there is does indicate that when a nerve is stimulated, it gives off calcium ions. Roeder (1930) studied the effect of stimulation on the calcium content of frog nerves. He did two sets of experiments. In the more extensive series he took 120 sciatic nerves, each attached to its gastrocnemius muscle; 60 nerves were stimulated, the other 60 served as con-trols. The experimental nerves were stimulated for eight hours, then the nerves were cut into three thirds; one third, the stimu-lated part (farthest from the muscle), a middle third, and a third third close to the muscle. The control nerves were similarly cut into thirds. In the controls the calcium content of all three parts was essentially the same. But in the stimulated nerves the region of stimulation lost calcium, and the part of the nerve close to the muscle gained calcium. The differences were striking; indeed there was twice as much calcium in the part of the nerve near the muscle as in the stimulated region. Lânczos (1935; 1936) stimulated the vagus sympathetic trunk leading to the frog heart, and she was able to show that as a result of the stimulation, calcium was released into the perfusion fluid. (She tested for calcium by using the heart itself as a means of assay. That ad-naline was not involved was indicated by the fact that the sub-stance given off was not destroyed by ashing). Rex-Kiss and Lissâk (1940) injected tetanus toxin into one leg of a cat, and then after 6-8 days compared the calcium content of the sciatic nerve on the side exposed to the toxin with the calcium content of the control nerve on the other side. The results were wholly consistent in tests on eight animals; in the presence of tetanus toxin the calcium content of the sciatic nerve decreased 16.0-38.6 per cent. Rex-Kiss and Lissâk assume that the tetanus toxin acts on nerve and not on muscle. This seems to be in line with known fact (Harvey, 1939; Ambache and Lippold, 1949), although the toxin may act on nerve endings as well as on the nerve fibers.

Is calcium involved when a nerve is stimulated by an electric


current? One of the important facts about the electrical excita-tion of muscle is that it occurs at the cathode. As shown in the last chapter, this is easy to demonstrate in muscle, for the muscle contracts where the stimulating current leaves it. For nerve the demonstration of electrical excitation at the cathode is not quite so simple. In 1858 Pflüger (1859) ran an electric current through a frog nerve and found increased sensitivity to chemical stimulation in the region of the cathode. The next year Bezold also passed electric currents through frog nerves, and from a study of the time required for the muscle attached to the nerve to contract, he concluded that excitation occurred only at the cathode. (This is when the electrical circuit is made; when the circuit is broken, a polarization current runs in the opposite direc-tion and excitation occurs at the new cathode, that is to say, the old anode ). This early work of Pflüger and Bezold was confirmed bv many subsequent investigators (for the older literature, see Cremer, 1909; some of the newer literature is referred to by Katz, 1939). Various distinguished workers, following the lead of Nernst, have attempted to calculate mathematically the time relations of electrical excitation of nerve. Some of these calcula-tions and the mathematical equations involved are rather com-plicated and, except to the workers intimately associated with the field, rather confusing. They are considered at some length by Rashevsky ( 1948 ). According to Rashevsky, "all ionic theories admit that cations are responsible for the excitation." As men-tioned previously, none of the mathematical theories tells us very much as to what happens when a nerve is excited. Some cation goes somewhere and does something, but what? All that the mathematical theories postulate is that the unnamed cation has some unnamed effect on the protoplasmic colloid.

Inasmuch as the cation that is responsible for excitation and response in muscle is certainly calcium, it would be hard for any-one imbued with the methods of thought of general physiology to think that some other cation was involved in the excitation of nerve. And yet most physiologists are convinced that potas-sium is the excitatory ion for nerve, and that calcium, instead of being primarily responsible for the excitation of nerve, is the


ion most antagonistic to excitation. There are two reasons for thinking this, and at first sight, they both look good.

The first reason is that if there is an excess of potassium in the fluid surrounding a nerve, the excitability is increased, that is to say, the threshold drops. Actually in experiments of this sort in which excitability toward an electric current is tested, the situa-tion is complicated by the fact that one cannot be certain as to how the resistance of the nerve to the passage of the current changes as a result of immersion in one solution or another. But as a matter of fact, in general, potassium does increase the excit-ability and the response in various types of living systems. How-ever, the effect is not due to the entrance of potassium into the protoplasm, but to the release of calcium by potassium and the entrance of this released calcium. It is of the nature of most types of cells to be relatively impermeable to calcium ion. Thus, for example, although the protoplasm of sea urchin eggs is ex-tremely sensitive to calcium and undergoes a clotting reaction as soon as it comes into contact with appreciable amounts of this ion, sea urchin eggs can be kept in isotonic solutions of calcium chloride for seven or eight hours before enough of the calcium enters to produce any clotting effect.* On the other hand, if the eggs are placed in solutions which contain no calcium at all, such as a solution of sodium chloride or potassium chloride, and if the eggs are washed in such solutions several times to remove traces of calcium, then in a matter of a few minutes, the proto-plasm of the sea urchin eggs undergoes a clotting reaction such as is always caused by contact with calcium ion. What happens is that the sodium ion and the potassium ion are able to release calcium from its bound state in the cortex of the cell, and this free calcium then enters the cell interior. Much of the confusion about the action of various cations is due to this peculiar effect. Knowledge of it can explain much (for discussion, see Heilbrunn, 1952a, Chapter 33). As a matter of fact, for nerves, there is a consistent body of evidence to show that the increase in excita-

* The entrance of calcium into the e g g of the sea urchin Arbacia can readily

be detected b y virtue of the fact that the red pigment granules of this e g g break

d o w n in the presence of small amounts of calcium.


bility produced by potassium is transitory. The first increase in excitability is soon followed by a sharp decrease in excitability (Jahn, 1924; Blumenfeldt, 1925; Höber and Strohe, 1929; also references cited by Höber and Strohe). This is illustrated in Fig. 43 taken from Blumenfeldt's paper. One is inclined to believe that when nerves are immersed in solutions relatively

2.2

F I G . 4 3 . The effect of KCl ( 2 ml. isotonic

KCl + 8 ml. Ringer's fluid ) on the threshold of

stimulation of frog nerve. T h e minimal intensity

required to produce a response is the rheobase,

the amount of current required is the quantity

( B l u m e n f e l d t ) .

rich in potassium, the first effect (increase in excitability) is due to the passage of calcium from the cortex to the interior of the fiber, the subsequent decrease in excitability to the entrance of potassium ions into the interior.

The second reason for believing that it is potassium rather than calcium that is responsible for excitation in nerve is more cogent than the first. For many years it has been known that when nerves are immersed in solutions of oxalates or citrates, they become excited; moreover, strangely enough, this excitation produces a rhythmic series of impulses. Something of the sort was noted by Loeb in 1901, and it is also mentioned by Fessard (1936); more complete descriptions are given by Brink and Bronk (1937), Monnier and Coppée (1939), and Monnier (1947). A similar phenomenon occurs in muscle, either in the absence of calcium, or more strikingly in the presence of phos-


phates, oxalates, or citrates, salts which precipitate or inactivate the calcium ion. When muscle is exposed to solutions of such salts the whole muscle presents a shimmering appearance. This is due to the fact that the individual fibers contract rhythmically and individually, that is to say, they do not contract in unison, so that although there is a good deal of contraction on the part of the individual fibers, the muscle as a whole hardly shortens at all. This peculiar behavior was described first by Biedermann ( 1881 ) ; there have been many other descriptions by later authors. Presence of nerve endings is not necessary for the behavior, for it can take place in muscles in which the end plates have been paralyzed by curare. However, according to Ashkenaz (1938b), the presence of a number of fibers is a necessary condition; twitching never occurs in isolated muscle fibers. This leads to the suspicion that the effect may be an electrical one, for, accord-ing to the theory developed in Chapter 4, loss of calcium from the outer part of a cell could cause a drop in potential there. Actually it does have this effect in muscle ( Kuffler 1944 ). How-ever, this electrical interpretation of the effect of oxalates and citrates on muscle ( and nerve ) is almost certainly not the whole story, and fortunately another and a more interesting explanation is at hand.

What we are trying to understand is the reason for the strange fact that oxalates and citrates cause repetitive impulses to arise in nerve. In the search for an explanation we shall not attempt, as some authors, such as Monnier, have done, to explain the behavior of the nerve in terms of proteins and lipids. Rather, in accord with one of the major premises of this book, we shall attempt to interpret the behavior of nerve protoplasm in terms of the behavior of cells whose protoplasm is much easier to study than that of nerve.

As will be shown in detail in Chapter 10, and more in its proper relations, the protoplasm of the immature eggs of marine invertebrates is stimulated to begin the process of maturation by the release of calcium ion from the cortex of the immature egg. This released calcium activates a proteolytic enzyme system which is believed to be a primary factor in the initiation of the


maturation division. In the eggs of the worms Nereis and Chae-topterus, the first indication that stimulation (by released calci-um) has been successful in evoking a proper response, is the disappearance of the membrane that surrounds the large nucleus or germinal vesicle of the immature egg, that is to say, the break-down of the germinal vesicle. According to Heilbrunn and Wilbur ( 1937), if immature Nereis eggs are exposed to ultraviolet radiation, the germinal vesicle breaks down, but if the eggs be-fore exposure to irradiation are immersed for a few minutes in an isotonic (0.35 molar) solution of sodium citrate before irradia-tion, then germinal vesicle breakdown is inhibited. (The effect of the sodium citrate is reversible, for on return to sea water from the citrate solution, the eggs recover their sensitivity to the ultraviolet). In 1941 Wilbur was able to show that more dilute solutions of citrate were able to stimulate Nereis eggs and to cause a breakdown of the germinal vesicle in them. Preliminary immersion in stronger solutions could prevent this response. So, for example, in one series of six experiments, a solution composed of 15 parts of 0.35 molar potassium citrate and 85 parts of sea water caused germinal vesicle breakdown in 99 per cent of all the eggs which were so exposed. On the other hand, when in these six experiments, the eggs were first treated for 6 min. with 0.35 molar potassium citrate, and were then exposed to the more dilute solution of potassium citrate, less than 1 per cent of all the eggs showed germinal vesicle breakdown. (And, as in the previ-ous studies with ultraviolet radiation, the inhibiting effect of the stronger citrate solution is reversible). Thus one and the same substance, potassium citrate, can both act as a stimulating agent and as an inhibitor of stimulation.

Now let us apply this knowledge to the nerve. In the first place, we must remember that nerve fibers are rather imperme-able to calcium ion, so that when a nerve is immersed in oxalate or citrate solutions, presumably all the calcium does not leave the nerve protoplasm. What happens apparently is that the citrate or oxalate liberates calcium into the interior of the nerve fiber, just as one would expect a salt containing sodium or potassium to do. But at the same time the citrate or oxalate removes calci-


urn from the outer part of the cortex, and this might well have the effect of creating a calcium gradient from the interior of the cell to the outside, in other words, the effect of the calcium precipitating or inactivating ion is to suck calcium out of the center of the cell. Thus in one and the same substance we have something which can both favor excitation and prevent it. Such a substance would indeed almost be necessary to explain the repetitive nature of the nerve response. Indeed, instead of the action of citrates and oxalates being a potent argument against the type of theory we have been supporting, actually this action can perhaps best be interpreted in terms of our theory. For at the present time there is no other explanation as to how oxalates and citrates can produce the effect and countereffect necessary for a rhythmic series of responses.

Before leaving this subject it should be pointed out that there are some nerves which depend for their excitability on the presence of calcium ion in their environment. Presumably these are nerves whose protoplasm is relatively permeable to calcium ion. According to Van Harre veld (1936), if crayfish nerves are placed in solutions of isotonic sodium chloride, they rapidly lose their power to respond. If the irritability of these nerves is to be maintained, calcium ion must be added to the surrounding medium (potassium and magnesium ions are relatively unim-portant). Also nerve fibers of the autonomic system become less irritable or completely lose their power of response when the calcium ion content of the fluid bathing them is decreased by the injection of citrates or oxalates (Gley and Bouckaert, 1927a, b; Bouckaert, 1927; Houssay and Molinelli, 1928 ). This is true both for fibers of the parasympathetic system and the sympathetic system; the experiments were done on the nerves of frogs, dogs, and rabbits. According to Houssay and Molinelli, when calcium chloride solutions were injected so as to restore the calcium content of the blood, the nerves recovered their irritability.

After this lengthy diversion, necessary in order to show that in so far as the protoplasm is concerned calcium is not (as many believe) an inhibitor of excitation, let us return to the main thread of our argument. The stimulating agents which cause


excitation in nerve can be assumed to release calcium just as they do in other types of protoplasm, and what direct evidence there is for nerve supports this assumption. The fact that electric excitation occurs at the cathode indicates that some cation is involved. There is no direct evidence as to which of the four cations present in nerve is responsible for the excitation, but if there is any consistency in protoplasmic behavior in so far as excitation is concerned, that cation should be calcium. Now let us inquire briefly into the consequences of calcium release from the cortex.

The first effect would be the production of an action potential. This is on the basis of the theory of bioelectric potentials de-veloped in Chapter 4. And secondly, release of calcium from the outer region of the cell would result in an increased passage of cations both into and out of the cell. For it is the calcium ion that is primarily responsible for the semipermeability of the plasma membrane. ( In so far as the passage of ions is concerned, the cortex constitutes what is probably the most important part of the plasma membrane). Actually there is good evidence to show that excitation does cause an increase in the permeability of the membrane or membranes surrounding the nerve fiber. Thus Lullies (1930) and Cole and Curtis (1939) showed by elec-trical measurements that excitation is accompanied by a sharp drop in the resistance of the nerve fiber to the passage of ions. Isotope studies also show that following stimulation there is a marked increase in the permeability of the nerve fiber to ions (Keynes, 1951a, b ) .

It might be argued that this increase in permeability is the essential factor in the excitation of nerve and, as a matter of fact, this point of view has often been held. But when a nerve is stimulated by an electric current, excitation begins at the cathode and it is to the cathode that calcium, the permeability-decreas-ing cation, migrates. This would tend to stiffen the membranes at the cathode rather than to weaken them, and it would decrease their permeability. When an electric current is sent through a cell, the cell membrane does indeed weaken at the anode, pre-sumably from the loss of calcium there. At any rate this is true of


ameba protoplasm (Mast, 1931). (The increases in permeability observed by Cole and Curtis and Keynes were not increases that were measured at the stimulating cathode alone. )

Our trend of thought would lead us to the conclusion that in excitation it is not only the outer boundary of the nerve fiber that is affected, but the interior protoplasm as well. The strongest indication that this may be the case comes from studies on the effects of anesthetics and narcotics on nerve (see below). But is there any evidence that the interior protoplasm of nerve cells and nerve fibers is changed in any way by excitation, and can colloidal changes in the protoplasm be detected?

There is indeed some such evidence, although it is perhaps rather scanty and not overly impressive. The earliest evidence comes from Russia. There, students of ganglion cells in the autonomic system that innervates the heart found that stimula-tion of these cells caused changes that they interpreted as being coagulative (Federow, 1935; Smitten, 1946). Both of these authors used an interesting preparation described by Gramenizki (or Gramenitzky) in 1934. In this preparation the auricle of a frog heart is stretched so that the individual cells (in a living condition ) can be observed under high powers of the microscope. In the normal resting condition the protoplasm of the ganglion cells is apparently fluid; if the cells are pierced with a micro-needle, the protoplasm flows out. After injury or after electrical stimulation the protoplasm becomes much more opaque. Accord-ing to Smitten, "It was ascertained that the protoplasm of the living nerve cells represents a very liquid sol which is able under the influence of injurious factors to pass instantly into the state of a highly viscous gel." Stimulation with an electric current also produces gelation and these gelations are believed to be reversible.

In crustacean nerve fibers D. K. Hill and Keynes (1949) found that electric stimulation caused the fibers to undergo a sharp increase in opacity. However, in later papers D. K. Hill ( 1950a, b ) concluded that the opacity change might be due to minute changes in the volume of the fibers. Bryant and Tobias (1952) have also reported opacity changes as a result of stimulation.


Whether these differences in opacity are due to colloidal change is still uncertain, but it is perhaps worth mentioning that when muscles respond to stimulation, they tend to become opaque (see Chapter 7).

Definite evidence that stimulation can cause a gelation of nerve protoplasm is given by the studies of Flaig (1947). When the giant nerve fiber of the squid is cut, protoplasm flows out of the cut end. But according to Flaig, when the nerve is stimulated, the protoplasm is no longer able to flow. Flaig attributes this inability to flow to a sharp increase in protoplasmic viscosity. After stimulation has ceased the protoplasm is able to flow again. There is no definite proof that these colloidal changes which occur in nerve protoplasm as a result of excitation are definitely related to the nerve impulse. Certainly there does not appear to be a sudden change from sol to gel and back again with each impulse of a rapid succession of impulses. But neither do such changes occur in muscle when it responds to repetitive stimula-tion. For if muscle is subjected to electrical stimuli of high fre-quency, the protoplasm shortens and stays shortened.

Any satisfactory theory of the nerve impulse would have to account for the action of anesthetics and narcotics in preventing the excitation of nerve cells and nerve fibers. For if these agents can interfere with the mechanism of excitation, then an under-standing of that mechanism should be able to interpret the nature of the forces which prevent the mechanism from functioning. Indeed one of the best tools that the physiologist has at his com-mand is his ability both to initiate vital processes and to prevent them. If we know which agents start and stop a process, and if we can ascertain also why they are able to start and stop, then we should be well on the road to a proper understanding.

For many years the commonly held interpretation of excitation has been based on an outworn theory that has survived much too long, a theory which is not in accord with our modern factual knowledge of the cell. According to this theory, the nerve (or muscle ) fiber has around its periphery a double layer of electric charges, negative inside and positive outside. Thus this layer is said to be "polarized." Because of this polarization, according


to the theory, there is a difference in potential between the intact membrane and any part of the fiber membrane which has been injured so that its polarization is lost. This is the reason for the injury potential. Excitation is thought of as having a "depolariz-ing" effect, as evidenced by a drop in the injury potential.

Now if excitation causes depolarization, and if this is the pri-mary factor in excitation, then any agent which inhibits excita-tion should prevent depolarization. But this is quite the opposite of the truth, for fat solvent anesthetics instead of preventing depolarization, actually cause it (Alcock, 1906; Voelkel, 1921). These anesthetics quite definitely depolarize any region of the nerve thev come in contact with. And the effect is the same, no matter whether the anesthetic is in a concentration so dilute as to cause a heightened excitability, or whether it is in a concentra-tion sufficient to cause anesthesia.

In other words, the fat solvent anesthetics act on the outer protoplasmic membrane or membranes of the nerve fiber in the same way that stimulating agents do. The reason for this effect and its relation to the general theory of anesthesia will be discussed in a later section (see Chapter 13). If fat solvent anesthetics do not prevent the excitatory changes in the plasma membrane and cortex of the nerve fiber, where then do they act? Obviously if they do not act on the outer region of the fiber, they must act on the inner protoplasm. Presumably in nerve, fat solvent anesthetics prevent the coagulative changes in the interior protoplasm. Thus their action on nerve protoplasm would be the same as that on other types of protoplasm (see Chapter 13 ), and it is not necessary to assume one kind of action on nerve and a totally different sort of action on other types of living material.

Our knowledge of what happens when nerves are excited is still not nearly as clear as we would like it to be. This is partly due to the difficulties attendant on studying the protoplasm of most types of nerves. Partly also to the fact that most nerve physiologists have either been content with mathematical formu-lations that tell nothing about the intimate nature of the processes involved, or have been willing to confine their thinking to the re-


suits of exact measurements on a restricted range of phenomena. But although our knowledge of nerve excitation is still very

fragmentary, the broad outlines of a proper theory are now beginning to emerge. What evidence there is indicates that when nerve is aroused by any one of the various agents which can excite it, calcium is set free from the outer region of the nerve fiber. This release of calcium is an important factor in the origin of the action potential. The calcium entering the protoplasm in the interior of the fiber changes this protoplasm from a fluid to a more solid condition. Fat solvents could exert an anesthetic action by preventing this gelation.

9.

In living cells generally, excitation or response in one part of a cell is followed by a wave of excitation or response that travels to other regions of the cell. This, for purposes of definition in relation to the present discussion, we shall call conduction. In addition, in many types of cells, excitation or injury produces effects on adjacent cells or even on cells at some distance away. This we shall call transmission. Actually the distinction between conduction and transmission is perhaps not wholly justified. Both conduction and transmission are phenomena which occur in lower organisms as well as in higher animals, but because of their interest to the medical profession, most of our knowledge has come from studies on higher animals.

A wave of excitation may pass around, along, or across a cell, and this occurs not only in nerve and muscle, but also in other types of living cells. Thus in egg cells, entrance of a spermatozoon at a given point is usually followed by a wave of reaction that passes around the periphery of the cell and prevents the entrance of spermatozoa other than the one that started the reaction. Moreover, in the long cylindrical cells of Nitella and other similar algae, local excitation or injury can initiate a wave of response that can pass along the length of the cell.

In the conduction of an impulse along a nerve or in the con-duction of a wave of excitation along the fibers of a muscle, electrical phenomena play a leading role. As, progressively, each part of a nerve or muscle fiber is excited, the action current of this part stimulates the region immediately ahead of it in the path along which the wave of excitation is travelling. This point of view was for many years supported by a number of leading physiologists and has now been generally accepted. For one

148

CONDUCTION AND TRANSMISSION

9. CONDUCTION AND TRANSMISSION 149

thing, the action current of nerve or muscle is strong enough to act as a stimulating agent, a fact known originally to Galvani. From our point of view, there is no need to discuss the phenome-na of conduction due to action currents, for the information about this type of conduction is presented in some detail in most of the recent books on physiology. The theory of the action current was discussed in Chapter 4. Granted the fact that it exists, this in itself is sufficient to account for the wave of excitation. In medullated nerves, insulated as they are by their medullary sheath, the excitatory wave apparently jumps from one node of Ranvîer to another and what happens between the nodes is still somewhat uncertain. Perhaps along these internodal distances, conduction of another type may occur.

There is a strong probability that all types of conduction are not wholly dependent on electrical potential differences or elec-trical currents. When the surface or the cortex of a cell is affected in one way or another, substances can diffuse into the cell interior within a small fraction of a second. There might also be diffu-sion from one part of the interior to another. In so far as the usual types of conduction are concerned, this could only be important for relatively short distances. In long cells or cell processes, such as muscle fibers or nerve fibers, diffusion from one part of the cell to a distant part would take a very long time.

The passage of a wave of excitation as a result of a moving sequence of action currents can carry over from one cell to another, or even from one tissue to another. This, according to our rather arbitrary definition, would involve transmission. As long ago as 1882, Hering showed that an impulse travelling along one nerve could jump across to a second nerve laid alongside it. In relatively recent years there have been various repetitions of Herings experiment. Similarly Kühne (1888) showed that if two frog sartorious muscles were pressed tightly together, excita-tion of one muscle was communicated to the second. If.the two muscles were separated by a thin rubber membrane or if action currents were short circuited with gold leaf, there was no trans-mission of excitation. However, Kühne could never quite exclude the possibility of chemical influence of one muscle on the other,


for his efforts to lead off action currents from one muscle that would stimulate another were never quite satisfactory.

Let us now consider the process of transmission as it occurs normally from one neuron to another, or from a nerve to a muscle. In the early part of the century it was generally assumed that when a nerve excited a muscle, the only way that such excita-tion could be transmitted from nerve to muscle was by an electric current. This assumption was implicit in Ralph Lillie's thinking about excitation (see, for example, Lillie, 1923). And yet, as far back as 1874, du Bois Reymond, after naming the junction between nerve and muscle the "end plate," wrote as follows concerning the possible mechanism of transmission across this junction: "Von bekannten Naturprocessen, welche nun noch die Erregung vermittlen könnten, kommen, soviel ich sehe, in Frage nur zwei. Entweder müsste an der Grenze der contractilen Substanz eine reizende Secretion, in Gestalt etwa einer dünner Schicht von Ammoniak oder Milchsäure oder einem anderen, den Muskel heftig erregenden Stoffe stattfinden. Oder die Wirkung müsste elektrisch sein."

In 1921 Otto Loewi published his important discovery that the vagus nerve acts on the muscle of the frog heart by liberating an inhibitor substance, which he later showed to be acetylcholine. (Loewi has described his work on acetylcholine in two lectures published in 1945). Following this discovery of Loewfs, there has been an enormous amount of work on acetylcholine, on the enzyme or enzymes which synthesize it, and the enzyme or en-zymes which destroy it. Indeed acetylcholine has become one of the magic words of modern physiology. It is believed to be produced not only by the parasympathetic fibers of the vagus, but by all other parasympathetic nerve fibers as well. Moreover, motor nerves produce acetylcholine at their endings in the mus-cles, that is to say, at the end plates.

Nor is acetylcholine the only substance produced at the end-ings of nerves, or at synapses. Nerve fibers of the sympathetic system produce either adrenaline, or a related substance, nor-adrenaline, or both. In this book we could scarcely attempt to survey the vast body of literature that is concerned with the


acetylcholine and adrenaline produced by nerves at their contact with other neurons or with muscles.

Following the discovery of the fact that acetylcholine was released at nerve endings, there were vigorous and violent dis-cussions as to whether the transmission across a synapse or an end plate was electrical or whether it was chemical. For the most part the argument seems to have been decided in favor of those who urged the chemical theory. There are various rea-sons for this. Back in 1874 du Bois Reymond questioned whether the action potential of a nerve or the end plate potential would be sufficient to cause an excitation of muscle fibers. Kuffler, the leading student of end plate potentials, has indeed pointed out that the many branches of the nerve fiber at the end plate would reduce the potency of the electric current to the point where it could not produce an effect on the muscle (see Kuffler, 1948, 1952). Moreover, no electrical theory has been able to explain the paralyzing effect that curare has on end plates, and there are better possibilities in the way of a chemical explanation.

Thus, in so far as our present knowledge goes, it is correct to assume that, as originally stated by Dale, Feldberg, and Vogt (1936), it is the release of acetylcholine at the endings of motor nerves that is the primary factor in the transmission of excitation from nerve to muscle. (For a review of more recent literature, see Riker, 1953. ) But the mere mention of the magic substance acetylcholine is scarcely a solution of the whole problem. We can of course maintain that acetylcholine initiates a potential dif-ference and that this then stimulates the muscle to contract. But how then explain the very queer fact that acetylcholine can make a skeletal muscle contract and that the very same substance can prevent contraction of heart muscle and keep it in a relaxed state (Hunt, 1915; Burgen and Terroux, 1953), and can also cause relaxation of the smooth muscle in the walls of blood vessels (Smith, Miller, and Graber,)?* Some attempts at explaining the action of acetylcholine on muscle have centered around the idea

* Various other references might be cited. According to Cullis and Lucas

( 1 9 3 6 ) , acetylchol ine acts on the heart of the embryo chick before the heart

has any nervous connections.


that acetylcholine initiates an action potential. This is possible enough, but if it is so, we must then explain why the action potential causes contraction in one type of muscle and relaxa-tion in another; or why, moreover, small doses of the substance can increase the excitability of the vagus and larger doses act in quite the opposite way ( Barone, 1932 ).

Strange as it may seem, what is probably the correct explana-tion is simple enough. Acetylcholine acts by releasing calcium from the cortex (or outer region) of the cell; it is thus a sub-stance which tends to free calcium, and by the same token tends to prevent protoplasmic constituents from combining with calci-um. In cells with relatively impermeable membranes, cells such as striated muscle fibers, the action of acetylcholine is confined to the surface and cortex. The calcium released as a result of the action of acetylcholine enters the interior of the muscle fiber and causes the protoplasm to shorten (see Chapter 7). On the other hand, the muscle fibers of the heart and the smooth muscle fibers lining the blood vessels are protoplasmic systems that are relatively permeable, and when they are exposed to acetylcholine, this substance rapidly enters the interior protoplasm. There again it prevents the calcium from uniting with the protoplasmic con-stituents that tend to combine with it; and because of this, the contraction of the muscle, dependent as it is on calcium ion, is prevented.

Actually there is some evidence to show that calcium is related to the action of acetylcholine and much more evidence to show that calcium is essential for the transmission of excitation from nerve to muscle. Fatt (1950) showed that acetylcholine caused "depolarization" at the end plate; and from the discussion in the last chapter, it seems clear that this could be due to the release of calcium from the outer region of the cell (or the end plate). Punt ( 1942 ) found that in the absence of calcium, acetylcholine was no longer able to cause contracture of the rectus abdominis muscle of the frog, and Koshtojanz (1944) showed that if this muscle was previously treated with oxalate solutions, it failed to respond to acetylcholine. Also in rabbit hearts previously treated with atropine, McNamara, Krop, and McKay ( 1948 ) showed that


calcium potentiates the stimulating effect that acetylcholine has on such a preparation.

That calcium is necessary for the transmission of excitation from nerve to muscle is one of the best known facts in physiology. In 1894 Locke immersed the sartorius muscle of a frog with its attached nerve in a solution of 0.6 per cent sodium chloride. Within 15 or 20 min., the muscle would no longer respond when its nerve was stimulated by single shocks from an induction coil, although it would still respond to repetitive shocks. After 1-2 hr., even repetitive shocks given to the nerve failed to elicit a response of the muscle. If then a trace of calcium chloride was added to the solution, the muscle responded again to stimulation of its nerve. The lack of calcium acts on the end plate, for it has no effect either on nerve or muscle. Since Locke's original experiment there have been many confirmations of his results. In the course of another investigation Heilbrunn and Ashkenaz (1941) showed that when the indirect excitability of a muscle was lost by immersion of a nerve-muscle preparation in sugar solutions, this excitability could be restored by the addition of calcium ion. Heilbrunn and Ashkenaz cited twelve papers in which Locke's work was confirmed, and there have been at least three other papers that also confirm Locke (Cowan, 1940); Coppée, 1946; Cicardo and Beninson, 1952). Thus there can be no doubt but that calcium plays an important role in the trans-mission of an impulse from a motor nerve to a skeletal muscle.

These facts all lend support to the view that acetylcholine can make a muscle fiber contract by releasing calcium from its pe-riphery and can also prevent the contraction of a muscle fiber by preventing calcium from combining with the protoplasmic constituents in the interior.

But what about adrenaline? The nerve fibers of the sympa-thetic system, where they come into contact with the smooth muscle fibers they innervate, release adrenaline. How does this act; does it also liberate calcium? When adrenaline is injected in-to an animal, according to some authors, the calcium of the blood goes up; according to other authors, it goes down; still others claim that it remains constant. But about one point there is


agreement. In 1928 Lawaczek found that adrenaline caused an increase in the ultrafiltrable calcium in the blood serum; it thus is able to liberate calcium from combination with protein. A similar result was reported by Hermann ( 1932 ) and also by Rosso (1934). So that, presumably, adrenaline can act like ace-tylcholine in releasing calcium from the cortex of the muscle fiber and that is why adrenaline can cause a muscle fiber to contract. Certainly more knowledge concerning this action of adrenaline is needed, but the knowledge that we now have is consistent with the point of view that we have been maintaining.

It would be wrong to suppose that the only protoplasm capable of releasing substances important in influencing the protoplasm of other cells and tissues is the protoplasm of nerve fibers. Indeed it seems probable that all cells can deliver either to their im-mediate environment, or to the blood stream, potent substances of one sort or another. For if the colloidal reactions of protoplasm are like the colloidal reactions of blood, as we have taken such pains to show (in Chapter 6), then the clotting of protoplasm in a cell or a group of cells should produce thrombin-like sub-stances capable of exerting a coagulative effect on the protoplasm of other cells. This leads to another aspect of the broad subject of transmission, an aspect not ordinarily included in discussions of the subject.

Injuries to one part of an animal can have effects on cells and tissues throughout the body. Because of its clinical importance, this phenomenon is best known in man. In primitive times, when man lived even more dangerously than he does now, he no doubt frequently suffered severe injuries of one sort or another. Under these conditions he might well collapse. The nature and causes of such collapse have no doubt been studied from the very begin-ning of medical science (for a historical discussion, see Wiggers, 1950). But in spite of its importance the subject is still largely veiled in mystery.

The term "shock" has been used, and is still used, to cover a wide variety of pathological conditions, inspired by many dif-ferent types of injury. This meaning has to some extent survived, and has to some extent been superseded. In 1908 Meitzer defined


shock as "a state of general apathy, reduced sensibility, extreme motor weakness, great pallor, very rapid small pulse, thready soft arteries, irregular gasping respiration and subnormal temper-ature." And in his book on traumatic shock, published in 1945, Piulachs defined shock as a sudden and intense drop in all the vital functions, in irritability, motility, mental function, respira-tion, circulation, and heat production. But because in man any failure of the blood to flow properly has an almost immediate effect on the brain, and because also loss of blood, as in severe hemorrhage, can cause typical shock symptoms, there has been an increasing tendency to define shock as a circulatory failure. So, for example, in Sodeman's "Pathological Physiology," pub-lished in 1950, shock is considered a disease of the circulatory system, and it is also so considered in various standard texts on medicine (for example, Cecil and Loeb, 1951). Indeed some medical dictionaries define shock as "a condition of acute peripheral circulatory failure." As a criterion of traumatic shock, physicians emphasize the drop in blood pressure and also the decrease in blood volume. Both of these changes are easy tc measure and they therefore have a practical appeal.

From a practical standpoint, it is perhaps well for the physician and the medical scientist to concentrate on the circulatory aspects of shock, but there is an unfortunate tendency to confuse cause and effect and an apparent willingness on the part of some workers to begin and end their physiological investigations with studies on the blood and the circulation. But the cells and the protoplasm in them are primarily responsible for the life and the death of an organism. And although anyone who has ever studied animals in shock is aware of the fact that the circula-tion may be greatly involved, it must also be recognized that shock can occur without any very striking alterations either in blood pressure or in blood volume (see, for example, Meitzer, 1908).

And certainly, from the standpoint of the general physiologist, shock, in many cases at least, is not due to a circulatory disturb-ance. This will become very clear as our discussion proceeds. Unlike the clinician, the general physiologist is interested in


studying a phenomenon in its simplest terms, in as uncomplicated a form as possible. From his standpoint a frog is in many ways a more interesting animal in which to study shock than is a mammal. In a frog impairment of the circulation does not have the immediate and drastic effects that such impairment has on a mammal. Cut the heart out of a frog, and the muscles and nerves of the animal, in spite of the absence of circulating blood, will continue to function for a considerable period of time. If a frog's head is cut off, in the absence of its brain it may continue to live for weeks. On the other hand, if a frog is given a sharp blow on the head, it will lie immobile on its back, incapable of turning over; in this condition the frog is insensitive and responds only to very strong stimuli. It may never recover, although its heart may continue to beat long after the reflexes have ceased to function in normal fashion. Obviously some substance has gone out from the injured head or brain to the rest of the animal. If the legs of frogs are scalded, the animals die. However, if after scalding the legs are tightly ligatured at the upper ends of the thighs, death occurs only much later, after gangrene has set in (Debie, Lafontaine, and Willot, 1946). Removal of the ligature results in death of the frog. This would seem to mean that a toxic substance is emerging from the injured tissues of the burned legs, but it might also be argued, that after removal of the ligature, blood escapes from the injured vessels of the legs. However, Debie, Lafontaine, and Willot found that if they removed the ligature for as much as 9 hr., and then replaced it, the frogs lived as long as if the ligature had never been removed at all. In 9 hr., any fluid loss from the damaged blood vessels should have been completed. Thus these experiments indicate that burn shock in frogs is not due to a loss of blood. Also in mammals, loss of blood is not the sole cause of shock. This is shown clearly by the work of Haist and Hamilton ( 1944 ). They clamped the legs of rats for 12-15 hr., preventing circula-tion through them. When the clamps were removed, all the rats died from shock; their average survival time was about 3 hr. On removal of the clamps, the legs swelled up; this swelling process was complete in 2 hr. If at the end of this 2 hr. period,


the clamps were replaced, most of the rats survived. Here again if death were due to fluid loss, this loss should have been com-plete in the 2 hr. that the clamps were released.

It is thus clear that the fluid-loss theory of shock, a theory that has had wide favor in the medical profession, is certainly not a completely adequate theory. In frogs and rats, and pre-sumably in other animals as well, injured cells give off substances which have marked effects on various other cells of the organism. The existence of such substances has been known for a long time, and there has been speculation concerning them for at least a hundred years. In the literature, the substances given off by injured tissues and believed to be responsible for shock are referred to as "toxic factor." Almost all of the studies on toxic factor have been made on mammals, but as has been indicated, there is just as clear evidence for the existence of toxic factor in frogs as there is for its existence in higher animals. And indeed the production of potent toxic substances by injured cells is no doubt a phenomenon of broad and general significance. Thus in crabs, any injury of an appendage results in a dropping off of the appendage. This is called autotomy, and there is a rather extensive literature on the subject. (A useful paper is that of Wood and Wood, 1932). Autotomy may be due to the formation of injury substances by the injured appendage. This is suggested by the work of Hopkins (1948). He injected ex-tracts of injured frog muscles into the legs of crabs and in this way he was able to produce autotomy. In worms, if one part of the animal is scalded, the whole animal dies, and death seems to be due to the production of toxic factor by the injured region. This follows from the work of Chaet, who thus far has published only a preliminary report of his experiments ( 1951 ). Much more work should be done on the substances produced by injury in various types of lower organisms. Even in animals without circu-lation, injury of one part of the animal can affect other parts of the organism, and in plants also injury substances are important. The injury substances both of plants and animals can stimulate cells to divide. Some of the information in this field will be discussed in the chapter on cell division.


Many excellent books have been written about shock, and the authors of these volumes have struggled valiantly to make sense out of an enormous, controversial, and often confusing literature. Relatively recent books include those of Moon (1942), Davis ( 1949 ), and Wiggers ( 1950 ). In his book on the "Treatment of Burns," Harkins (1942) presents an interesting and scholarly survey of the literature on burn shock. In this chapter, no attempt will be made to review, even in the briefest possible fashion, the vast amount of work and of thought that has been expended on the shock problem. But because shock is a broad biological phenomenon, or at least is related to phenomena that occur not only in mammals, but in many relatively low types of organisms as well, we shall attempt an interpretation in terms of all living material. In other words we shall adhere to the standpoint of the general physiologist rather than to that of the clinician.

In attempting to interpret a phenomenon, a favorite method of the general physiologist, a method he shares with scientific workers in all fields, is to inquire as to the nature of all the agents which can make the phenomenon appear. If all these agents can be shown to have some one effect in common, then perhaps we can arrive at a conclusion as to the basic cause. One difficulty in such an approach to the shock problem is the fact that in higher animals one and the same agent sometimes produces shock and sometimes is entirely without effect. Thus, according to Wiggers (1950), one investigator of shock was led to conclude that the only way to obtain a standard procedure for obtaining shock in dogs was to create standard dogs. And if there is varia-tion in the various individuals of a given species, the variation becomes much greater when different species are studied. Thus, although the guinea pig is very sensitive to histamine shock, the rat is relatively insensitive, and the frog scarcely seems to be affected by histamine at all. To the biologist or the general physiologist, this would mean that histamine is not the all-im-portant substance it was once thought to be; indeed its effects are very likely due to secondary reactions.

The following list of agents or conditions which induce shock is not a complete list, but rather it is an attempt to show the


wide variety of agents which can induce shock or a shock-like state:

1. A crushing injury can cause shock and this is commonly known as traumatic shock. If the muscles of a dog are crushed, the animal goes into shock. A blow on the head can cause shock in a man or a frog. This type of effect is sometimes called "head injury.'' The head region is more sensitive to traumatic shock than is any other part of the body.

2. Strong electrical currents can cause shock. In this type of shock also, the head region, that is to say, the brain, is more sensitive than other parts of the body. The reason for this greater sensitivity of the brain tissue will become apparent later.

3. Burns can cause shock. The effect can be produced by short exposures to temperatures far above those the animal is accustomed to, or by longer exposures to more moderate temper-atures. Sun stroke and heat prostration can in most cases be considered as a form of shock.

4. Exposure to excessive roentgen radiation causes X-ray sick-ness, which can be considered as a form of shock.

5. Surgical operations can cause shock. This may be a form of traumatic shock. Also manipulation of the intestines may cause shock.

6. If the legs of a rabbit, a rat, or a frog are bound with ligatures so that the circulation is cut off, when the ligatures are released, the animals go into shock. This effect may be due to tissue injury resulting from anoxia in the parts of the animal deprived of circulation.

7. Excessive hemorrhage can cause shock. This may in part be due to the anoxia resulting from decreased blood flow, but anoxia is apparently not the only factor ( see below ).

In all the types of shock considered so far, the tissues of the shocked animals can be considered to have been injured in one way or another, so that it can be postulated that an injury sub-stance is responsible.

8. Anaphylaxis. This is a process that has been widely studied by immunologists. If a foreign protein or other antigen is injected


into an animal, the animal becomes sensitized, so that some time later a second dose of the antigen can cause death. When an anaphylactic reaction occurs, the animal shows many of the symptoms of shock. Anaphylactic shock has been studied in mammals, birds, and frogs. Probably in anaphylaxis, the organ-ism develops an enzyme capable of acting on the foreign protein, so that anaphylactic shock may be similar to the shock produced by proteolytic enzymes.

From a theoretical point of view, the more interesting types of shock are those caused by the injection of various substances, for if we knew all the substances which could induce shock, we might be able to deduce the nature of their action. In the follow-ing enumeration of substances which can induce shock, no attempt will be made to present an absolutely complete list.

9. Histamine. For a time it was commonly believed that histamine was the substance primarily responsible for various types of shock, including anaphylactic shock, and including also allergic reactions, which can perhaps be regarded as a mild form of shock. The guinea pig is very sensitive to histamine, but the rat responds only to rather massive doses, and the frog, as stated previously, is almost completely insensitive.

10. Adrenaline. Like histamine, this is a basic substance. According to Bainbridge and Trevan (1917), adrenaline can cause shock, and this was confirmed by Erlanger and Gasser (1919). If adrenaline can cause shock, this is perhaps an ex-planation of the fact that violent nervous stimulation, stress, or strong emotion can cause shock, for under these conditions adre-naline enters the blood stream from the medulla of the adrenal gland. Adrenaline shock may in part be due to déficiences in circulation caused by the constrictive effect of adrenaline on the blood vessels, but this presumably is not the complete explana-tion.

11. Protamine. A third type of basic substance which can cause shock is protamine. At any rate the injection of protamines can cause a sharp drop in blood pressure and other effects similar to those which occur in shocked animals (Thompson, 1900; Allen and Egner, 1948).


12. Insulin. That an excess of insulin in the blood stream can cause shock is well known. The effect is associated with a drop in the sugar content of the blood, and ingestion of sugar soon relieves the shock symptoms. The effect is complicated. For it is hard to believe that lack of sugar can in itself produce a state of shock.

13. Thrombin and thromboplastin. According to Freund (1920), if freshly clotted defibrinated rabbit blood is injected into a rabbit, it goes into a state of severe shock—often it dies. Injection of thrombin also causes shock (see Nolf and Adant, 1954; for literature references, Tagnon, 1945). Tissue extracts containing thromboplastin likewise cause shock. This subject is discussed at some length by Moon (1942); see also Mills (1921), Mylon, et al (1942).

14. Proteolytic enzymes. In view of the fact that thrombin is a proteolytic enzyme and that proteolytic enzymes clot blood (and protoplasm), it is not surprising that proteolytic enzymes can produce shock. Trypsin, chymotrypsin, papain, all can cause shock. So too can the proteolytic enzymes of snake venom. Kal-lfcrein (or callicrein) is a proteolytic enzyme which is very potent in producing shock (for literature references, see Heil-brunn, 1952b).

15. Peptone. For seventy-five years it has been known that the injection of small amounts of peptone in solution caused shock symptoms. For references to some of the literature, see Nolf and Adant, 1954.

On the basis of our knowledge of cells and protoplasm, can any general theory be proposed which would account for the fact that when living tissues are injured, they produce substances which depress the activity of other tissues? If it is true that both stimulation and injury produce in protoplasm a clotting reaction similar to that which occurs in blood, then it is logical to suppose that just as the clotting of blood produces a clotting (and pro-teolytic) enzyme, so in protoplasmic clotting an enzyme is formed which induces clotting in the protoplasm of cells not previously injured. In this way the effects of injury of a given tissue could be transmitted to other tissues by way of the circulât-


ing blood. Let us see how this type of explanation can fit the known facts.

In the first seven cases of shock enumerated above, there is in every instance cellular injury. The effect may be many-sided. Thus, when the circulation is cut off by tourniquets, greater effects are produced if the tourniquets are applied tightly enough to cause tissue injury as well as stoppage of circulation. Stoppage of circulation can cause an increase in the thromboplastin content of muscle (Stoner and Green, 1947). This may be the reason why when the amount of blood is greatly decreased as a result of severe hemorrhage, the blood clots more rapidly. Actually there is such an increase in the coagulability of the blood follow-ing hemorrhage ( Cannon, 1939 ).

If the point of view that we have outlined is correct, then it is easy to understand why thrombin and thromboplastin can cause shock. And in view of the fact that proteolytic enzymes, in general, act like thrombin both on blood and on protoplasm, we can also understand their action in producing shock. If in the protoplasm, there is an equilibrium between clotting factors and anticlotting factors (see Chapter 6), then any neutralization or inhibition of anticlotting agents would favor the activity of the proteolytic clotting agents. This being the case, there is a ready explanation of why basic protamine can cause shock, for even as a salt (chloride or sulfate) it can neutralize heparin and make it inactive. Perhaps histamine and adrenaline can act in somewhat the same way. These basic substances could enter cells from their combination with acid, just as ammonium hy-droxide enters cells from solutions of ammonium chloride (com-pare Jacobs, 1922). However, the action of adrenaline may also be due to the fact that indirectly or directly it may activate clotting enzymes.

The action of peptone can also be explained if we assume that as soon as it enters the blood stream it causes an increase in proteolytic activity. This assumption is based on the fact that the tissues of animals can produce adaptive enzymes just as yeast cells or bacteria do. This assumption is backed up by observations that indicate that there is an increase in the pro-


teolytic activity of liver tissue following the injection of peptone (Weimar, 1955). In some experiments of Beraldo (1950), there is also an indication that peptone can cause the activation of proteolytic substances in dog blood. It is also logical to suppose that the anaphylactic reaction is due to the appearance of pro-teolytic enzymes in the blood.

Why insulin acts as it does is still a mystery, but perhaps if more attention were given to the effects insulin might have on the colloidal condition of protoplasm, we might get some clue as to the nature of its action on cells. The fact that insulin promotes glycogenesis is perhaps important, for glycogenesis might tend to prevent the sugars that enter the cell from being transformed into heparin. In this way the proteolytic activity of the protoplasm might be enhanced.

In all attempts at an interpretation of shock phenomena, we must never forget the importance of homeostatic reactions. When thrombin or thromboplastin is injected into the blood stream of an animal, the blood although first more readily coagulable, soon coagulates more and more slowly, so that in deep shock the blood is commonly incapable of coagulation. This is, to some extent at least, due to the increase in the concentration of heparin and heparin-like substances in the blood stream (Jaques and Waters, 1941; Allen and Jacobson, 1947). In hibernating animals, in which all vital functions are depressed just as they are in shock, the blood clots only very slowly, and this is apparently due to the presence of heparin or heparin-like substances ( Suomalainen and Lehto, 1952). When an animal is thrown into shock, the first effect may be what one would expect from the entrance of thrombin or thromboplastin into the blood; that is to say, the blood would clot more rapidly; then as a result of a homeostatic reaction, heparin may be set free. Actually there is some evidence that this sequence does really occur. Thus in birds, Gahringer (1926) showed that anaphylactic shock first caused the blood to become more rapidly coagulable, after which it slowly became less and less coagulable. The same sequence also occurs when shock is induced by tourniquets (Mylon et al., 1942), and also in shock produced following electrical injury (Moreau, Balis-


tocky, and Heilbrunn, 1948 ). In the chapter on Cellular Homeo-stasis, Chapter 14) it will be shown that a proteolytic (clotting) enzyme could very well induce the release of heparin.

In many types of shock, severe death or injury is accompanied by a loss of blood from the blood vessels. Autopsies show hemor-rhages in various parts of the body. This can easily be demon-strated in frogs dying of shock. In such animals droplets of blood emerge from various organs and tissues and form petechiae, blood may flow from the mouth, or the stomach may be distended with blood that has escaped from its vessels. Such escape of blood is doubtless due to the leaky condition of the capillaries and small blood vessels. Ordinarily capillaries are able to seal themselves in the same way that a cell does, somewhat after the fashion of a self-sealing inner tube of an automobile tire. This sealing reaction may well be due at least in part, to the clotting of blood just as soon as it emerges. But perhaps in the presence of heparin, this clotting does not occur. This would account for the leakiness of the capillaries in shocked animals. However heparin may also act in other ways. With an ingenious method Rieser ( 1955 ) has shown by direct measurements that in shocked animals, and also in animals into which heparin has been injected, the breaking strength of the capillaries is markedly decreased.

It is obvious that much more needs to be known about what happens to cells when they are injured, and we need to know more also about the nature of the substances produced as a result of the injury and how these substances act on other cells. Cer-tainly a rational theory of shock and allied phenomena can only be arrived at when we consider what happens to cells and proto-plasm as well as what takes place in the blood of shocked animals. The theory outlined above gives a basis for future work. Eventu-ally it should be possible to interpret shock in terms of the known properties and the known behavior of protoplasmic colloids.

In this chapter no attempt has been made to present a com-plete and exhaustive discussion of our present-day knowledge concerning conduction and transmission. Rather, as in other sec-tions of the book, our effort has been concentrated on the en-deavor to show in so far as possible how knowledge of the col-


loidal reactions of protoplasm can throw light on problems that have for many years puzzled the physiologist and the pathologist. Perhaps in the not too distant future we will know much more than we now do about the mode of action of adrenaline and of the various other substances that play a role in shock. All these substances produce their effect by acting on protoplasm and any reasonably complete theory must consider the changes that occur in the protoplasm that produces the humoral substances as well as the changes that occur in the protoplasm on which they act.

10.

A living cell can divide to form two cells. The process is not a simple splitting of a droplet of protoplasm into two smaller droplets. Indeed the division of a cell is an extremely intricate process, a process in which there is an elaborate mechanism to insure an equal distribution of the nuclear material to both daughter cells.

Since the process of cell division first came to be recognized some eighty years ago, there has been a vast amount of study devoted to it. And in recent years, as the interest in the solu-tion of the cancer problem has increased, this study has become more intense.

Cancer is uncontrolled cell division. Ordinarily in higher animals there is little or no cell division in most of the organs and tissues of the body. Search the brain, the muscles, or the pancreas of a normal adult animal and one can examine section after section without seeing a single cell in division. But should a tumor develop in any of these organs, there would be an abun-dance of dividing cells.

At the present time enormous sums of money are being spent in the hope of finding some solution to the cancer problem. But in spite of glowing reports in the newspapers, the tragic truth is that progress has been slow. Beyond any doubt the billions of dollars spent in the hope of finding a cure for cancer have not on the whole been wisely spent. The fault to some extent lies with the men who have had charge of the disposition of the funds available for cancer study. Often enough they have given money to their friends or to each other; rarely have they taken true cognizance of what constitutes real scientific achievement; too often they have been more impressed with glowing (and

166

CELL DIVISION

10. CELL DIVISION 167

often extremely inaccurate) reports in newspapers than with careful, sincere, scientific publication; almost never have they been willing to offend or refuse to support those who often by devious means have become powerful factors in the petty politics of the scientific world.

The earlier studies of cell division were concerned mainly with the form changes that occur during the division process. The various parts of the mitotic apparatus: spindle, asters, centro-somes, and chromosomes, were carefully described, mostly on the basis of what could be seen in fixed and stained preparations. On the basis of these visual observations, elaborate theories were proposed; rarely were these tested, indeed, most of them were incapable of test. In his book "Mitosis" which, as its subtitle indicates, is primarily concerned with the movement of chromo-somes in cell division, Schräder (1953) has reviewed facts and theories in this field. But in his concluding statement, Schräder admits that "The present survey of past attempts to solve the puzzle that is mitosis may well seem disheartening. Not one of the many theories that have been broached has in it the definite promise of a final solution." For additional information on some aspects of cell division, Hughes' book, "The Mitotic Cycle" may be consulted. It was published in 1952.

Some things are known: the appearance of the spindle in animals and plant cells, the fact that fibers reaching from pole to pole of the spindle may (rarely) be seen in living preparations, although ordinarily they are not visible, the fact that at the point of chromosomal attachment to spindle fibers there is a small granule, variously called kinetochore, centromere, or any one of 25 other names. (Schräder regards this structure as especially important ). We know that centrosomes and astral rays can often enough be seen in living cells. Also that the movement of chromo-somes in the anaphase is slow, of the order of magnitude of 1 μι/min.; sometimes as fast as 3 μ/min.

In recent years cytochemical techniques have provided addi-tional information. The chromosomes are now definitely known to be largely deoxypentosenucleoprotein; the mitotic spindle can be isolated and its protein composition examined (Mazia and


Dan, 1952). But although the cytologists have been much im-pressed with this cytochemical information, it has until now been of little help in the interpretation of mechanism. Indeed it is in a certain sense merely an expansion of our static morphological knowledge, and the fact that a particular part of a cell is com-posed of one chemical or another does not help us very much in explaining the dynamic forces that come into play when a cell divides. Eventually, perhaps when we know more about the chemical changes that occur in division, such information may be more helpful.

The division of a cell is a physiological process, and in order to interpret it properly, we must think in physiological terms. In the first place, from the standpoint of a general physiologist or a cell physiologist, it is important to study favorable types of material. Much time can be wasted by attempting to do experi-ments on material that is not suited for experimentation. Thus, for example, there are disadvantages in studying the physiology of cell division in tissues in which the various dividing cells are out of phase, that is to say, tissues in which the dividing cells are in various stages of the division process. For many kinds of work (though not all), it is a great advantage to have all the cells dividing synchronously. That is why marine eggs are so valuable for the physiological study of mitosis. When sea urchin eggs or eggs of the worm Chaetopterus are fertilized, they can be depended on to go through the various stages of cell division almost simultaneously, so that the time that elapses between the cleavage of the fastest egg to divide and the slowest may be only a matter of two or three minutes.

Secondly, it should be emphasized that the division of a cell can be considered as a response to a stimulus, just as the contrac-tion of a muscle is. Indeed the response to stimulation in an egg cell is very similar to the response of other types of living cells. This is clearly shown by the fact that the same anesthetics or narcotics that prevent response in muscle and nerve also prevent cell division. (For a discussion of anesthesia or narcosis, see Chapters 12 and 13.) The essential point is that the theory of what makes cells divide should not be very different from the theory of what makes muscles contract or nerves respond.


From the standpoint of a physiologist, one of the most essen-tial problems of cell division is the problem of what makes a cell divide and what prevents it from dividing, and this is a problem that beyond all others has a significance for the cancer problem. But of course this is not all there is to the physiology of cell division. Once a cell has begun to divide, we need to know as much as we can about the forces involved in various stages of the division process. In the solution of these problems, anything that we can learn about the form changes, the chemical changes, or the physical changes that occur in various parts of the dividing cell is important. But for the limited purposes of this book, we shall emphasize most the problem of what makes a cell divide and what stops a cell from dividing, for the solution of this problem is related to other aspects of our subject, some of which have already been discussed and some of which will be discussed in subsequent chapters.

But before beginning our physiological discussion, it will be necessary to mention a few facts about the morphological changes that occur during the process of cell division. Most of what we know has been learned from a study of fixed and sec-tioned material, but there is much that can also be learned from observation of living cells. By studying sections, the early cy-tologists were able to describe in some detail the sequence of stages that followed one another in the course of the division process. The facts are to be found not only in books on cytology, but also in many elementary textbooks, so that only brief com-ment is necessary here. Commonly the nucleolus disappears, then the nuclear membrane; the mitotic spindle becomes visible, and the chromosomes line up in an equatorial plane along it. Definitions of stages vary, but we can call this stage in which the chromosomes are in the equatorial plane of the spindle, the metaphase; the stages that immediately preceded it constitute the prophase. The chromosomes split either in the metaphase or more typically in the prophase. For each of the original chromosomes, there are now two daughter chromosomes. During the anaphase these migrate toward the poles of the spindle. Then during the telophase the chromosomes tend to vesiculate,


or at any rate to lose their sharp contours; at about this time also, in animal cells, there is a constriction at the outer boundary of the cell, and a division plane extends across the cell, bisecting the spindle at right angles. The cytologists pieced together these stages from studies of fixed material in which there were often all stages present. To be certain of exact time sequences, it is a simple matter to prepare fixed and sectioned material of divid-ing marine eggs, fertilized at a given time, allowed to develop at a given constant temperature, and then fixed at every minute during the period between fertilization and cleavage. Such a study was made by Heilbrunn and Wilson (1948) for the Chaetopterus egg, and their paper can be consulted for details. This study was made in order to interpret properly the time sequence of protoplasmic viscosity changes in relation to the various stages of mitosis. A byproduct of the study was the observation (not strongly emphasized) that in late anaphase the cytoplasmic granules appear to push their way into the equatorial plane of the spindle. This confirms an older observa-tion of Conklin (1902) on Crepidula eggs. He found that in late anaphase stages, yolk granules of the cytoplasm are to be found "in a plane running right through the middle of the spindle." Two of Conklin's drawings are shown in Fig. 44. There is thus evidence that in late anaphase, not long before the cell divides, the spindle breaks in the middle, or at least loses its rigidity in this region. This evidence is supported by studies with the polarization microscope (see below).

Details of changes occurring within the cell during the process of mitosis can certainly best be observed in fixed material, prop-erly sectioned and stained. But there are some features that can be seen most clearly in the living. If the egg of the sea urchin Arbacia is closely watched during the period between fertilization and cleavage, it can be noted that a narrow, clear streak that extends almost the entire distance across the egg be-comes visible about 21 min. after fertilization (at about 21° C ) . This is the first evidence of the mitotic spindle. Gradually the long, narrow streak shortens, and at the same time it widens in the equatorial plane. No structures can be seen in it. At the

171 10. CELL DIVISION

ends of the spindle are clear spherical areas, the centrospheres, and from these centrospheres there extend radiating lines com-posed of rows of granules. These rows of granules represent either

(a)

(b)

F I G . 44 . Sections through eggs of Crepidula

plana; (a) shows a late anaphase stage of the

first c leavage division; (b) a similar stage of the

second c leavage division. In both cases yolk

granules can be seen in the equatorial plane of

the spindle.

the astral rays themselves or the spaces between the astral rays; the latter interpretation is the usual, and probably the correct one.


In many eggs the astral radiations, or rather the rows of granules, can be seen more clearly than they can in the Arbacia egg. Whether or not they extend to the periphery of the cell is hard to determine by direct visual observation. An indication that they do is the fact that after the spindle has formed, the surface of the egg typically ceases to be smooth and becomes somewhat crenated (Heilbrunn, 1920a). This is believed to be due to the fact that the astral rays exert a pull on the periphery of the cell. More direct evidence in favor of this assumption is the fact that when eggs with a spindle are centrifuged, the contour of the cells becomes violently distorted, presumably because of the pull of the spindle on the periphery.

Two or three minutes before a sea urchin egg or a clam egg divides, it can be seen to elongate. This process of elongation has indeed been observed for various types of cells. The Germans call it "karyokinetische Streckung"—in English it can be called "mitotic elongation." Churney (1936, 1940) has made a careful study of mitotic elongation in the egg of the sea urchin Arbacia. The process takes place rather rapidly. If we conceive of a cell in mitosis as being held together by the mitotic spindle with its astral rays pulling on the cortex or periphery; then if suddenly there is a weakening of the spindle in its equatorial plane, we would certainly expect that the cell would elongate in the direc-tion of the spindle. Actually the spindle does weaken in its equatorial plane, as has already been noted.

There is another phenomenon that can readily be observed in many living cells in the course of the division process, although not in marine egg cells. As far back as 1879, Permeschko, observ-ing the course of mitosis in cells in the tail of a salamander, noted that shortly before the cells divided into two, numerous delicate protoplasmic processes were extruded around the periph-ery of the cell and were just as rapidly withdrawn again. Similar observations have been made by various students of cells in tissue culture. Thus, for example, Strangeways (1922) observing em-bryo chick cells during the process of division, noted that during the anaphase, "small balloons of cytoplasm project from the surface of the cell . . . these remain for a few seconds and then


collapse. This movement continues for about 6 minutes, new balloons being formed as the others collapse." While these move-ments continue, the cell begins to divide. Similar observations were made by Bucciante (1927) also on tissue culture cells. One of Bucciante's figures is shown in Fig. 45. He regarded the movements as ameboid. Various students of tissue culture have made motion pictures of these movements and they have often

F I G . 45 . Amebo id processes in a dividing tissue culture cell.

The cell is a myoblast ( B u c c i a n t e ) .

been shown. The existence of these movements, often referred to as "bubbling" movements, at the periphery of the cell in divi-sion is a strong indication of the relative fluidity of the surface protoplasm at the time of division. Presumably as the cell prepares for division and as it actually divides, the outer region, or cortex is in a rather fluid state, for otherwise it would resist the extrusion of bubbles or pseudopodia-like processes.

Finally brief mention should be made of what is at the present time a favorite method of study. In various parts of the world, students of cell division have used improved methods of polariza-tion microscopy in studies of the dividing cell. The pioneer in this field of study was Schmidt. As early as 1936 he observed the birefringence of the mitotic spindle, and he noted also that as the spindle elongates it separates into two biréfringent parts with an isotropic gap in between (see Schmidt, 1939; this paper has references to earlier publications ). Other studies on the bire-


fringence of the spindle include those of Inoué and Dan ( 1951 ), Inoué (1951, 1953), and Swann (1951). In his 1953 paper, Inoué presents illustrations of spindles of eggs of the worm Chaetop-terus and of pollen mother cells of the lily. These are supposed to show spindle fibers, and no doubt they do in the original photo-graphs, but the reproductions in the journal are not, to my mind, very convincing. Moreover, in order to see the spindles clearly, Inoué was obliged to centrifuge the cells, and the strain caused by the centrifugal force might well induce birefringence. In addition the Chaetopterus eggs were centrifuged in the presence of sugar solutions, and in our experience such solutions are not without effect on the physical properties of the protoplasm even when they are used in isotonic concentration—the solutions that Inoué used were hypertonic. As a matter of fact, the question as to whether spindle fibers are or are not visible is of no great interest to a physiologist, for whether or not the gel that is the mitotic spindle is split now and again into fibrillar elements is not necessarily important for the machinery of the dividing cell. More important is the fact, observed originally by Schmidt and confirmed by Swann, that in the late anaphase the spindle loses its birefringence at the equator. Actually, as noted earlier, the breakdown of the spindle at the equator was noted by Conklin in 1902 and by Heilbrunn and Wilson in 1948 in their studies of fixed and stained preparations, for these authors saw evidence that cytoplasmic elements had invaded the equatorial region of the spindle during the late anaphase. Probably in the cytological literature, there are other observations of the same sort.

Obviously studies of the morphology of the dividing cell and its parts are of genuine interest but, although they provide a convenient basis for speculation as to the forces involved in the division of the cell, they give no clue as to what makes the cell divide and what prevents it from dividing. And these are questions of primary importance for the physiologist.

What types of factors do make cells divide? In the first place, injury can be a potent stimulus to cell division. If a tree is cut or wounded, a callus often forms at the site of the injury; if a shrub is cut down to the ground, it will rapidly grow up again,


and this growth is primarily due to cell divisions. In animal tissues, wherever regeneration is possible, the cell divisions responsible for the regenerating growth are the direct result of injury. Thus if the tail of a fish is cut off, if a flatworm is cut into two or more pieces, if a large fraction of the liver of a rat is cut out, the cells in the surviving injured tissues immediately respond by beginning to undergo cell divisions. This phenomenon is an example of homeostasis at the cellular level (compare Chapter 13). But our immediate concern is not so much that regulation occurs, but rather the nature of the regulatory process. Un-doubtedly injury substances are involved, but what is the nature of these substances that can cause a cell to begin the process of cell division? As shown in earlier Chapters (see Chapters 5 and 6), when a cell is injured, its protoplasm undergoes a clotting reaction, and in the course of this reaction, which is similar to the clotting of blood, substances are produced which can induce or promote clotting in uninjured protoplasm.

There are two types of experiments which, more than any other, have been used in the attempt to understand the nature of the stimulus that makes a cell divide. In the first place, many investigators have studied the initiation of cell division in the eggs of marine invertebrates. And secondly, ever since the discovery in 1915 that cancer could be produced in the ears of rabbits by long continued application of a coal-tar distillate, numerous workers have attempted to discover exactly which chemical compounds could induce cancerous growths to make their appearance.

First as to the marine eggs. This type of material has many obvious advantages. From animals such as sea urchins, worms, or clams, great numbers of eggs can be obtained. These are typically spherical, of a convenient size for microscopic study, but not too small for a physiological study of their protoplasm. More-over, when they are made to divide by the addition of sperma-tozoa, in most cases the eggs all divide in synchronous fashion. Thus the properties of the cell and the changes that occur in the course of the division process can be investigated much more readily than if the eggs were in all different stages of division, as they are in tissues such as the tip of a plant root.


If sea urchin eggs are placed in hypertonic solutions and are then returned to sea water, many of them undergo cell division. This was first shown by Morgan in 1897. In a preliminary note published in 1898, Morgan wrote, "if unfertilized eggs of Arbacia are put into sea water, to which 1.5 per cent sodium chloride has been added and left there from one to three hours, they will, when returned to ordinary sea water, begin to segment after about half an hour." Morgan's complete paper, containing details of his work with hypertonic solutions containing an excess either of sodium chloride or magnesium chloride, was published in 1899; this paper contains many cytological details. No doubt inspired by Morgan's work, Loeb began his famous experiments on arti-ficial parthenogenesis, experiments in which he showed dramati-cally that the action of the spermatozoon in initiating cleavage and subsequent development could be duplicated by various types of artificial treatments. Loeb's work is summarized in his book "Die chemische Entwicklungserregung des tierischen Eies" published in 1909, and then in English translation as "Artificial Parthenogenesis and Fertilization" in 1913.

Following Loeb, many workers published papers on artificial parthenogenesis, papers which showed that cleavage and often later development as well, could be initiated by a variety of chemical and physical agents. At first there was a tendency to believe that only specific types of chemicals could produce results. In his earliest experiments, Loeb himself felt that the magnesium ion had a special virtue, but he soon found that any relatively innocuous substance in hypertonic solution could in-duce cleavage. And not only hypertonic solutions were effective. Indeed the list of chemical and physical agents which could make the egg cell divide grew ever larger. Eggs of one sort or another can be induced to divide by treatment with acids or alkalies, by exposure to heat or cold, by fat solvents of various sorts, by isotonic solutions of potassium chloride, and by ultraviolet radia-tion. How can all these diverse agents act? Do they all have a similar effect on the egg protoplasm? And what could this pos-sibly be?

Loeb's ideas as to the nature of the effect produced by parthen-


ogenetic agents changed from time to time, but eventually he came to believe that the primary effect was of the nature of a "cytolysis," and that this cytolysis in some way or another caused an increase in the rate of oxidation of the egg cell. By cytolysis, Loeb meant a vacuolization reaction; such a vacuolization is shown in Fig. 27 (p. 79), which was copied from one of Loeb's figures. In general, when eggs are exposed for too long a time or to too high a concentration of a parthenogenetic agent, they com-monly do undergo what Loeb called cytolysis. Thus, to produce cleavage or parthenogenetic development, exposures to partheno-genetic agents must not be too long, or they must be followed by a corrective agent. A successful treatment, Loeb thought, in-volved only a superficial cytolysis. Loeb's ideas as to the nature of the cytolytic action were not always very clear; in general, he seemed to think of it as a liquefaction, which it certainly is not. Nor was Loeb very clear as to why cytolysis should produce an increase in oxidative processes; what he did do was to offer evidence that this did actually happen. But his evidence was not very sound, for it was based on measurements made with Winkler's method of determining oxygen in solution, and this method, as is well known, gives false results if organic substances are present in solution (compare Heilbrunn, 1915b). Such sub-stances are present in sea water in which sea urchin eggs have been suspended, and they are present in especially large amounts in sea water in which cytolyzing eggs have been suspended.

And yet, in spite of the criticisms that could be raised against Loeb's experimental work and his theories, and the number of such possible criticisms is great, there is nevertheless a certain amount of truth in Loeb's theoretical interpretations. For what he called cytolysis, which is a vacuolization reaction, does indeed occur if eggs are overexposed to agents which tend to make them divide. When these agents act efficiently without causing the death of the cell, it might perhaps be more correct to speak of their action as being an incipient cytolysis (vacuolization) in-stead of a superficial cytolysis. The essential point is that all parthenogenetic agents do actually produce within the eggs a protoplasmic clotting, and this clotting carried to excess typically


causes extensive vacuolization. But how and why do the various diverse treatments that cause eggs to divide produce this clotting reaction?

In the light of our present day knowledge of protoplasm, this information is not too hard to arrive at. In Chapter 3 it was shown that the outer cortex of the cell is composed of a gel that owes its stiffness to the presence of calcium in it. This is true for the ameba and for the egg of the worm Chaetopterus, and it may be rather generally true. In the presence of oxalate in the environment, the cortex loses its rigidity, so too it becomes less rigid if calcium ion is displaced by an excess of potassium ion (Heilbrunn and Daugherty, 1932; Wilson and Heilbrunn, 1952). Cold and heat both liquefy the cortex. The effect of cold can be explained by the fact that at lower temperatures, proteins tend to bind less cation; heat apparently acts by affecting the lipid part of the lipoprotein cortex. Ether also liquefies the cortex both of the ameba (Daugherty, 1937) and of the Chaetopterus egg (Wilson and Heilbrunn, 1952); it presumably acts on the lipid part of the cortex just as heat does. Indeed Wilson and Heil-brunn were able to show that every one of the agents listed above as being able to cause egg cells to divide causes a liquefaction of the cortex of the Chaetopterus egg. And, for the most part, it is easy to understand why. Some of the reasons for this behavior have already been cited. Moreover, ultraviolet radiation, which liquefies the cortex both of the ameba ( Heilbrunn and Daugherty, 1933) and of the Chaetopterus egg (Wilson and Heilbrunn, 1952), is known to release calcium from combination with pro-tein. According to Anslow, Foster, and Klingler (1933), ultra-violet radiation frees cations from amino acid combination by breaking the carboxyl bond.

That calcium can induce division of sea urchin eggs is sup-ported also by the work of Hollings worth, published in 1941. She found that if sea urchin eggs were left for some hours in isotonic calcium chloride solutions, the calcium eventually seemed to penetrate the eggs, especially if the sea water was made slightly alkaline. As a result of the entrance of calcium ions, the eggs showed rather a high percentage of cleavages.


If the various agents which induce egg cells to divide actually do release calcium into the egg interior, this should, of course, induce clotting, for, as has been shown in Chapters 3 and 4, the clotting of protoplasm is dependent on calcium ion. Hence we would expect that the treatments which are successful in causing cells to divide would cause some sort of a viscosity increase in the protoplasm. This has been clearly demonstrated for Chae-topterus eggs. Heilbrunn and Wilson (1955a) have shown for the Chaetopterus egg that hypertonic solutions, isotonic potas-sium chloride, cold, heat, acid, alkali, ether, and ultraviolet radia-tion, under conditions in which these agents cause the division of the cell, all produce an increase in the viscosity of the protoplasm in the interior of the egg cells. Such an increase in viscosity must not be too great, for then the egg is killed. In the sea urchin egg also, Heilbrunn showed long ago (1915a) that various types of parthenogenetic agents cause a sharp increase in the viscosity of the protoplasm.

If protoplasmic clotting is similar to blood clotting, then just as it is possible to cause the clotting of blood by thrombin in the absence of calcium, so it should be possible to cause proto-plasmic clotting and cell division by substances produced when protoplasm is clotted. This seems to be true; at any rate Harding (1951) found that substances obtained from the protoplasm of heat-killed muscle cause the initiation of cleavage in the eggs of the sea urchin Arbacia. This is illustrated in Fig. 46. Harding found also that the injury substances which induced cleavage caused a marked increase in the viscosity of the egg protoplasm. This viscosity increase occurred some minutes after the eggs were removed from the solutions containing the injury substances and returned to sea water. Figure 47 indicates that there is a correlation between the length of exposure necessary to produce cell division and the viscosity change in the protoplasm.

If the eggs of various marine invertebrates are compared, it will be noted that there is a wide variation in the effectiveness of various parthenogenetic agents in inducing cell division. Sea urchin eggs respond very favorably to hypertonic solutions, and such solutions cause high percentages of the treated eggs to


F I G . 4 6 . Cell division resulting from treatment of Arbacia eggs with

extracts of injured tissues.

favorably to heat whereas sea urchin eggs show very little re-sponse to heat treatment; in this they differ from Chaetopterus eggs. Such variations in behavior are easily understandable. For in all cases the treatments involved must be of sufficient intensity or duration to cause a mild clotting reaction, but not a reaction strong enough to cause death. And some types of eggs are more readily killed by certain treatments than are other types of eggs.

But whereas the eggs of various invertebrates on the whole be-have in more or less similar fashion, the frog egg at first sight is totally different. This egg, much larger than the eggs we have been considering, is rather insensitive to any and all of the treat-

divide. Hypertonic solutions are also very effective for Chae-topterus eggs. On the other hand, such solutions do not have much of a parthenogenetic or mitosis-inducing effect on eggs of the starfish or of the worm Nereis. These eggs respond very


ments that cause marine eggs to divide. This fact has caused students of frog egg parthenogenesis to wonder whether there was one type of parthenogenesis for the sea urchin egg and another for the frog egg.

Exposure time, minutes

F I G . 47. Viscosity and per cent of c leavage for

various t imes of exposure to extracts of injured tissue.

Viscosity determinations were 2 5 min. after removal

of the e g g s to sea water. A V indicates that the viscosity

was be low a g iven point, an inverted V that the viscosity

was higher.

In order to make a frog egg divide (and begin its develop-ment), it is necessary to pierce the egg with a fine needle. More-over, such a needle prick is vastly more effective if the needle before entering the egg is dipped either in blood serum or in tissue extracts or homogenates. The prick of a needle would cause a surface precipitation reaction; in other words, it would initiate a clotting reaction. And the fact that blood serum or tissue extracts favor the response of the egg is easily explained, for both blood serum and tissue extracts contain substances which favor not only blood clotting, but protoplasmic clotting as well. In testing the cleavage-inducing potency of various granular fractions of tissue homogenates, Shaver ( 1953 ) has found in his studies on the frog egg that "some preliminary experiments on the clotting ability of granule fractions have indicated that, in general, there is a correlation between their thromboplastic and


cleavage-initiating properties." In other words, if Shaver's ex-periments are correct, the potency of a material derived from protoplasm depends on its efficacy in promoting a clotting reac-tion.

Thus, for frog eggs as well as for marine eggs, we have a logical theory to explain why so many diverse types of agents are all effective, and it is a theory that offers an interpretation that is in accord with the known properties and the known behavior of the protoplasmic colloid. Whether the theory is correct or not, it is the only theory that at the present time can offer any sort of an explanation of the established facts. Other theories of the initiation of cell division have largely faded from the picture. Thus, it can no longer be maintained that the initiation of cell division is primarily due to an increase in the permeability of the plasma membrane of the cell. This theory, strongly supported in the early years of the century, especially by the researches of R. S. Lillie, could scarcely explain the fact that cold and also calcium salts cause an initiation of cell division. It would be hard to prove that cold increases permeability, and calcium does the very opposite. Indeed, Lillie, the chief protagonist of the theory, abandoned it in 1941 in favor of the idea that an increased acidity of the protoplasm was primarily responsible for the impetus to cell division. Such a theory could scarcely serve to explain the action of the diverse treatments that cause cells to divide. But it should be remembered that clotting of protoplasm may in itself cause an acidification ( see Chapter 6 ). Also a release of calcium from the cortex of the cell might cause an increase in permeabili-ty, for, as is commonly recognized, calcium is the ion which more than any other is responsible for the semipermeability of the plasma membrane. Also it could scarcely be argued that all the agents which initiate cell division directly cause an increase in the activity of oxidizing enzyme systems. And yet, as noted previously (see Chapter 7), clotting could in itself cause an increase in the rate of oxidative processes. For dividing cells, this idea is supported by the work of Rapkine (1931); see below.

The fact that we can explain the initiation of cell division in terms of colloidal change and that this explanation fits all the


facts thus far known is important, but what is equally important is that this theory also offers an interpretation of mechanism. Granted that the permeability of the cell increases following suit-able stimulation by physical or chemical agents, or granted that following such stimulation the oxidative processes increase, how can either of these processes be translated into a machinery for making the cell divide? But on the basis of the colloidal theory, we can indeed postulate such a mechanism. The clotting reaction which is induced by all the agents that make the cell divide is directly connected with the origin of the mitotic spindle. For the mitotic spindle can be considered as having been formed from the protoplasm as a result of a clotting process.

This view is in accord with the facts that are known about the physical changes that occur in the protoplasm during the course of the mitotic process. For, as we have seen, the mitotic spindle is a solid structure that arises out of the fluid protoplasm; its appearance is preceded by a gelation in the protoplasm, the so-called mitotic gelation. That such a gelation actually does occur can be clearly demonstrated. If the viscosity of the proto-plasm of sea urchin eggs is measured at frequent intervals be-tween fertilization and cleavage, it can be seen that soon after fertilization there is sharp increase in viscosity, an increase in which the viscosity is doubled or tripled. This viscosity increase precedes the appearance of the mitotic spindle. One can con-clude that the viscosity increase, dependent as it probably is on the clotting or partial gelation of some constituent of the proto-plasm, results in the formation of the mitotic spindle. Once the spindle has appeared, the viscosity drops again. These viscosity changes have been described by Heilbrunn ( 1920a ) and by Wil-son (1950). Similar viscosity changes have been described for other types of cells and, indeed, it seems clear that in cells generally, the appearance of the mitotic spindle is preceded by an increase in protoplasmic viscosity. The viscosity curve for the egg of the clam Cumingia is shown in Fig. 48, and that for the egg of the worm Chaetopterus in Fig. 49.

There is a difference in the curves for the Cumingia egg and the Chaetopterus egg, for in the curve for the Cumingia egg


20

18

16

14

12

10

8

6

4

2

o — o ^ ν ν ν ν V V V ν ν ν V V V V χ o ^ X

I I V V V

6 . ( Λ Λ\

i Λ Λ Λ Λ » Μ Λ Λ Λ Λ Λ Λ »

«Ι Λ Λ ° M V V aV

Λ Λ Λ Λ Λ Λ Χ Ι Λ Λ Λ Λ \ Λ

Χ / " Λ Λ Λ

Λ Λ Λ

Λ Λ Λ

10 15 20 25 30 35

Time, minutes after fertilization

40 45 50

F I G . 48 . Viscosity changes in the protoplasm of the Cumingia eggs

during the interval b e t w e e n fertilization and first c leavage (Heilbrunn, 1921).

5 10 15 20 25 30 35 AO 45 50 55

M I N U T E S AFTER FERTILIZATION

F I G . 49 . Protoplasmic viscosity changes in the e g g of Chaetopterus

during the time b e t w e e n fertilization and first c leavage (Hei lbrunn and

Wilson, 1 9 4 8 ) .


the viscosity rises and falls during the maturation divisions, whereas in the curve for the Chaetopterus egg, no detectable change occurs during the maturation divisions. This difference is due to the fact that in the relatively small Cumingia egg the maturation spindles occupy a rather large fraction of the egg volume; on the other hand, the maturation spindles of the Chae-topterus egg are relatively small. In the Chaetopterus egg, studies of sections of eggs fixed at minute intervals have shown that a viscosity increase precedes the appearance of the spindle; then just as the spindle becomes visible, the viscosity drops again. In the sea urchin egg, appearance of the mitotic spindle is also preceded by a sharp increase in the viscosity of the protoplasm ( Heilbrunn, 1917, 1920a ). During this period in which the proto-plasmic viscosity is increasing, there is, according to Rapkine (1931), a marked and progressive decrease in the free SH groups in the protoplasm, an indication that clotting processes in the protoplasm are accompanied by an oxidative process. This idea is also supported by Mazia, who has found that during the forma-tion of the mitotic spindle, SH groups are converted to S-S groups (Mazia, 1954).

In the discussion thus far, we have been considering what happens when an egg in sea water is so treated that it begins to divide. There is also another type of initiation of mitosis that occurs in the eggs of marine invertebrates and, indeed, in all types of eggs. When an egg is in the ovary, usually division is inhibited; typically the egg is immature, that is to say, it has not yet begun its maturation divisions. Although the sea urchin egg matures while it is in the ovary, most types of eggs undergo maturation divisions only after they leave the ovary. In some cases mere release from the ovary is sufficient to start the matura-tion mitosis. Thus in the Chaetopterus egg, as soon as the egg is discharged from the ovary, the large nucleus or germinal vesicle breaks down and the first maturation division begins. Figure 50 shows two Chaetopterus eggs, or rather parts of them. The egg to the left still has an intact germinal vesicle, the egg to the right has had its germinal vesicle break down. In sea water, when the germinal vesicle of the egg breaks down, the first maturation


division begins, but this division proceeds only as far as the metaphase stage and it remains in this stage until either fertiliza-tion or some artificial stimulus provides the impetus for a con-tinuation of the mitotic process. This is one type of behavior and a fairly common one. An equally common type of behavior is exemplified by the egg of the worm Nereis or the egg of the clam Spisula. These eggs when shed into sea water from the ovary are in the germinal vesicle stage just as the Chaetopterus eggs are. But for Nereis and Spisula eggs, entrance into sea water does not provide the stimulus necessary to initiate the first

F I G . 5 0 . T w o Chaetopterus

eggs , one wi th germinal vesicle

intact, the other with the ger-

minal vesicle broken down.

maturation division. Only when the spermatozoon enters or when the eggs are artificially stimulated does the germinal vesicle break down. Thus in the two types of eggs the division process may be blocked either at the germinal vesicle stage or at the first maturation division metaphase. In our previous discussion of the Chaetopterus egg, we have already considered the nature of the stimulus that starts this egg dividing after it has stopped in the metaphase stage of the first maturation division. Here, as in the sea urchin egg, a release of calcium from the cortex initiates a clotting reaction in the protoplasm of the cell interior.

Let us now consider the nature of the stimulus that causes a breakdown of the germinal vesicle, that is to say, a breakdown of the membrane surrounding this large nucleus. In view of the fact that in most types of cell division the nuclear membrane breaks down in the early stages of mitosis, it is of interest to inquire as to the nature of this reaction and the nature of the forces which are responsible for it. Whether breakdown of the


membrane of the germinal vesicle is induced as a result of its leaving the ovary or only after that, either by fertilization or by artificial activation, presumably the fundamental nature of the stimulus is the same.

Take first the case of the Nereis egg. This egg was carefully studied by F. R. Lillie and his drawing of the egg is reproduced in Fig. 51. The nucleus of the immature Nereis egg breaks

F I G . 5 1 . Uninseminated e g g of Nereis

(Li l l ie , 1 9 1 9 ) .

down as a result of artificial stimulation. One of the simplest ways of accomplishing this is to subject the egg to ultraviolet radiation. As already noted, the effect of this radiation is to release calcium from the cell cortex. A clear demonstration of the fact that it is the release of calcium that initiates the break-down of the nuclear membrane is provided by some experiments of Heilbrunn and Wilbur ( 1937 ). They found that if Nereis eggs were first treated for a few minutes with citrate solutions so as to remove or bind the calcium of the cortex, they were then in-capable of response to ultraviolet radiation. ( Citrate also prevents the breakdown of the germinal vesicle as induced by potassium chloride or sodium chloride). The effect of citrate is reversible, for on return of the eggs to sea water, they are again susceptible to the action of the ultraviolet. But it must not be supposed that the release of calcium has a direct effect on the nuclear mem-


brane, for if the egg cells are immersed in calcium solutions and then broken so that the nuclear membranes come into contact with the calcium, the membranes do not break down. What presumably happens is that calcium liberated from the cortex activates an enzyme system which dissolves the membrane. This is in line with our basic hypothesis that the primary fact of stimulation is the activation of a clotting enzyme which is at the same time a proteolytic enzyme.

Support for this idea comes from the work of Goldstein ( 1953 ) on the Chaetopterus egg. This egg, it will be remembered, be-comes activated, that is to say, begins the maturation division process, as soon as it leaves the ovary and enters sea water. As in the case of the Nereis egg, citrate solutions prevent germinal vesicle breakdown, and in this case also the effect of the citrate is reversible, for if following citrate treatment the eggs are placed in sea water, germinal vesicle breakdown then occurs. According to Goldstein, within 2 min. after the eggs enter sea water, there is a liquefaction of the cortex. Such a liquefaction, as our earlier discussion shows, would indicate a release of calcium. Goldstein believes that this released calcium activates a proteolytic enzyme system, and from eggs frozen 5 min. after they were shed into sea water, he was able to prepare a protease. On the other hand, if eggs were frozen 14 min. after shedding, the homogenates of such eggs showed no proteolytic activity. Goldstein believes the egg protects itself against excessive proteolytic activity by a homeostatic response. This is an example of cellular homeostasis, to be discussed in a later chapter. That the activation of a protease could account for the dissolution of the nuclear mem-brane is indicated by the fact that when the eggs are immersed in solutions containing crystalline trypsin, the germinal vesicle breaks down. It might be argued that this effect of trypsin is an indirect one and that the trypsin has a digestive action on the surface of the cell. However, trypsin causes a breakdown of the germinal vesicle even when cells are immersed in trypsin solutions made so strongly acid that they could have little or no effect on the exterior of the cell. (In interpreting this experi-ment, it should be remembered that strongly dissociated acids do not enter cells readily ).


When a sea urchin egg or a mature Chaetopterus egg is ex-posed to chemical or physical agents which incite it to divide, the protoplasm in the interior of the egg cell undergoes a clotting reaction. But immature Chaetopterus eggs with the germinal vesicle intact have rather viscous protoplasm, and this becomes decidedly more fluid when the germinal vesicle breaks down. This is true not only of the Chaetopterus egg, but of other marine eggs as well (for example, the eggs of Nereis and Spisula). How can this be explained? It might be thought that as the nuclear mem-brane breaks down, its fluid contents or some substance dissolved in the nuclear fluid causes the drop in viscosity. But, as a matter of fact, in the Chaetopterus egg the decrease in viscosity occurs before the nuclear membrane breaks down. Thus in the im-mature Chaetopterus egg, release of calcium from the cortex causes not only a dissolution of the nuclear membrane but also a liquefaction of the protoplasm. Apparently calcium entering a cell can cause either gelation or liquefaction. At first sight this may seem strange, but it should be remembered that a protease can act either as a clotting enzyme or as a dissolving enzyme. Indeed there is some evidence that thrombin can both clot blood and liquefy the clot after it has formed. This very sort of thing appears to happen in the Chaetopterus egg. Figure 52 shows a curve of the viscosity of Chaetopterus egg protoplasm in the time immediately following entrance into sea water. The value at zero time was obtained by centrifuging parapodia of the worm; these contained ovarian eggs. In order to make rapid measurements, the centrifuge force used in the tests was four times as great as that used ordinarily (for further details, see Heilbrunn and Wilson, 1955b).

From the discussion thus far, it is apparent that we are now in a position to interpret the strange fact that so many diverse agents can induce cell division in marine eggs. All of these agents act as stimulating agents do generally ( see Chapter 12 and 13), and the effect that they produce can readily be trans-lated into the formation of the mitotic spindle (as well as the dissolution of the nuclear membrane). Let us consider now the other type of study that has attempted to determine the nature


of the impetus to cell division. There has been an enormous amount of work on the carcinogenic activity of various types of compounds. In his "Survey of Compounds Which Have Been Tested for Carcinogenic Activity" published in 1951, Hartwell

120

100

> - 8 0

(Λ ο Ο 6 0

4 0

2 0

I • I • I • I • I I ι L_

0 1 2 3 4 5 6

T I M E IN M I N U T E S

F I G . 5 2 . Viscosity of Chaetopterus e g g protoplasm

during the interval b e t w e e n entrance of the e g g into

sea water and breakdown of the germinal vesicle.

Measurements were m a d e at 2 1 ° . At this temperature

germinal vesicle breakdown occurs 7 min. after the e g g

has entered sea water.

listed 2055 papers on the subject. The authors of these papers studied 1329 chemical compounds, of which 377 were found to cause tumors. When this work began, there was a strong belief that certain specific chemical compounds were effective, and chemists were strongly of the opinion that if they could deter-mine the nature of the chemical structure in active carcinogens, this would of itself solve the problem of why they caused cancer.


But in spite of the fact that some of the best organic chemists in this and other countries attacked the problem assiduously, the results have been disappointing. For just as it was found in the study of the impetus to cell division in marine eggs that the effectiveness of a compound could hardly be interpreted in terms of chemical relationships, so, in the study of carcinogens, a bewildering list of dissimilar compounds is now known to be potent. Among the substances which have been found to cause cancer are the following: benzopyrenes, cholanthrenes, benzan-threnes, dibenzacridenes, dibenzcarbozoles, sex hormones (such as the estrogens and testosterone), sterols, azobenzenes, various oils (such as oil of camphor and oil of eucalyptus), carbon tetra-chloride, urethane, chloroform, acetylaminofluorine, naphthyl-amine, tannic acid, thorotrast (colloidal TI1O2), arsenic, beryl-lium, selenium, and nickel. This is indeed a heterogeneous group of substances. In addition, roentgen rays, radium, and ultraviolet rays all can cause cancer.

Small wonder that students of cancer have racked their brains for an explanation. For a discussion of the action of carcinogenic agents, Greenstein's authoritative book on the "Biochemistry of Cancer" should be consulted. In the first edition of this book, published in 1947, Greenstein devoted 72 pages to the subject; in the second edition published only seven years later, the discus-sion had grown to 140 pages. Moreover, in the introduction to the chapter that deals with carcinogenesis with extrinsic factors, Greenstein writes that "it would be obviously impossible, short of writing an encyclopedia, to discuss this phase of cancer re-search in its entirety." Presumably all the agents which cause cancer induce certain unique or special chemical reactions, but as to what these are the evidence is not clear, and Greenstein sums up the situation by stating that "it must reluctantly be concluded that few if any decisive facts emerge." What is the reason for this failure of so many capable workers to reach any-thing like a solution of the problem? One difficulty lies in the fact that the cells which have been used in studies of carcino-genesis are not cells whose physical or colloidal properties are easy to determine. Clinical scientists have been loath to use the


methods and the reasoning of the general physiologist who is ever ready to search for the simplest type of protoplasmic material and to apply the knowledge gained from it.

What little information there is concerning the changes that result from treatment with carcinogens favors the idea that the effects of carcinogens on the tissue cells of higher animals are similar to the effects produced in egg cells by agents which make them divide. Suntzeff and Carruthers (1943, 1944) found that some carcinogens cause a release of calcium. Calcium release is also caused by ultraviolet rays and by roentgen irradiation (for discussion and many references, see Heilbrunn and Mazia, 1936). Moreover, according to Lucké, Parpart, and Ricca (1941), the potent carcinogen 2,5,6-dibenzanthrene causes vacuolization ( cytolysis ) of sea urchin eggs, and there is an extensive literature to show that both ultraviolet and roentgen radiation cause vacuolization in various types of living cells. Also the fact that injury substances, as we have pointed out, can make sea urchin eggs divide finds its counterpart in the old observation that chronic injury leads to cancer. In the older literature on cancer, this was a point that was often stressed. Thus Lumière (1929) in his book on "Le Cancer. Maladie des Cicatrices" names 27 older authorities on cancer who have supported this point of view and these 27 are but a sample. Lumière himself cites 44 kinds of evidence in support of the same idea. However, in modern books on cancer, the subject of injury and irritation as a cause of cancer has been pushed in the background in favor of a vast array of newer facts, perhaps less pertinent. Thus in the huge volume on "The Physiopathology of Cancer" edited by Homberger and Fish-man and published in 1953, there is only scant mention of this aspect of the subject. And yet cancer of the stomach, which according to Palmer (1951), kills more people than any other cancer of the body, seems always to be associated with a chronic irritation. The fact that injury and injury substances induce cell division and cause cancer is readily understandable on the basis of the theory that has been previously outlined in this chapter.

In this chapter, our interest has been focussed on the nature


of the factor or factors which induce a cell to begin the process of division. As yet we have not inquired at all into the forces involved in the various stages and aspects of the process. For one reason or another, chromosomes move from the equatorial plane of the spindle toward the poles. Also in animal cells— although usually not in plant cells—the division of the cell is climaxed by a constriction which divides the cell into two. Much has been written both about the movement of chromosomes and about the constriction of the cell. Actual pertinent data that would help to explain these important aspects of cell division are not very impressive. This, however, has not deterred theoreti-cally minded cytologists and cell physiologists from engaging in a wide variety of guesses.

Because of the lack of useful physical data, no attempt will be made here to interpret the movement of the chromosomes. The mechanism of constriction, that is to say, the actual division of the cell into two halves, can perhaps be interpreted without too much difficulty. In the first place, it seems to be fairly well estab-lished that the division of the cell depends on the mitotic ap-paratus, spindle and asters, although according to Mitchison (1952, 1953) and Swann and Mitchison (1953) these structures need not persist through the final stages of the division. When-ever, by the application of strong centrifugal force, the mitotic spindle is pulled loose from its moorings and made to lie in another part of the cell from where it was anchored originally; no matter where the spindle is, constriction of the cell, that is to say, the division furrow, is always at a right angle to the axis of the spindle.

Thus until genuine contradictory evidence is presented, we can with reasonable assurance assume that the mitotic apparatus is responsible for the constriction of the cell into two. The reason for this is not hard to understand. The evidence presented at the beginning of the chapter indicates strongly that the astral rays are attached to the cortex of the cell. Hence we have a system in which the cortex, the astral rays and the spindle form one tensile system—the spindle pulls on the astral rays, the astral rays on the cortex—all the tensions act against each other and


there is an equilibrium all around. If now at some point there is a break in the integrity of the tensile system or even a weaken-ing of it, the whole system will stretch to find a new equilibrium in the same way as a stretched rubber band will elongate if any-where along its length there is a break or a weakening. Now, as pointed out previously, studies with both the ordinary light microscope and with the polarization microscope indicate that in the late anaphase there is a break or weakening at the equa-torial plane of the spindle. Cytoplasmic granules can be seen to invade this region of the spindle; also the birefringence of this region diminishes or disappears. Granted such a break in the spindle, there should be an immediate elongation of the spindle and, indeed, of the whole cell. This is the mitotic elongation. The easing of tension as a result of the break would pass as a wave from the equator of the spindle out toward the poles of the spindle, from there to the cortex opposite these poles, and then around the cortex. This could well account for the division of the cell. As a matter of fact, a drop in the tension of the outer membranes of cell in the regions opposite the poles of the spindle would ipso facto cause a constriction to occur at a point equi-distant from these regions. This is indicated by the model ex-periments of McClendon (1913) and Spek (1918). These workers found that in an oil drop suspended in a liquid of the same specific gravity, if the interfacial tension was lowered at two points opposite to each other on the spherical surface of the drop, there would be a division of the drop in a plane equidistant from the two points at which the tension was lowered. Thus a break in the spindle along its equatorial plane could in itself result in the division of the cell.

11.

On the basis of what it is that makes a cell divide, we should be able with some assurance to predict what it is that stops a cell from dividing. In this chapter an attempt will be made to discuss the problem of the suppression of cell division both from the theoretical and the practical aspects. On the clinical side this problem is of primary importance. For if we knew how to stop the division of cancer cells without inflicting too much injury on other cells of an organism, we would be in a position to cure cancer.

Naturally there has been no lack of effort to promote the search for suitable antimitotic substances. Some of this effort has been expended in haphazard surveys of all sorts of substances. This hit or miss method, although it has often proved very valuable in various aspects of clinical research, has little appeal for a theoretical biologist. Certainly if every known substance in all possible concentrations were tested for its effect on cancer cells, eventually it would be possible to arrive at a cure for cancer. The amount of labor involved in such a series of tests would be stupendous. Clearly it is an advantage to know in which direc-tion it is wisest to expend our efforts.

At the present time there are two major schools of thought on the subject. There should perhaps be more. The need for a cancer cure is so essential that no plan of attack should be ne-glected. Because of the great discoveries in biochemistry in recent years, chemists have played a leading role in the search for antimitotic agents. To a chemist the obvious method of attack is to find some way of preventing the accumulation in a cell of those substances necessary for cell division. One could, of course, deprive a cell or an organism of all nutrient substances; under

THE SUPPRESSION OF CELL DIVISION

195


these conditions it might be thought that cancer cells would be deprived of the energy necessary for cell division. But obviously starvation is not a cure for cancer, for physicians are well aware of the fact that in starved or semi-starved patients, cancer cells do not stop their growth and divisions. Hence in recent years the search has been for agents which will block certain metabolic pathways. Unfortunately cancer cells, like cells generally, show considerable adaptability—if one metabolic pathway is blocked, another is apt to take over. At the present time the research on metabolic pathways, although it has yielded very valuable infor-mation for the theoretical biochemist, has not been successful in providing a cure for cancer. Perhaps in the future success will be attained; certainly it is the ardent hope that it will, but in the meantime it is scarcely wise or proper for workers in this field to make extravagant claims. As a matter of fact, such claims are almost never made by laboratory workers, who for the most part are well acquainted with the difficulties of the situation. This does not prevent enthusiastic reports and prognostications in the press, reports based on glowing information provided by profes-sional promoters whose function it is to obtain funds for an insti-tute or institutes supposedly dedicated to the task of discovering the truth about cancer. Obviously an institute for cancer needs funds, but the amount of money available is limited, and it is certainly unfortunate if, as a result of overstatement, an undue fraction of the total support available goes to projects that are not nearly as promising as their promoters say they are.

When a cell or a group of cells in the brain or the liver start to divide so as to produce a neoplastic growth, that is to say, a cancer, this impetus to cell division can scarcely be due to a change in the sustenance of the cells. Obviously the impetus to division comes from some change in the colloidal machinery. The preceding chapter suggested what the nature of this change might be. On the basis of this information, we should be able to discover ways of preventing or blocking the divisions. This line of reasoning opens up the possibility of a second type of research in the quest for antimitotic agents. It is this second type of research that we shall emphasize, not because it is necessarily

11. THE SUPPRESSION OF CELL DIVISION 197

more important than the first type of research mentioned in earlier paragraphs, but because the ideas that underlie this point of view fit in with the fundamental thesis of this book, which is concerned with the colloidal mechanisms of the protoplasm rather than the chemical reactions occurring in it.

In the first place, if the impetus to cell division is due to stimuli much like those which cause a muscle to contract or a nerve to become aroused, then it is to be expected that the anesthetics which prevent the response of muscle or nerve will also prevent the cell from dividing. This is indeed the case. As far back as 1904 Fühner studied the effect of fat solvent anesthetics on sea urchin eggs and determined the concentrations necessary to prevent cell division. Similar studies were made by R. S. Lillie in 1914. He studied a large group of fat solvent anesthetics and compared the concentrations necessary to prevent cell division (reversibly) with the concentrations required to prevent muscu-lar contractions in the larvae of the worm Arenicola. There was a remarkably close agreement. Lillie's explanation of the effect of the anesthetics was that they had some action on the plasma membranes of the cells. His reason for believing this was that, as he had shown in 1912, fat solvent anesthetics prevented the cytolytic effect of isotonic salt solutions on unfertilized eggs. This may have been a reasonable view in 1914, but at present it would be difficult to understand what relation there might be between the plasma membrane and the vacuolization reaction which stu-dents of marine eggs call cytolysis. What we do know is that cytolysis is due to a clotting of the protoplasm (see Chapter 5), so that the fat solvent anesthetics presumably acted to prevent this clotting reaction. Heilbrunn ( 1920a, and b ) tested the effect of various fat solvents on the colloidal properties of the proto-plasm of fertilized sea urchin eggs. All of these fat solvents prevented the mitotic gelation, and it is thus easy to understand why they should tend to prevent cytolysis, for vacuolization or cytolysis is the end result of the clotting reaction in protoplasm. Chloral hydrate and urethane in solution act like the fat sol-vents do.

As was noted in the preceding chapter, cell division is a process


that involves first a sharp increase in the viscosity of the proto-plasm and then a return of the protoplasm to its original fluid state. Both increase and decrease are essential; if either is inter-fered with, the cell cannot divide. Most antimitotic agents act by preventing the increase in viscosity, but hypertonic solutions and chloretone, and also potassium cyanide, all of which prevent cell division in the Arbacia egg, act by keeping the protoplasm of this cell in a highly viscous state. These viscosity-increasing agents tend to prevent the protoplasm from returning to the con-dition it was in before it was stimulated. It is as though the effect of the stimulation were exaggerated. If a comparison were to be made with muscle, we might say that the twitch of a muscle can be prevented either if the muscle is kept in a relaxed state or if it is kept in a contracted state.

The action of cyanide is hard to understand, for different marine eggs act very differently. In work thus far reported only in a brief preliminary note, Wilson and Heilbrunn ( 1953 ) found Chaetopterus eggs about five hundred times less sensitive than Arbacia eggs. Thus in 10"3 molar KCN, Arbacia eggs are unable to go through the entire process of cell division although they are able to go through the final stages. On the other hand, for Chae-topterus eggs a concentration of 5 χ 10~3 molar must be used to obtain the same result. And in Chaetopterus eggs, instead of gela-tion being fostered or enhanced by the cyanide, the mitotic gela-tion is entirely inhibited. Similarly in the egg of the clam Spisula, which is intermediate between the Arbacia egg and the Chae-topterus egg in its sensitivity to cyanide, mitotic gelation is in-hibited by cyanide. This variable sensitivity to cyanide is well known. Thus Robbie (1949) in studying the respiration of various invertebrate tissues found that they showed a thousand-fold variation in their sensitivity to cyanide. He could find no adequate explanation for this, and was forced to conclude that it "may be indicative of some as yet unknown variable in the cellular respiration system." The variation in the colloidal be-havior of protoplasm in relation to cyanide may in part be ex-plained by the variation in the effect of cyanide on various prote-olytic enzyme systems. As is well known, cyanide is generally


thought to favor the action of cathepsin and papain (see, for example, Purr, 1935) but it tends strongly to inhibit the action of trypsin (Sugai, 1944). Indeed the early work of Willstätter, Grassmann, and Ambros ( 1926 ) indicated that although cyanide activated some plant proteases, it definitely inhibited others. If proteolytic enzymes are responsible for the clotting of proto-plasm, and if cyanide acts differently on different enzymes, we can understand the variability in the behavior of protoplasm toward cyanide. Cyanide also produces variable effects on blood clotting. Dilute concentrations of cyanide prevent the coagula-tion of lobster blood (Loeb, 1903), but they seem to have rela-tively little effect on the clotting of human blood (Kühnau and Morgenstern, 1934). Perhaps when we know more about the relation of oxidation systems to the clotting of blood and proto-plasm, we will be better able to interpret the effect of cyanide on cell division and on the colloidal properties of protoplasm. In one way or another sulfhydryl groups seem to be involved, but exactly how they are involved is not at present known.

Interesting as are the effects of fat solvents and cyanide, these substances are of no practical value in the control of cancer. For fat solvents not only prevent cell division, they may also initiate it; and to put it mildly, cyanide is not a very healthy curative agent. We shall pass therefore to the consideration of other antimitotic agents. It should, of course, be remembered that any agent which kills cells stops them from dividing. We are concerned not with substances toxic generally, but with sub-stances which have a more or less specific effect on cell division. Such substances may prevent cell division without killing the cells.

The best known and the most widely studied of these sub-stances is colchicine. Originally used as a remedy for gout, this alkaloid, a phenanthrene derivative, was found, more or less by accident, to prevent cell division. Interest in colchicine was enhanced by the discovery that by suppressing cytoplasmic divi-sion in plant cells it could induce polyploidy and as a result it could produce new mutants of interest to horticulturists. Partly for this reason, the literature on colchicine is vast. A bibliography


published in 1947 has a list of papers which covers nearly fifty pages of fine print (Eigsti, 1947; Dustin, 1947); almost all of these papers were published in the ten years immediately prior to 1947. Various authors have attempted to apply our knowledge of the antimitotic activity of colchicine to the practical treat-ment of cancer. Experiments on mouse tumors have not been verv successful, primarily because colchicine is a toxic substance and in order to obtain any effect with it, it is necessary to use concentrations just below those which are lethal. According to Lambers (1951), if as little as 0.012-0.015 mg. of colchicine are injected intraperitoneally into mice daily, the mice are poisoned and die. Colchicine causes hemorrhages to occur, and in this wav its action resembles that of bacterial extracts and bacterial polysaccharide preparations, to be discussed later. The fact that hemorrhages are induced leads to the suspicion that colchicine may act as a blood anticoagulant. This seems to be borne out by the work of Loicq (1937); he found that some hours after colchicine was injected into rabbits, the blood clotted more slowly.

In so far as its effect on the colloidal properties of protoplasm is concerned, it has been convincingly shown by Wilbur ( 1940 ) that colchicine prevents mitotic gelation in the sea urchin egg. This supports an earlier conclusion arrived at by Beams and Evans ( 1940 ), and there has also been some additional ( although not very strong) evidence presented by S wann and Mitchison ( 1953 ). In plant material, it is not certain whether or not colchi-cine has the same effect in preventing mitotic gelation that it has on animal cells. In studies on cells of the aquatic plant, Elodea, Staffelt ( 1949 ) found that after long periods of exposure, colchicine caused an increase in viscosity. His measurements began only after an exposure of twenty hours and they were not made on dividing cells.

If we adopt a rational approach in our search for antimitotic substances, and if, as we have shown, the appearance of the mitotic spindle and the division of the cell are always preceded by a gelation which is causally related to the formation of the spindle, and if this gelation is similar in a colloid chemical sense


to the gelation which occurs in the clotting of blood, then we might expect that substances which prevent blood clotting would also be antimitotic agents. Heparin does indeed prevent cell division. This was shown very clearly for tissue culture cells by Fischer (1930, 1936). Heparin also acts as an antimitotic agent for marine eggs (Heilbrunn and Wilson, 1949). In these eggs, as was to be expected, the heparin prevents the mitotic gelation, and their protoplasm remains fluid. One difficulty with experi-ments on the effect of heparin on the division of egg cells is that, presumably because of the large size of its molecule, it diffuses into the interior of the cells rather slowly. This is especially noticeable in the case of frog eggs. There the thick layer of jelly may also be a strong factor in delaying diffusion. But the heparin solutions do prevent cell division in frog eggs after they have had time to diffuse. This is indicated by the extensive experiments of Kaye and Lowell, as yet unpublished.

There is no doubt but that heparin can prevent the division of tumor cells in tissue culture, but can it prevent the division of tumor cells when the tumors are in the intact animal? In other words, is heparin perhaps useful as a therapeutic agent? The first experiments in this direction were made by Goerner (1931); he became interested in the problem as a result of reading Heil-brunn's 1928 monograph. Goerner found that if pieces of rat carcinoma tissue were exposed to a 0.1 per cent heparin in 0.9 per cent sodium chloride solution for twenty-two hours and then transplanted they were not able to produce neoplastic growths, whereas similar pieces which had been exposed to a 0.9 per cent sodium chloride solution produced large-sized tumors. In the next year, extensive experiments were performed by Zakrzewski (1932). He studied the effect of heparin on the growth of rat and mouse sarcoma, using thousands of animals in his experi-ments. Intravenous injections, intraperitoneal injections, and in-jections into the tumor tissue were all effective in reducing the rate of tumor growth. Zakrzewski made sketches of the size of the tumors in experimental and control animals and he also kept records of the number of days the animals survived. The tumors he used were very malignant, so that in the case of the


mouse tumor, no single animal, out of 3000, survived. His first experiment was done with 50 mice of which 25 were controls. Five days after implantation of the tumors, he injected intra-venously 0.1-0.5 ml. of 0.1 per cent Hynson, Westcott, and Dunning heparin into 25 mice, and he repeated such injections each successive day. The heparin injections definitely retarded the tumor growth, and there was also a definite increase in the longevity of the mice which had been given heparin. The survival time of the control animals varied from 19 to 30 days, the average was 25 days; the experimental mice lived 25-37 days for an average survival time of 31 days. This work of Zakrzewski was repeated by Balazs and Holmgren ( 1949 ), and they obtained similar results. In one of their experiments they used 150 mice, injecting 0.3 mg. of sodium heparinate intraperitoneally in 50 of them, a similar amount of another mucopolysaccharide in each of another batch of 50 animals; the remaining 50 mice served as controls. The survival both of the heparin-treated mice and of the mice treated with the other mucopolysaccharide was signifi-cantly better than that of the controls. However, in 1952 at the Roscoe B. Jackson Memorial Laboratory in Bar Harbor, Kreisler, interested in heparin as a result of the work of Heilbrunn and Wilson, attempted to discover whether this anticoagulant would influence the growth of a tumor graft. Apparently unaware of earlier extensive attempts to answer this question, Kreisler did an experiment on only 15 mice, made no measurements, followed the growth of the tumors merely by "manual palpation," and could find no effect of the heparin. In my laboratory, we have been able to confirm both that heparin has a strong inhibiting effect on the growth of tumor cells in tissue culture, and also that the growth of tumors in vivo is retarded by heparin. Bocher (1952), in an unpublished master's thesis, showed the effect of heparin on tissue cultures of tumor cells, and Muriel Lippman has obtained strong evidence that heparin has a marked effect on tumor growth. What Miss Lippman did was to implant ascites tumors into mice, and then subsequently inject 0.1 per cent heparin intraperitoneally. Such injections lowered the mitotic index of the tumor cells, and they also had a marked effect


in inhibiting the growth of the tumors (Lippman 1955). It is easy to measure the size of an ascites tumor by determining the volume of ascites fluid. In Miss Lippman's experiments, there was a decided difference in the volume of the tumors in the control animals and the tumors of mice which had for several days received injections of small amounts of heparin. The control mice died with huge tumors; the animals that had re-ceived heparin injections died also, but the tumors in them were sometimes so small as to raise a question as to the cause of death. Was it due to the tumor, to heparin, or to some secondary cause? There is a possibility that after the tumors have grown in mice for a time, they might produce sufficient protein to induce anaphylactic shock. If this were the case, some agent which would prevent such shock might help to prolong still more the life of the cancerous mice treated with heparin.

Heparin, or some substance very like heparin, might also be a factor in the antimitotic action of roentgen radiation on cancer cells. Of all known antimitotic agents, roentgen radiation is still the best and of course the most widely used agent in the treat-ment of cancer. A strange fact about the action of X-rays is that if one tumor of an animal is irradiated, another tumor at some distance from the first and not directly exposed to the rays may also show regression (Hevesy, 1945; Holmes, 1949). Obviously, in order to produce this effect, some substance, presumably with antimitotic action, must be given off from the irradiated tissues. This substance could well be heparin or a compound similar to heparin, for according to Allen and his collaborators ( 1947, 1948, 1951), when dogs are exposed to whole body irradiation, heparin, or some substance that acts like it, is liberated into the blood stream. The direct action of roentgen radiation on dividing cells might also involve heparin. This is a conclusion reached by Wilson (1950). He irradiated sea urchin eggs, and then after they were fertilized, he followed the colloidal changes occurring in them. In eggs given 10,000 roentgen units, the mitotic gelation proceeded as rapidly as it did in the control (nonirradiated) eggs. But subsequently to this, the liquefaction that follows the mitotic gelation was markedly delayed. This is shown in Fig. 53. The


delay in the liquefaction may be due to the fact that as a result of the irradiation heparin has escaped from the eggs. Actually irradiation may cause a breakdown of heparin-protein combina-tions (Kelly, 1951). Also it should be noted that sea urchin eggs are rich in heparin; much of it is released from the egg at

300 ρ-

ω °.200

U J

>

κ 100h-

12 18 24 30 36 42 48 54 60 66 72 78 84 90 M I N U T E S A F T E R F E R T I L I Z A T I O N

F I G . 5 3 . Viscosity of the protoplasm of Arbacia eggs irradiated with

1 0 , 0 0 0 r and then fertilized. T h e dotted line shows the viscosity of the

control, untreated eggs ( the final rise in viscosity at c leavage was not

p l o t t e d ) . The solid line shows the viscosity of the irradiated eggs .

the time of fertilization, for it is known to constitute the essential part of the so-called fertilizin ( see Immers and Vasseur, 1949 ).

In studying the antimitotic effect of heparin on marine eggs, it was soon found that heparin itself has a much less potent action than some substances of a heparin-like nature ( see below ). This is presumably correlated with the fact that heparin has a large molecule and enters cells with some difficulty. If we could find substances which act like heparin, but which enter cells more readily either because they have a lower molecular weight or because they are more lipid soluble, such substances should prove to be more potent antimitotic agents.


If our general theory of the protoplasmic colloid is correct, if all cells contain substances which favor protoplasmic clotting as well as those which inhibit or retard it, then by proper extrac-tion methods we should be able to obtain substances of the sort required. If a wide enough range of living materials could be tested, it might indeed be possible to find substances of some use in cancer therapy.

That there is hope in this direction is indicated by the early work of the physician Coley in and around New York City (see Nauts, Swift, and Coley, 1946; Nauts and Coley, 1947; Nauts, Fowler, and Bogatko, 1953). For many years Coley treated cancer patients with extracts of various bacteria, and beyond any doubt he was able to obtain some remarkable cures. One of the bacteria useful in providing potent extracts was Serratia marcescens. From this bacterium, Shear and his associates at the National Cancer Institute were able to extract a substance which has a marked effect in causing regression of tumors in mice, a substance which they identified as a polysaccharide (Shear and Turner, 1943; Hartwell, Shear, and Adams, 1943). Shear's polysaccharide causes hemorrhages to occur in the tumors, and this behavior can certainly be correlated with the fact that it acts like heparin in preventing the clotting of the blood.* Actually, as Most (1951) showed, it is much weaker than heparin in its effect on blood clotting. But it is much more powerful than heparin in its effect on protoplasm. Heilbrunn and Wilson ( 1950a, b ) in studies on the Chaetopterus egg found that Shear's polysaccharide in dilute solution (0.15 per cent) could inhibit cell division. Such concentrations completely prevented the mitotic gelation and kept the protoplasm fluid. Actually Shear's preparation of his polysaccharide contains protein and lipid as well as polysaccharide (see especially Rathgeb and Sylvén, 1954a, b ) . The lipid constitutes 11-15% of the whole. An even higher percentage of bound lipid is found in a preparation from Escherichia coli, a preparation which also causes hemorrhage and

* Substances similar to heparin m a y act m u c h more strongly than heparin

itself in causing leakiness of b lood vessels. This is a subject that needs further

investigation.


regression of mouse tumors (Ikawa et al., 1952). It is quite likely that the lipid constituent of these extracted substances enables the potent polysaccharide to enter cells. In work on similar substances from ovaries (see below), there is an indica-tion that pure preparations of polysaccharides are less potent as antimitotic agents than preparations from which all of the protein and lipid have been removed.

Although Shear's polysaccharide has been used clinically, it is more toxic for men than it is for mice (Holloman, 1947; Oakey, 1947 ). The strong possibility exists that there may be antimitotic polysaccharides that are relatively less toxic for human beings. Certainly a search for such substances is worth pursuing.

At the University of Pennsylvania and at the Marine Biological Laboratory in Woods Hole a small group of investigators, mostly graduate students, has for a few years been actively engaged in this type of work. Starting with the thesis that protoplasm, like blood, is poised in a delicate balance between the factors that tend to cause clotting and those that tend to prevent it, we began a search for naturally occurring substances that would keep the protoplasm fluid and inhibit protoplasmic clotting. As a test material we used eggs of the worm Chaetopterus. As Figure 49 shows, when these eggs are fertilized, preparatory to the appearance of the mitotic spindle there is a sharp increase in viscosity. At 21° the protoplasm is relatively viscous for the period between 30 and 40 min. after fertilization. In testing for anticlotting and antimitotic substances, we made centrifuge measurements of the protoplasmic viscosity at this time. We were interested not in any and all substances, which by virtue of a toxic action of one sort or another, prevented cell division, but only in those substances which behaved like heparin and Shear's polysaccharide in keeping the protoplasm fluid and preventing the mitotic gelation.

Early in our search we discovered that the ovaries of various invertebrates were rich in the type of substance we were looking for. Ovaries of sea urchins, sand dollars, starfish, worms, clams, and lobsters all contain anticlotting, antimitotic substances. That ovaries should be rich in antimitotic substances is obvious from


the fact that eggs remain in the ovary for long periods of time without undergoing mitotic divisions. In very many cases, as soon as an egg is released from the ovary, it begins a maturation division. This is true of eggs from many diverse types of organ-isms, both vertebrate and invertebrate. Of the various inverte-brate ovaries we studied, the starfish ovary gave us the most powerful extracts, and we first concentrated our attention on these extracts.

An extract from starfish ovaries, made simply by allowing the cut-up ovaries to stand in acidified sea water, will stop cell division in both Chaetopterus and Arbacia eggs (Heilbrunn, Wilson, and Harding, 1951; Heilbrunn et al., 1954). More potent extracts can be obtained if the ovaries are homogenized before being extracted and these extracts, crude as they are, can be diluted at least 200 times and will still prevent cleavage in 100 per cent of the Chaetopterus eggs exposed to them. The extracts from the starfish ovary exert a very strong liquefying effect on the protoplasm, and this effect is not only on the interior proto-plasm, which is prevented from undergoing gelation, but also on the cortex. If the potent substance present in the extracts is present in high concentration, as in undiluted extracts, the cortex is so completely liquefied that the egg is destroyed. The active substance in the extracts is apparently an acid polysaccharide, and is thus akin to heparin. The evidence for this is many-sided; some of this evidence will be presented briefly, additional details are in the original papers. The extract is strongly metachromatic, but the metachromatic reaction disappears in the presence of sufficient protein, just as the metachromatic reaction of heparin does (Kelly, 1951, 1955). As Dunn has shown in some as yet un-published experiments, if the extract is salted out with varying concentrations of ammonium sulfate, the activity appears in the globulin fraction. If this fraction is resuspended in dilute sodium chloride solution and digested with trypsin, the resultant solu-tion is metachromatic and (after boiling to destroy trypsin) shows an anticoagulant action on sheep plasma. It also prevents cell division in Chaetopterus eggs. The active substance is heat stable; although when combined with protein, its activity


is lost after an exposure to 80° C. An important point is that the activity is destroyed by periodate. The dialysis behavior of the extract is similar to that of heparin. The extract gives an ultra-violet absorption spectrum similar to that of heparin. This is illustrated in Fig. 54a, in which the absorption spectra of heparin and two samples of dilute starfish ovary extract are compared. Unfortunately the ultraviolet absorption spectrum of heparin is not a very distinctive one. It is interesting to note that the general shape of the curves in 54a is rather similar to the curve obtained for the absorption spectrum of the substance previously men-tioned as being obtainable from extracts of Escherichia colt, a substance which causes regression of mouse tumors and which also contains polysaccharide combined with lipid. The absorption spectrum of this bacterial polysaccharide is shown in Fig. 54b.

Of all the liquefying, antimitotic agents we have tried on Chae-topterus eggs, the starfish ovary extract is certainly the most potent. It is effective also for sea urchin eggs, for eggs of the clam Spisula, and for starfish eggs. But it has no very great effect on frog eggs, nor does it have as strong an antimitotic action on embryo mouse cells in tissue culture as does ordinary commercial heparin. Thus it seems probable that the heparin-like substance in the starfish ovary extract is more potent on the protoplasm of invertebrate animals than it is on the protoplasm of vertebrates. This is borne out by the fact that although ordinary heparin stops frog heart in diastole, our starfish ovary extract is incapable of producing such an effect; on the other hand, the starfish ovary extract does stop the clam heart in diastole. Thus, obviously, the starfish extract is not very promising as a cure for cancer.

We were led then to inquire into the possibility of obtaining extracts from the ovaries of vertebrates, extracts which would perhaps act on mammalian cells. First we made extracts of the ovaries of various fishes. We did this because it is known that the ovaries of some fishes contain very potent substances which appear to be similar to the active substance of the starfish ovary extract. There is a vast literature on the poisons of fish ovaries, a literature that goes back several hundred years. Some of the key references to this literature are given by Heilbrunn et al., 1954;


F I G . 5 4

( a ) Ultraviolet absorption

spectra of heparin and t w o

samples of starfish ovary extract.

T h e open circles show the ab-

sorption spectrum of sodium

heparinate; the c losed circles

and the triangles show the ab-

sorption spectra of the starfish

ovary extracts ( Hei lbrunn et al.,

1 9 5 4 ) .

0.65

0.55

0.45

0.35

0.25

0.15

0.05 240 260

Wave length, πιμ

300

( h ) Ultraviolet absorption

spectrum of the active sub-

stance from an extract of

Escherichia coli (Ikavva et al.y

1 9 5 2 ) .


see also Yudkin, 1944, 1945). The most famous of the poisonous fishes are those belonging to the genus Tetraodon ( and the family Tetraodontidae ) ; these fishes are found in Japan, where they are called "fugu," and many Japanese have died as a result of eating them. The poison of the fugu is found especially in the ovary (although it is also present in the testis and the liver). Fugu poison or tetrodotoxin has been studied by various chemists who have attempted in one way or another to purify it. Doubt-less the different preparations are not identical one with the other. Chemists working for the Sankyo Company of Japan, a company which sells tetrodotoxin, have prepared a crystalline tetrodotoxin which has an amazingly high toxicity, but whether this represents the substance originally present in the fish ovaries is not certain. The earlier studies of fugu poison indicated that it was a carbohydrate of no very great molecular weight, containing both nitrogen and sulfur, and precipitable by alcohol. These studies showed also that it prevented the clotting of bird and mammalian blood, and that it stopped the frog heart in diastole, just as heparin does. Because of the similarity in the behavior of fugu ovary extract and heparin, we were led to expect that from the ovaries of tetraodont fishes, we might be able to obtain ex-tracts which would exert an antimitotic action.

This indeed proved to be the case. In 1953 Couillard began working on extracts of the ovaries of the American tetraodont, Sphaeroides maculatus, the common puffer or blowfish. So far only preliminary reports of his work have been published ( Couil-lard, 1953, 1954 ). From ovaries of the puffer, Couillard was able to obtain extracts which prevent cell division in starfish and Chaetopterus eggs. These extracts show anticoagulant activity, both for blood and for protoplasm, and they prevent mitotic gelation in Chaetopterus eggs.

If it is generally true, as we have suggested, that ovaries of various animals may be rich in anticoagulant, antimitotic sub-stances, then we might expect that other species of fish would also have ovaries containing such substances. This was indeed found to be the case. Up until the present, fourteen species of fishes have been investigated, all that were readily available at


Woods Hole. In every single instance, when the ovaries were extracted with acidified sea water, extracts were obtained which when neutralized had a marked effect in retarding or preventing division of Chaetopterus eggs. As was to be expected, some fish yielded more powerful extracts than others. Always the extracts kept the protoplasm of the eggs in a fluid condition; that is to say, they prevented mitotic gelation. So far, only a preliminary report of this work has appeared (Heilbrunn, Wilson, and Lippman, 1953 ). The fact that extracts of all the fish ovaries we examined acted in the same way as extracts of the puffer ovary led us to wonder if there might not be evidence of the toxicity of ovaries of fishes other than those belonging to the family Tetra-odontidae. Such literature there is, and it is very voluminous; it goes back 400 years. An interesting older book on the subject is that of Autenrieth, published in 1833. We rather suspect that the ovaries of most fish have toxic substances in them, but that these ordinarily are incapable of passing through the wall of the intestine.

Nor is the presence of anticlotting, antimitotic substances con-fined to the ovaries of fishes. They were found also in the ovaries of frogs and salamanders, and in the ovaries of dogs, cows, pigs and sheep. Indeed in every case in which the ovary of an animal, invertebrate or vertebrate, was extracted with acid sea water, we were able to obtain substances which showed the behavior we were looking for.

The fact that the ovaries of all sorts of animals are rich in anti-mitotic substances suggests that perhaps one or another of these substances might be of use in the treatment of cancer. At the present time, with the aid of a skilled chemist, Dr. Robert J. Rutman, we are investigating the possibility of obtaining from cow ovaries substances which will tend to prevent cell division in mouse tumors. The work has not progressed very far, and as yet only rather crude extracts have been studied, but the indica-tions are that we can obtain from these ovaries substances which act like heparin in lowering the mitotic index in tumor cells and in lengthening the survival time of mice into which ascites tumors have been implanted. With better extracts from cow ovaries and


perhaps also with extracts from the ovaries of other large mam-mals, we should be able to obtain substances which act more strongly than heparin in controlling the growth of tumors.

Nor are ovaries the only possible source of antimitotic sub-stances. If our basic thesis is correct, all types of living material should contain them. An interesting source is muscle. In the chapter on muscular contraction (Chapter 7), it was pointed out that when frog muscle is stimulated to fatigue, it gives off to the surrounding medium, heparin or a heparin-like substance. Hence it might be expected that the fluid bathing fatigued mus-cles would have antimitotic properties. And indeed it does. See Heilbrunn and Wilson ( 1955c ). If Chaetopterus eggs are used as test objects, it is simpler to use muscles of an animal like the lobster, for the medium surrounding lobster muscles is osmoti-cally not very different from sea water. However, lobster muscle cut out of the body into sea water does not behave well. Ac-cordingly the tail muscles of lobsters were stimulated repeatedly by inserting electrodes into the base of the tail. After about fifteen minutes, the muscles were thoroughly fatigued and no longer responded to stimulation. They were then cut out into sea water, teased apart, and allowed to remain in contact with the sea water for thirty minutes. Control muscles from a lobster not exposed to electrical stimulation were given similar treat-ment. Then the antimitotic potency of the sea water which had stood over the fatigued muscles was compared with the anti-mitotic potency of the sea water which had stood over the non-fatigued muscles. Both the sea water from the fatigued muscles and that from the control muscles showed an antimitotic effect, but this was much more marked in the case of the fatigued mus-cles. Thus the sea water from the fatigued muscles could be diluted two or even four times and still produce as great an effect as the undiluted extract from the control muscles. In six experiments in which the sea water from fatigued muscles was tested, in four cases it completely prevented cleavage, in the remaining two cases cleavage occurred in only 1 and 2 per cent of the eggs. On the other hand, the control sea water from nonfatigued muscles in the six experiments on the average per-


mitted 54 per cent cleavage. The difference thus is very striking. Similar results were obtained with frog muscles. At first these

were fatigued in frog Ringer's solution and then this was evapo-rated down so that its tonicity was like that of sea water. But such evaporation is difficult to control accurately. Hence another technique was employed. The heart was cut out of a frog and then all the leg muscles were stimulated. These were then cut out and extracted in sea water, and the extracts compared with those from control nonfatigued muscles. In three experiments of this sort that were tried, it was clear that the fatigued muscles gave off about twice as much antimitotic substance as did the control muscles. Thus the results both with frog muscles and with lobster muscles indicate that when a muscle is stimulated to fatigue, it can give off substances which prevent the division of cells. Moreover, centrifuge tests showed that the sea water which had been in contact either with the lobster muscles or with the frog muscles in all cases tended to keep the protoplasm fluid. This effect was decidedly more pronounced when the sea water had fatigue substances in it.

If tired muscles give off substances which prevent cell division, then it might be thought that vigorous exercise would cause a decrease in the rate of cell division in the intact animal. This appears to be the case. In 1948 Bullough, after noting that the amount of cell division in the epidermis of mice was less during periods when the mice were active, placed mice in a revolving box which forced them to remain active. In such mice, the num-ber of cell divisions in the epidermis was much less than it was in the controls. This is illustrated in Fig. 55. The mice which were exercised from 11 o'clock in the morning until 5 o'clock in the afternoon showed very much less cell division than did the controls. Bullough in interpreting his experiment concluded that it "seems to indicate that excessive exercise, or heightened metabolic rate, results either in the production of a mitosis-depres-sing substance which takes some hours to be eliminated, or in the using up of some mitosis-stimulating substance which takes some hours to reform in sufficient quantity." Our work would seem to be more consistent with the first of these two possibilities.


This opinion is buttressed by the fact that Bullough and Green ( 1949 ) found that when mice were exposed to tourniquet shock, the mitoses in the epidermis steadily decreased in number until after some hours there was no cell division at all. This can easily be interpreted in the light of our knowledge of shock, for as shown in Chapter 9, in a state of shock, heparin or heparin-like

substances appear in the blood, and these could prevent cell division.*

The fact that muscular activity of an animal lowers the rate of cell division in its tissues would seem to indicate that muscular exercise might exert an antimitotic effect on tumor cells, and that exercise would exert a retarding effect on the growth of a tumor. An attempt to show this was made by Heilbrunn, Hala-ban, and Wilson in 1952. They divided cancerous mice into two groups; one group was put in cages provided with exercise wheels, the other group were in cages without exercise wheels. When mice were given an opportunity to exercise on the wheels,

* However , Bul lough and Green bel ieve the effect to be due to a drop in

metabolism.

F I G . 55 . Number of mitoses in the ear epidermis of exercising mice as

compared wi th the number in control mice . T h e mitoses counts were m a d e

in sections 1 cm. in length.


10

g 8

I 6

<υ i 4

ζ 2

0 4 8 12 16 20 24 28 32 36 40

Days

F I G . 5 6 . T h e effect of exercise on the survival t ime of

cancerous mice .

this tumor when the mice were made to exercise and when they were not. In the mice that had had the exercise the tumors did not grow as large as they did in the controls. Rusch and Kline believed that these results were due to a lack of nutriment and they cite older experiments to support the view that a limita-tion of food can restrict tumor growth. It might perhaps be true that rather vigorous exercise would benefit cancer patients; this would bear looking into.

All in all, the facts that we now know about the suppression of cell division can readily be interpreted in terms of the general theory of the initiation of cell division as we have outlined it. Thus there is a rational basis for the study of the chemotherapy of tumors in terms of a search for agents which will properly influence the colloidal properties of the protoplasm. This is a line of research that has not to any great extent been followed in the

their survival time was a little longer. The results of one such ex-periment are shown in Fig. 56. More striking results were ob-tained by Rashkis (1952). Instead of merely allowing his can-cerous mice to exercise when they so desired, he forced them to exercise by making them swim. There is also an older paper of Rusch and Kline (1944). They studied mice which had been implanted with a fibrosarcoma and they measured the growth of


past. Much more effort has been expended in an attempt to block various metabolic pathways, and as a result our knowledge of the course of metabolism has improved. There is also hope that metabolic studies may provide useful practical information that will help cancer sufferers. But as yet, in spite of glowing reports to the press by professional promotors and press agents, no outstanding success has been obtained.

From a theoretical standpoint, it would be interesting to know why blocking this or that metabolic pathway would prevent cell division. There is a large literature on the effect of pteroylglu-tamic (folic) acid antagonists in causing stoppage of cell division, but how this effect is produced is not very clear, at least not to the biologist or cell physiologist concerned with the mechan-ism of cell division. In view of the fact that proteolytic enzymes may act as clotting agents and so cause mitotic gelation, it is interesting to note that according to Guest, Ware, and Seegers (1947), pteroylglutamic acid deficiency is accompanied by an increase in antifibrinolysin activity. Fibrinolysin is a proteolytic enzyme found in many types of cells; thus folic acid deficiency may act by tending to inhibit those protease enzymes which cause the mitotic gelation.

What we need is more knowledge on the why and wherefore of the suppression of cell division. If such knowledge were to be gained, the study of the chemotherapy of cancer might be less haphazard and more successful.

12.

The question of how protoplasm is excited or aroused and how such excitation can be harmlessly prevented is as funda-mental as any question the biologist or physiologist has to face. For if we knew the answer to this question we would be in a position to understand a great deal about the dynamics of living protoplasm, the mechanism of the vital process.

In early chapters we have from time to time had to consider the nature of excitation in one type of living system or another. For we could scarcely approach the problem of muscular con-traction, of nerve response, or of cell division without inquiring into the reason why the muscle is induced to shorten, the nerve to respond, or the cell to divide. In this chapter we shall try to consider not merely one type of living tissue or another, but all living material, in the hope of arriving at a correct general theory, a theory which would apply to all types of irritable protoplasm.

Is such a general theory possible? Apparently it is, for in widely diverse types of living cells and in widely different organ-isms much the same agents act as stimulants and much the same agents prevent a response to stimulation. And there are similari-ties also in the manifestations of the excitatory process. This will become clearer as we proceed.

In our discussion we shall first present in broad outline the facts that are known about stimulation and anesthesia. Beyond any doubt it is necessary to understand this factual knowledge in order to interpret it. This seems obvious. And yet time and again in recent years, chemists with scarcely any knowledge of the elementary facts about stimulation and anesthesia have not hesitated to offer theoretical interpretations of these processes. Unfortunately these theories have been taken too seriously by

217

STIMULATION AND ANESTHESIA


biologists and physiologists, for we are apt to be too much impressed by highly technical language we do not understand. In order for a chemist to explain anesthesia, no matter how ex-cellent a chemist he is, he must know what it is he is trying to explain.

First as to definitions. These are difficult. In the past, among general physiologists, leading authorities in the field of stimula-tion and anesthesia have been uncertain as to what they would call stimulation, excitation, response, anesthesia, and narcosis. Actually the working physiologist, for any particular experiment, is never very much at a loss in his use of terms. It is only when an attempt is made to provide definitions that will cover all pos-sible types of living material and all possible conditions that difficulties are encountered. So, for example, what do we mean when we talk about the anesthesia or narcosis of a bacterial cell? It is very easy to say that all protoplasm is irritable. This is a concept we teach to beginning students of biology, and these beginners can glibly repeat what we have taught them. But what is it that we mean? It is indeed hard to say. For without more knowledge than we now have, what do we know about the response of the epithelial cell of an animal or of the chlorophyll-containing cells in the leaf of a plant?

It is safe to say that when most physiologists think of stimula-tion and response, they are thinking of living systems in which the response is easily observed and measured. There can be no question about the response of a muscle or a nerve. Also it is clear that the same agents which cause the easily studied response of nerve and muscle have a comparable effect on some other living systems. Consider an ameba, for example, or a plant cell in which the protoplasm is streaming around a central vacu-ole. The movement of the ameba, hither and yon, is going on rather constantly. There may be internal stimuli involved but we do not know what they are. But if an ameba is subjected to a sudden mechanical impact, if it is exposed to an electric shock or to the rays from an ultraviolet lamp, the movement stops and the ameba tends to round up. Thus the types of stimu-lating agents which cause a response in nerve and muscle also

12. STIMULATION AND ANESTHESIA 219

make the ameba respond, but instead of making it move, they stop its motion. The same sort of phenomenon occurs in the streaming protoplasm of plants. Take a Nitella cell, for example. This is a long, cylindrically-shaped cell. In it the protoplasm travels round and round incessantly. But expose the Nitella cell to sudden heat or cold, to electric shocks, to mechanical stimula-tion, or to potassium chloride, and the movement will stop. More-over, at the point where these stimulating agents act, the proto-plasm becomes negative. This has been repeatedly demonstrated by Osterhout and his co-workers (see, for example, Osterhout, 1931; Osterhout and Hill, 1935). In the diagrams shown in Fig. 57, a stimulus applied to the cell at a point (A) causes negativity

Excited Resting

Stimulus remcwe

A-

"Recovered Excited Resting F I G . 57 . Diagram to s h o w arrangement for the s tudy of potential

differences in the cell of Nitel la ( O s t e r h o u t ) .

there. When the stimulus is removed, the electric disturbance moves along the cell; the stoppage of movement in the Nitella cell is truly a response, for the agents which cause it are, in general, the same as those which arouse a nerve or a muscle and, moreover, the electrical changes induced by these stimuli are the same. Indeed, for many years it has generally been recognized by capable physiologists that in the case of the proto-


plasmic streaming or cyclosis of plant cells, stimulation causes stoppage and an electrical action potential, and anesthesia is the prevention of such response ( compare Osterhout, 1933, Osterhout and Hill, 1933). And yet papers have appeared in scientific journals in which authors, apparently ignorant of older literature and concepts, have regarded the stoppage of streaming or cyclosis as anesthesia. Such misconceptions can only lead to confusion. There aie also other instances in which the concept of anesthesia has been perhaps loosely used. A resting cell is at all times undergoing oxidative processes. If these oxidative processes are interfered with, can the resultant depression of oxidation be con-sidered as a type of anesthesia or narcosis? Arguments might be presented either way and, of course, any investigator can make his own definitions; but, in general, in irritable living systems which can be best studied and best understood, anesthesia or narcosis is usually regarded as the prevention of excitation. And a mere slowing or stopping of respiration or a shift in metabolic pathways can scarcely in the strict sense be regarded as anesthe-sia. Hence it is perhaps unwise to consider the stoppage of luminescence in a bacterium as evidence of true anesthesia, for this type of bioluminescence does not occur as a response to stimulation and it is essentially a manifestation of an oxidative process proceeding along a certain pathway.

In what has gone before, I have used the term anesthesia as general physiologists have been in the habit of using it for many years. This is not the way the word is used by medical men. Thus a standard medical dictionary defines anesthesia as "loss of feeling or sensation" and narcosis as "a state of profound unconsciousness." And Harris ( 1951 ) in his recent book on "The Mode of Action of Anaesthetics" states that anesthesia "means the abolition of conscious sensation, and perforce should be ap-plied exclusively to the higher forms of life." If the medical dictionary definitions were to be followed, we general physiolo-gists would be deprived of any word to describe what for so many years we have referred to as anesthesia. And, on the other hand, if we were to follow Harris, we would have to give up the word anesthesia and use only narcosis. Some writers, impressed


by the medical point of view, have done this. Harris defines narcosis as "the reversible depression of the autonomic activity of cells which is produced by narcotic drugs." This definition seems to show a lack of understanding of the point of view of the general physiologist and is scarcely acceptable.

Perhaps the greatest physiologist of all time was Claude Bernard. It was he who first emphasized the fact that anesthesia was a universal phenomenon, not confined to the brain and the nerves. In 1875 he wrote: "Le anesthésique nest donc pas un poison spécial du système nerveux; il anesthésie tous les tissus en engourdissant, en arrêtant momentanément leur irritabilité." Thus according to Claude Bernard, anesthetics affect all living tissues and rob them of their irritability.

This is the concept of anesthesia that has been held by many general physiologists, including such authorities as R. S. Lillie and Osterhout. It should, however, be pointed out that in his thinking Claude Bernard did not always adhere strictly to the concept outlined above, or at least his conception of irritability was not very clear. For at times he used the word anesthesia in a broader sense and he regarded it as involving not merely a loss of irritability but a temporary inhibition or retardation of some vital function such as photosynthesis or fermentation. In order to define our terms, we shall therefore speak of anesthesia in a strict sense as involving a loss of irritability, a reversible loss of the power to respond to a stimulus; and anesthesia in a broad sense as being the reversible loss or weakening of some vital function. In this broad sense an inhibition of respiration or bioluminescence could be regarded as anesthesia. It would be well if we had two different words to define the two types of anesthesia; but as in so many instances, our language has not been able to keep up with our scientific thinking and we are, moreover, handicapped by the fact that the words we use in physiology have already taken meanings in common speech. As we come to learn more about vital processes we may find that the distinction between anesthesia in the strict sense (or true anes-thesia) and anesthesia in the broad sense is not as sharp as it may at first sight appear to be. For even such processes as


photosynthesis and fermentation can be conceived of as involving a response to internal stimuli. But for the present, and as long as we have no concept of what such internal stimuli may be, it seems wisest to define our terms as we have done. True anes-thesia, the reversible prevention of response, is a general phenom-enon which can be studied in many different types of living matter. If we learn to explain it, we may then go on to consider why other vital functions such as metabolism, functions not usually dependent on external stimuli, can be reversibly retarded or inhibited.

After this lengthy semantic digression, let us now inquire into the nature of the agents which arouse or excite protoplasm and the nature of the agents which prevent such excitation. In earlier chapters we have discussed the types of effective stimuli which cause muscle cells to contract, nerve cells or nerve fibers to become aroused, or egg cells to divide. By and large, there is a basic and fundamental similarity in the stimuli that are effective for these different types of living material. And, as already noted, the same stimulating agents provoke a response in an ameba or in the streaming protoplasm of a plant cell. Mechanical impact, an electric shock, cold, heat, and ultraviolet radiation all typically provoke a response in protoplasm. Visible light is rarely effective, although in special cases the protoplasm may have pigment which absorbs the visible rays and is sensitive to them. Various chemical substances can also act as proto-plasmic stimulants; hypertonic solutions are usually effective, also the potassium ion. There is a rather variable sensitivity to other chemical agents and this is due, in part, to the variable permea-bility of different types of cells. Thus calcium ion, if it can enter a muscle cell, will make it contract, and some types of smooth muscle respond in this way; on the other hand, striated muscle is relatively insensitive, for its plasma membranes do not allow the calcium ion to enter. In general, it can be said that the agents which act as stimuli on one type of living material are usually effective on other types of living material, although, of course, each type of material has its own characteristic sensitivity.

As to the anesthetic agents, much has been written. This is


partly due to the fact that various types of anesthetics are useful in surgical operations and are, therefore, of importance both from a clinical and from a commercial standpoint. Year after year new anesthetics are discovered. Moreover, chemists and chemically-minded physiologists have been interested into inquiring into the chemical nature of substances with anesthetic action in the hope of learning from the structure of these compounds why it is that they act as they do. As was to be expected, it has been possible to show that certain types of molecular configuration lead to more powerful anesthetic action. There is a vast literature in this field, a literature that is discussed in books on pharmacology and in books on anesthesia. Long ago it was shown that in a homologous series of alcohols, increase in number of carbon atoms causes an increase in potency. This rule applies not only to alcohols but to other types of organic compounds as well. There are also other well-established facts. Compounds with branched-chain struc-ture are usually less active than those with straight chains. The substitution of a halogen (chlorine, bromine, or iodine) for a hydrogen atom increases the potency of an anesthetic.

Many different types of organic compounds have an anesthetic action. A partial list includes alcohols, aldehydes, ketones, esters, barbituric acid derivatives, carbamates, and alkaloids. What do all these compounds have in common and why do some of them have a greater potency than others? These are questions that have worried chemists and pharmacologists for many years. Ob-viously all the various organic substances that can produce anes-thesia are not similar in their chemical constitution. But from a physicochemical standpoint, they may indeed be similar in at least one respect. They all either dissolve fat or tend to become dissolved in fat and if their anesthetic potency is stronger, the stronger their affinity for fat. This is the basis of the famous Meyer-Overton theory, a theory that was developed independent-ly by H. H. Meyer and E. Overton at the turn of the century. Support for the Meyer-Overton theory comes from the fact that if one determines the partition coefficient between oil and water of various organic compounds, their anesthetic potency runs parallel to their tendency to dissolve in oil rather than in water.


Moreover, if as the temperature is raised the relative solubility in oil increases, then the anesthetic potency rises with increas-ing temperature; and if the oil solubility decreases with rising temperature, the anesthetic potency drops. More recently Fergu-son (1939) and Brink and Posternak (1948) have found a cor-relation between the thermodynamic activity of various organic substances and their potency as anesthetics. Although at first sight this point of view is very impressive it does not represent any advance over the older ideas of Overton and Meyer, for, in general, the thermodynamic activity runs parallel to the lipid solubility and, according to Κ. H. Meyer (1951b), the correlation between anesthetic power and thermodynamic activity is not as close as the correlation between anesthetic power and lipid solubility.

The Overton-Meyer theory has had a wide influence and rightly so. But it must be recognized that it is in no sense a theory of anesthesia, for it makes no attempt to explain why the lipid solubility of a substance enables it to prevent a cell from responding to a stimulus. Rather the Overton-Meyer theory is a statement of which types of substances can act as anesthetics. And in this too, it is not a complete theory. For although it may be generally true that many substances which dissolve in oil are anesthetics, it is equally true that many substances which do not dissolve in oil are also anesthetics. So, for example, distilled water sometimes acts as an anesthetic; thus hypotonic solutions act like ether in preventing the division of sea urchin eggs and, indeed, in the past distilled water has sometimes been used for surgical operations (see below). And perhaps the most general of all anesthetics is the magnesium ion. There is no better anes-thetic for most invertebrates and in vertebrates also, magnesium is an anesthetic both for nerve and for muscle.

If we are to seek to interpret properly the action of anesthetic agents, it is important to have a clear idea as to just which sub-stances and which agents do cause anesthesia, and do prevent an irritable protoplasmic system from responding to stimuli. And in any survey of anesthetic agents it is essential that the facts be observed objectively without any regard to preconceived ideas


or theories. This has not always been done. Thus one prominent physiologist insisted that magnesium was not an anesthetic be-cause it was not a fat solvent and, indeed, there has been a tendency among students of anesthesia to look the other way when magnesium was mentioned.

Ordinarily, for nerve or for muscle, it is a rather simple matter to determine whether a given agent is acting as an anesthetic. The nerve or the muscle is exposed to the agent and then a stimulus is applied. If there is no response, and if then the nerve or muscle becomes irritable again after the agent is removed, we can conclude that the agent causes anesthesia. But it must be remembered that although a nerve or muscle may not respond to a stimulus of a certain strength, this does not mean that the tissue has become completely nonirritable. For if the intensity of the stimulus is increased, it may then be possible to get a response from tissues that do not respond at all to somewhat weaker stimuli. Rarely indeed have tests been made to show whether or not anesthetized tissues continue to be nonirritable when very strong stimuli are applied. Thus, from a practical standpoint, what we mean by anesthesia is a condition in which the threshold for stimulation has been markedly increased. If enough work were done, we might find that even some of our best anesthetics might not completely prevent response if strong enough stimuli were applied. Of course, by increasing the con-centration of an anesthetic substance, we could raise the thres-hold higher and higher, but death might intervene before the tissue became completely incapable of response. As a matter of fact, this difficulty is not a serious one, for if in the light of future developments it becomes necessary to change our defini-tion of anesthesia, we can always do so. If we please, we can define anesthesia not as involving a complete loss of irritability, but as constituting a state in which the irritability is decreased to such an extent that much more potent stimuli than those which are ordinarily effective are required to provoke a response.

There is another point which should perhaps also be con-sidered. In muscle and nerve, the irritable systems we know best, response to one stimulus prevents, for a brief time interval, a


second response to another stimulus. This is the well-known phe-nomenon of the refractory period. Thus in one sense the re-fractory period represents a state of anesthesia, and agents which prolong the refractory period could perhaps be regarded as ex-erting an anesthetic action. Muscle sometimes remains con-tracted for long periods, as in contracture or tonus, and in this contracted state it can scarcely show a response to stimuli. Our difficulty here is largely a semantic one and, as is usual in such cases, we can easily settle it by sharpening our definitions. For our purposes, we shall not consider as anesthesia a condition in which a second response is inhibited because of the fact that the tissue is actually responding or has not yet recovered from an earlier response. This sharpening of our definition is in accord with customary physiological usage.

The following list is not exhaustive, but it covers the main types of agents which can act as anesthetics:

1. Fat solvents. These include a wide variety of chemically diverse substances.

2. Magnesium salts. 3. Water. If hypertonic solutions are stimulating agents, it

might be expected that hypotonic solutions would have an anes-thetic action. As already mentioned, hypotonic solutions act as anesthetics for dividing sea urchin eggs. Water is also an anes-thetic for Nitella, and when Nitella cells are immersed in distilled water they are no longer able to respond to electrical stimulation (Osterhout and Hill, 1933). According to Braun (1924), it has been known since 1869 that water can anesthetize nerves; he states also that water has occasionally been used in minor surgical operations.

4. Pressure. The fact that pressure could relieve pain was known even to the ancients. During the middle ages, the Arabs made use of this knowledge in their practice of surgery. Then in the 16th century the famous surgeon Ambroise Paré again introduced this method of relieving pain and since then at various times pressure has been advocated as being useful in surgical operations. It is well known that pressure can act as an anesthetic on nerves. High pressures of inert gases can cause anesthesia.


5. Cold. In the middle of the 16th century Thomas Bartholi-nus, a student of the Italian anatomist and surgeon, Marco Aurelio Severino, introduced the use of cold as an anesthetic; he had learned about it from his teacher. Then in 1807 when Napoleon invaded Russia his surgeons noted that amputations in extreme cold involved no pain. Recently surgeons have again come to make use of cold anesthesia.

6. Heat. Strangely enough, heat as well as cold can act as an anesthetic. According to Winterstein (1905), heat anesthesia can occur in many different types of animals, from jellyfish to frogs.

7. Anticoagulants. In some special cases, substances which prevent the clotting of blood and protoplasm can prevent re-sponse to stimulation. Thus heparin can act in this way, although usually it is unable to enter cells rapidly enough to produce an effect. This action of heparin and similar substances has already been mentioned in Chapters 7 and 11.

The above list is not a complete list. Moreover, no attempt has been made to cite more than a few references to the literature. Some additional references are cited by Heilbrunn ( 1952b ) ; also useful information can be obtained from the older books on anes-thesia, for example, the books of Gwathmey (1924) and Braun ( 1924 ). It will be noted that some of the agents we have listed as anesthetics had previously been described as stimulating agents. As a matter of fact, almost all anesthetics can under certain conditions have a stimulating action. This is a highly important point and will be developed later.

In any discussion of the various ways in which a state of anes-thesia can be produced, it is perhaps pertinent to mention the fact that conditions resembling anesthesia may occur in the normal life of an animal, or as the result of an injury. In hiberna-tion, in sleep, and in shock, animals become relatively insensitive to stimulation. These three types of phenomena will now be considered briefly. In all of them, the depression of irritability comes as a result of physiological changes which may involve the brain, the endocrine glands, or the circulatory system. And yet, although the origin of the depressed irritability may be dif-


ferent in one case or another, the fact that in every instance there is an increase in the threshold of stimulation necessary to produce response indicates that there are changes in the protoplasm of the irritable tissues, changes which are like those that occur in a state of anesthesia. Thus a complete theory of stimulation and anesthesia should eventually be able to explain what happens to the protoplasm of animals when they hibernate, when they sleep, or when they are thrown into a state of shock. The evidence that we have now is rather scanty, but it does indicate that both in hibernation and in shock there are liberated into the blood substances which are potentially capable of exerting an anesthetic effect on the irritable tissues of the body. First let us consider hibernation. During the cold months of the year, many animals go into an inactive state. This is true both of coldblooded and warmblooded animals. In such hibernating animals, the blood is known to contain substances which have an irritability-depres-sing or anesthetic action. Thus in the snail, Helix pomatia, Lustig, Ernst, and Reuss ( 1937 ) found a marked increase in the magnesium content of the blood during winter sleep. In the hibernating hedgehog, the magnesium content of the blood is two or three times what it is in the summer ( Suomalainen, 1939 ). In the same animal during hibernation the blood clots much more slowly than it does when the animal is active, and this is ap-parently due to the presence of heparin in the blood (Suoma-lainen and Lehto, 1952). Likewise in ground squirrels and hamsters, the blood clots much more slowly during hibernation (Svihla, Bowman, and Ritenour, 1951; Svihla, Bowman, and Pearson, 1952). It may well be true that the lowered irritability of the tissues of hibernating animals is due to the greater amount of magnesium and heparin in their blood. Heparin enters most cells slowly, but during the time that an animal is entering the hibernation state, there should be time enough for the heparin to enter the protoplasm of the irritable tissues of the animal and help make it dormant. This could constitute an explanation of why the protoplasm behaves as it does, and that is our particular interest. What causes the heparin to be released into the blood is something else again. Cold itself is certainly a factor, and the


endocrine glands also seem to be involved, especially the islands of Langerhans.

If winter sleep is to be interpreted on the basis of the presence of anesthetic substances in the blood, can a similar explanation be given for ordinary sleep? We are not concerned with the ques-tion of how sleep is originally induced. Apparently some part of the brain is involved. But if sleep to some extent resembles anes-thesia and if the sleep induced by opiates and barbiturates is like ordinary sleep, it is not unfair to assume that in ordinary sleep also, anesthetic substances are involved. Unfortunately this problem has scarcely been attacked at all. Thus in a rather recent extensive review on sleep (Kayser, 1948) it is hardly mentioned. Some authors have found an increase in the magnesium content of the blood during sleep or at least an increase in the Mg—Ca ratio (compare Cloetta, Fischer, and van der Loeff, 1934; Hop-kins, 1935; Suomalainen, 1939). However, these changes are not great and it is doubtful whether they would be sufficient to account for the depression of irritability during sleep. Further work is needed.

When animals are thrown into shock, they respond only with difficulty and usually only to relatively strong stimuli. Thus in shock also there is a lowered irritability of the various tissues that respond to stimulation. In this case the explanation seems clear. For in an earlier chapter (Chapter 9) it was noted that when animals are in a state of shock their blood may contain relatively large amounts of heparin. This is certainly true of anaphylactic shock (Jaques and Waters, 1941) and it is also true of peptone shock (Wilander, 1939). In various other types of shock, al-though heparin may not as yet have been demonstrated in the blood of the shocked animals, the fact that the blood becomes noncoagulable leads to the suspicion that either heparin or some substance like it is present. Heparin or some substance that behaves like it may also be involved in the collapse that follows when a man is given a violent blow on the head (or the solar plexus). Much has been written about "head injury," but little is known about the reason why consciousness is lost as a result of it. According to Moreau (1948), if frogs are hit on the head,


their blood becomes relatively noncoagulable. In this case also, heparin or a heparin-like substance may be involved.

It has taken us some few pages to consider the various types of substances and the various physical agents that are capable of anesthetizing protoplasm, as well as to attempt an interpretation, in terms of known anesthetic substances, of certain physiological and pathological states in which irritability is depressed. In the next chapter an effort will be made to explain these facts in rela-tion to the demonstrable properties and behavior of the proto-plasmic colloid. But we shall turn now to a consideration of some of the effects of stimulation and anesthesia on living systems.

First it is hardly necessary to remind the reader that com-monly at the point where response occurs there is a drop in electric potential. This action potential has been much studied in nerve and muscle. It is discussed in Chapter 4. There must be a definite relation between the effect of the stimulus and the origin of the action potential. For many years cell physiologists and nerve physiologists thought that they had discovered the relationship. For in nerve there is clear evidence that when excitation occurs there is an increase in the permeability of the plasma membrane at the point of excitation. Such a change in permeability was postulated in the Bernstein theory of the action potential. As shown in Chapter 4, this theory can no longer hold. Nevertheless, there is an abundance of evidence to indicate that very often stimulation does cause an increase in the permeability of the plasma membrane. For many years Höber in Germany and R. S. Lillie in America urged that this increase in perme-ability was the primary factor involved in causing all sorts of irritable systems to respond and that anesthetics acted by pre-venting this permeability increase. However, in his later years Lillie abandoned the theory, and Höber also was reluctantly forced to realize that the theory could no longer fit all the facts.

In 1916, in arguing for the permeability theory, Lillie brought out the fact that the magnesium ion, well known as a permea-bility lowerer, caused anesthesia and prevented response in irrita-ble systems. But although it is true that the magnesium ion does anesthetize, it is equally true, and has often been shown, that the


ion that antagonizes magnesium and restores irritability is calci-um, which lowers permeability even more than magnesium does. And this fact Lillie completely ignored. The ability of Lillie ( and also at times Höber) to ignore evidence contrary to their theory is an example of what I have enjoyed calling the Nelson complex. For like Lord Nelson at the battle of Copenhagen, physiologists sometimes have a blind eye which makes it possible for them not to see what they do not want to see. In addition to the fact that calcium can act as a stimulating agent, there are also various other arguments that can be used to disprove the perme-ability theory of stimulation and anesthesia. Thus it would be hard to understand why both cold and heat could of themselves both cause increased permeability, and yet both are stimulating agents (as well as anesthetics). Moreover, although Winterstein in 1926 in his remarkable book on anesthesia leaned toward the permeability theory, evidence since then indicates clearly that fat solvent anesthetics instead of lowering permeability, as the theory requires, often have the opposite effect (Höfler and Weber, 1926; Jacobs and Parpart, 1937; Bärlund, 1938). But our main point at this time is that excitation is often (although not always) accompanied by an increase in the permeability of the plasma membrane. And, as we shall see later, from a colloidal standpoint there is good reason why this should be so.

Secondly, it is clear that, generally speaking, response to stimulation is accompanied by an increase in respiration. Actual-ly öiis is one aspect of response. A muscle which is contracting uses more oxygen and gives off more carbon dioxide than a resting muscle; it also produces more heat. Similarly an excited nerve has a higher rate of metabolism than a resting nerve and gives off more heat. And, in general, apparently, although perhaps not always, a tissue when it is aroused has a higher rate of metabolism than when it is in a quiescent state. Clearly then, the response to stimulation must be able in one way or another to set off a mechanism which will increase the oxidation rate.

Many physiologists who have thought about stimulation and anesthesia—and which physiologist has not—have believed that the primary effect of stimulation, the essential causative effect,


is to increase the rate of metabolism, and the primary effect of anesthetics is to lower the rate of metabolism. This is a theory that it is easier to hold for anesthesia than it is for stimulation. For how conceivably could both cold and heat, acting as stimu-lants, directly increase the rate of action of metabolic enzymes? To explain response in terms of one of its manifestations seems illogical, and is almost certainly a confusion of cause and effect. With respect to the action of anesthetics, there is perhaps more reason to suppose that various fat solvent anesthetics owe their effect to a direct action on the respiratory enzymes of the cell. This is an idea that is, or at least has been, commonly accepted by biochemists, and it has been accepted the more readily be-cause they have had too little understanding of the physiological phases of the problem. The fact that an anesthetic like paral-dehyde causes a 3 per cent drop in oxidations can scarcely ex-plain its anesthetic action. For if the Qio of the oxidation reac-tions of the respiration of a cell is between 2 and 3, and if we assume an average Qio of 2.5, this would mean that a drop of temperature of a single degree Centigrade would cause a de-crease in the rate of respiration of about 10 per cent, and no one would ever claim that a drop of a degree in temperature could act as an anesthetic. The leading student of the effect of anes-thetics on cellular oxidations has been Quastel, and his work more than any other has had a marked influence on those who have thought about the reasons why anesthetics work. When Quastel found that ether in anesthetic concentration had no effect on the oxidation of sugar by brain tissue (Jowett and Quastel, 1937) he was able to turn his blind eye to this contradiction to his theory; but later, because of the fact that morphine had no effect on cell oxidations, Quastel (1951) gave up his oxidation theory of anesthesia—only to return to it again in 1954.* (Ghosh and Quastel 1954). Actually older authors, for example, Vernon

* In this later work, Ghosh and Quastel find a greater effect of anesthetics

w h e n the cells have b e e n aroused. This is only logical, for if, as shown in the

next chapter, the colloidal changes involved in excitation, may produce an

increase in oxidative processes, then the prevention of excitation and the colloidal

changes that accompany it wou ld tend to keep the rate of metabol ism low.


(1912, 1914), had shown that at least one protoplasmic oxidizing enzyme, an enzyme which we would now call cytochrome oxi-dase, was not affected by anesthetic concentrations of various fat solvents. And, indeed, the very first student of the effect of anesthetics on cells, Claude Bernard, showed clearly enough that the anesthetics do not act because of any direct action on the enzymes of a cell. Bernard (1875, 1878-1879) was interested in knowing why the fermentation of yeast stopped in solutions of fat solvent anesthetics. And so he tried the effect of these anesthetics on fermenting enzymes which he had extracted from yeast cells. In anesthetic concentration the fat solvents had no effect on the enzymes, and Bernard concluded that the slowing or stoppage of the fermentation reactions in the living cell must in some way be due to an action of the anesthetics on the proto-plasm. He then went on to study the effect of anesthetics on the colloidal properties of the protoplasm of muscle fibers; but at the time that Bernard worked, there was no knowledge of the theory of the colloid chemistry of protoplasm and no knowl-edge of the techniques necessary for the study of protoplasm from a colloidal standpoint, and hence Bernard reached the wrong conclusions as to what the anesthetics did to the proto-plasm. This was of course to be expected in Bernard's day, but it is rather surprising that within the past few years various authors, who seem to be almost as ignorant of the modern science of the colloid chemistry of protoplasm as Claude Bernard was, continue to further mistaken ideas and concepts (see, for ex-ample, Johnson, Eyring, and Polissar's book on "The Kinetic Basis of Molecular Biology" published in 1954).

Earlier in this chapter it was pointed out that some of the agents which act as stimuli for irritable tissues may also act as anesthetics. This is a remarkable fact and it cannot be over-looked. Cold and heat arouse muscle and nerve, but under some conditions they act as anesthetics for these tissues. Pressure also can act both as a stimulant and as an anesthetic. So can potas-sium. Perhaps even more interesting is the fact that dilute solu-tions of fat solvents ( and other anesthetics ) often increase rather than decrease irritability. During surgical operations, patients


under ether anesthesia not infrequently toss violently to and fro; this in technical medical jargon is called "jactitation." Also, in some cases, convulsions occur. Concerning such convulsions, Harris (1951) in his book on "The Mode of Action of Anaes-thetics," a book written from a clinical standpoint, writes, "The idiopathic* convulsions which have been reported with di-ethyl ether and most other anaesthetics in common clinical use have been attributed to a variety of causes." Harris himself, thinking in terms of modern mammalian physiology, inclines to the view that in one way or another the magic substance acetylcholine is involved. Not only do fat solvents sometimes cause convulsions; cocaine sometimes acts in the same way. And, as is common knowledge, a relatively small amount of alcohol can cause ex-hilaration and stimulation, whereas a larger amount can cause complete lack of consciousness.

As a matter of fact, the stimulating action of low concentra-tions of anesthetics is a phenomenon that has been known since the very beginning of the study of anesthesia. In the only treatise that presents a scientific consideration of the basic facts concern-ing anesthesia, Winterstein (1926), after a preliminary section devoted to a definition of basic concepts, begins by discussing what he calls the excitation stage of narcosis. Medical physi-ologists have attempted to interpret this excitation as being due to an effect of the anesthetics on the inhibitor centers of the brain. So, for example, if a man is stimulated by a relatively small amount of alcohol, his inhibitions are suppressed and he becomes overexcited. Perhaps to some extent this is true. But the excitation produced by low concentrations of alcohol and other fat solvents occurs in isolated nerves and muscles, that is to say, in the complete absence of a brain. This has been shown over and over again. Winterstein cites dozens of references, and some of these go back to the early days of physiological study. Thus von Humboldt in 1797 found an increase in the irritability of frog muscle in the presence of alcohol or ether,

* For the nonmedical reader, it should b e noted that "idiopathic" is defined as

'spontaneous" or "of unknown origin."


and this sort of observation has often been repeated since then, both for striated and for smooth muscle.

Nerve too is excited by low concentrations of anesthetics, and this also has been known for a long time. Winter stein cites eleven references, dating from 1879 to 1923, to show that dilute solu-tions of anesthetics increase the excitability of nerve. And this information goes much farther back than 1879, for Johannes Müller in the 1830's was well aware of it.*

Because of the fact that decidedly higher concentrations of ether and other fat solvent anesthetics are necessary to suppress the irritability of nerve and muscle than are required to rob the brain of consciousness, it is easy to understand why a patient under ether anesthesia may toss about or go into convulsions. For the concentration of anesthetic sufficient to anesthetize the brain cell may not be sufficient to anesthetize the muscles and, indeed, may be in the range that would excite them.

But our major concern is not with the clinical aspects of anes-thesia. The point to be emphasized is that, quite generally, dilute solutions of anesthetics, instead of acting in anesthetic fashion, have quite the opposite effect; that is to say, they decrease the threshold of irritable tissues instead of increasing it. This is a fact long known, well established, and almost completely ignored by those who have attempted to develop theories of stimulation and anesthesia. Thus if the primary action of anesthetics is on the oxidative enzymes of a cell, then by what physicochemical magic could we interpret why it is that a dilute solution of ether has an effect quite opposite from that of a more concentrated solution? It may be true that dilute solutions of ether and other anesthetics do actually increase the rate of metabolism, and Winterstein in his book presents some evidence that they do, but this is surely to be regarded as a result of stimulation rather than the cause of it. Unfortunately the chemists who have urged the oxidation theory of anesthesia have never bothered their

* See, for example , page 6 2 5 of the Engl i sh translation of Midler's famous book

on physiology, publ ished as the "Elements of Physiology" in two volumes in

London in 1838.


heads about how to explain the stimulating action of dilute solutions of anesthetics.

Nor have the adherents of the permeability theory ever made much effort to explain why dilute solutions of anesthetics stimu-late. We might be quite ready to admit that anesthetics either decrease or increase the permeability of the plasma membrane, but that one concentration of a fat solvent has one sort of an effect and a slightly higher concentration exactly the opposite effect is not easy to believe.

Thus the older theories have all failed. They have failed be-cause those who have believed in them have either not been aware of the known facts or have been willing to ignore those facts that contradicted their theories. A successful theory should explain not merely the action of some stimuli, but all of them; not merely the action of some types of anesthetics, but all of them. And it should certainly make an effort to interpret that enigma of enigmas, the fact that dilute solutions of anesthetics may act as stimulating agents. In the next chapter we shall attempt to develop such a theory.

13.

THE COLLOIDAL THEORY OF STIMULATION AND ANESTHESIA

Our knowledge of what happens when an irritable tissue is exposed to an effective stimulus is many sided, and we also know a great deal about how the response to a stimulus can be pre-vented. Some of this knowledge was briefly summarized in the last chapter. In this chapter we shall endeavor to make sense out of the facts that are known, and to interpret them from the standpoint of cellular physiology. In other words, we want to know not only how the protoplasm of a cell reacts to a stimulus, but why it does so; we want to know also how and why this response is prevented by anesthetics. And our theoretical inter-pretation should be consistent with what we know about the physicochemical properties of living protoplasm.

In the first place, it should be apparent that any proper theory should be able to explain both stimulation and anesthesia. For the two processes are like the two sides of a coin, and any one-sided explanation is certainly not satisfactory. Thus if we decide that anesthesia is due to a certain specific effect on some physical or chemical property of the protoplasm, then we should be able to show also that the opposite effect is produced by stimulation. But we must remember that anesthetics under certain conditions act as stimulating agents, so that at least, in part, the action of stimuli and anesthetics must have something in common.

An ideal theory must explain first why the various types of stimuli—cold, heat, pressure, electric shock, ultraviolet radiation, the potassium ion, all can produce the same sort of an effect on the protoplasm. Moreover, a theory should explain not merely the response itself, as for example, the contraction of a muscle, it should explain and interpret also the various phenomena that

237


accompany the response. These phenomena include the action potential, the increase in permeability that commonly occurs, and the fact that as a result of the response, the metabolism of the cell is thrown into high gear. Secondly, we need to know why anesthetics of all sorts are able to prevent irritable tissues from responding. And thirdly, we should attempt to understand why so often dilute solutions of anesthetic substances can induce a response or at least lower the threshold of various tissues so that they become more irritable.

In earlier chapters, especially those chapters in which muscular contraction, nerve excitation, and cell division were considered, explanations were offered in terms of the colloidal changes that occurred in these types of response. Now in this chapter a broad, general theory of response will be presented. But, of course, such a broad theory must lean on the facts as we know them for various individual tissues. Thus many of the points brought out in earlier chapters will have to be repeated. Only in this way can the full force of our argument be made manifest.

1. All of the usual stimulating agents cause a liquefaction of the cortex of the cell. Only in relatively few cells can the cortex be studied from a colloidal standpoint. In ameba the cortex is liquefied both by heat and by cold. This was shown very clearly by Thornton (1935); he made determinations of the viscosity of the cortex of Amoeba proteus at various temperatures. Thorn-ton's work has already been discussed and his temperature-viscosity curve for the cortex is shown in Fig. 10 on page 39. Likewise in the egg of the worm Chaetopterus, the rigidity of the cortex is greatly decreased both by heat and by cold. This was shown by Wilson and Heilbrunn (1952). They studied the amount of centrifugal force necessary to dislodge granules from the cortex. At a temperature of 38° C. this force is 40 per cent less than it is at 21° C. On the other hand, if the egg is suddenly exposed to a temperature of 0° C , the rigidity of the cortex also drops and the drop is just about as great as that which occurs in the heat.

Is there any logical reason why both heat and cold should liquefy the cortex? There is indeed. For the cortex almost

13. c o l l o i d a l t h e o r y 239

certainly contains both lipid and protein, both combined with calcium. And, as shown in Chapter 3, it is the calcium that keeps the cortex rigid. Now the effect of heat is to dissolve the lipid or to change it in such a way that the lipoprotein molecule is no longer as able to bind calcium as it does at lower temperatures. Certainly, as we shall see later, all fat solvents act in the same way as heat does. Also by actual chemical test of cell homo-genates it can be shown that both fat solvents and heat do actually release calcium from binding (Berwick, 1951; Weimar, 1953). On the other hand, if we assume that some of the calcium in the cortex is bound directly to protein, then a drop in temper-ature would free this calcium. For the cation binding power of a protein decreases with a decrease in temperature (Austin, Sunderman, and Camack, 1927 ).

Ultraviolet radiation acts in the same way that cold and heat do. If an ameba or a Chaetopterus egg is exposed to ultraviolet radiation, there is a definite liquefaction of the cortex. For the ameba this was shown very clearly by Heilbrunn and Daugherty (1933). In an extensive series of experiments, they found that when an ameba was exposed to the radiation of a mercury arc lamp, the viscosity of the protoplasm of the cortex dropped to about one-fourth of its original value. Similar results were ob-tained for the Chaetopterus egg (Wilson and Heilbrunn, 1952). If the rigidity of the cortex depends on the presence of calcium, then it is easy to understand why ultraviolet acts as it does, for ultraviolet radiation is known to break the carboxyl bond in amino acids (Anslow, Foster, and Klingler, 1933). This would release the calcium bound to protein and such a release does actually occur when proteins are exposed to ultraviolet radiation (Clark, 1923).

Thus heat, cold, and ultraviolet radiation all cause a liquefac-tion of the cortex. So too does mechanical impact. Angerer in 1936 bounced amebas about in a shaking machine and found that this agitation caused a very pronounced drop in the viscosity of the cortex of ameba. Angerer's results are shown in Fig. 58. From this figure it is evident that vigorous shaking can cause a drop of the viscosity of the cortical protoplasm so that it may

240 d y n a m i c s o f l i v i n g p r o t o p l a s m

become only about 5 per cent as viscous as it was before shaking began. The liquefaction induced in the cortex by the mechanical impacts which result from shaking is doubtless due to the fact that the cortical gel has thixotropic properties.

An electric current also causes a liquefaction of the cortex of an ameba. This is shown by the careful experiments of Angerer

I • ι • ι • • • ι • ι ι . ι • ι ι ι — ι — I

0 2 4 6 8 10 12 14 16 18 20 Agitation time, min.

F I G . 5 8 . T h e effect of agitation on the relative viscosity of the proto-

plasm of the cortex of Amoeba proteus. The three curves give results for

different rates of shaking. Viscosity measurements were begun 1 0 sec. after

shaking had ceased.

and Wilbur (1943). They exposed Amoeba proteus to direct currents and also to alternating currents; then a few seconds after the current had ceased to flow, they measured the viscosity of the cortex of the ameba with the centrifuge method. The results for the direct current are shown in Fig. 59. With greater current density, the liquefaction occurs more rapidly and is more pronounced. It is easy to understand why an electric current would tend to liquefy the cortex for it would remove calcium from it. At the anodal end of the ameba, this liquefac-tion is so great that the ameba breaks there. Alternating currents also cause a liquefaction of the cortex. Figure 60 shows Angerer

13. COLLOIDAL THEORY 241

and Wilbur's results for various densities of alternating current. For a given current density, the alternating current is even more effective in causing liquefaction than is the direct current. It is possible that the alternating current produces a local heating of the cortex and that this heat effect is partially responsible for the liquefaction.

CURRENT DENSITY ("DC")

e « 2 5 XMO"' AMPS./CM 1

x * l O X

O : 2 0 X "

o * 3 0 X "

6 0 8 0 100 120 1 4 0 STIMULATING TIME IN SECONDS

F I G . 59 . T h e effect of direct electric current on the relative viscosity

of the protoplasm of the cortex of Amoeba proteus.

In addition to the various physical agents which act as stimuli, there are a number of chemical substances which are capable of arousing nerve or muscle or other irritable tissues. Some of these chemical agents are rather specific in their action and stimulate only certain types of cells. Among the chemical stimulants which have an effect on diverse types of protoplasm is the potassium ion. It causes muscles to contract, it makes ameba stop and round up, and it induces some egg cells to divide. The potassium ion, by virtue of the fact that it displaces calcium from the cortex of cells, causes the cortex to become relatively fluid. In the


ameba this effect is very pronounced ( Heilbrunn and Daugherty, 1932) and it is also clearly demonstrable in the egg of Chae-topterus (Wilson and Heilbrunn, 1952).

There is thus sufficient evidence to show that the best known types of stimulating agents produce a liquefaction of the cortex and it also seems clear that by their very nature they should do

2 0 - ·:7 . 0 X

3u 60 90 120 150 180 2iO 2^0 STIMULATING TIME IN SECONDS

F I G . 6 0 . T h e effect of alternating current on the relative viscosity

of the protoplasm of the cortex of Amoeba proteus.

so. The liquefaction of the cortex is not the end result of stimula-tion. Rather it can be considered as one of the first evidences that a response has begun. Following the liquefaction, a train of consequences follows. Thus when a muscle contracts, it proba-bly is not a change in the cortex that is primarily responsible for the shortening, but a change in the interior protoplasm. Similarly the division of an egg cell may be initiated first by a change in the cortex, but the mitotic spindle which actually causes the cell to divide is formed in the cell interior. Some stimulating agents can bypass the first stage of stimulation, that


is to say, the liquefaction of the cortex, and can produce colloidal changes in the interior protoplasm of the same sort as those which ordinarily follow the liquefaction. For example, both for certain types of muscle and for some egg cells, calcium chloride can act as a stimulant and it does this without causing any cortical liquefaction. The reason for this behavior is clear enough. As we shall see, liquefaction of the cortex involves a release of calcium ion to the interior of the cell. It is this released calcium which makes a muscle contract or an egg cell divide. But it should be noted that the calcium itself is not potent unless it can initiate an enzymatic reaction. By introducing the proper enzyme system into cells we can also bypass the second step in the train of consequences that ordinarily follows liquefaction of the cortex. However, ordinarily stimuli of one sort or another cause a liquefaction of the cortex.

2. The liquefaction of the cortex is accompanied by a release of calcium and some of this calcium enters the cell interior. In the preceding section, it was pointed out that stimulating agents cause a liquefaction of the cortex because of the fact that by their very nature they would tend to free calcium from binding with protein or lipoprotein. That they actually do cause such a release has been proven for several types of irritable systems.

In the chapter on muscular contraction, a rather large body of evidence was presented to show that when muscles are stimu-lated, they give off calcium to the surrounding medium. Some of this evidence will now again be brought to mind, although for a more complete statement, the reader should refer back to Chapter 7. If many muscles of a rabbit's body are stimulated electrically, the calcium content of the blood may increase by as much as 50 per cent. Muscles isolated from the body, when they are stimulated, give off calcium to the surrounding medium. Thus, for example, a study with radioactive isotopes showed that this was so for frog muscles. In experiments on the effect of stimulating agents on the homogenates obtained from muscle, Weimar (1953) found that these agents caused a release of calcium.

Thus we know with some degree of certainty that stimulation


does cause a release of calcium from muscular tissue. We do not know that this calcium comes from the cortex. Also there is no great amount of evidence to show that the free calcium, which we assume to have come all or in large part from the cortex, finds its way into the cell interior. According to Weise (1934), resting muscles of the rat contain no free, diffusible calcium, but after they have been fatigued they then contain appreciable amounts of free calcium.

When a nerve is stimulated, there is also apparently a release of calcium. At any rate, several authors have published data which indicate such a release. For further discussion, see Chap-ter 8. In sea urchin eggs also, when the cell is stimulated to divide by the entrance of the spermatozoon, there is a release of calcium. Thus in 1937 Mazia found that although in the un-fertilized Arbacia egg, there was only approximately 0.0005 molar calcium present in diffusible form in the protoplasm; after fertili-zation there was about three times as much. Presumably this increase in free calcium was caused by a release of calcium from the cortex, but Mazia had no way of proving this.

In cells of the aquatic plant, Elodea, Mazia and Clark (1936) were able to show that stimulation by electric shocks, by me-chanical impact, by ultraviolet radiation, or by temperatures of 40-55° C , all caused a release of calcium. In these Elodea cells the vacuoles in the center of the cells contain oxalate which serves as a useful indicator for calcium. Following stimulation, calcium oxalate crystals appear in the vacuoles. If, however, be-fore exposure to stimulating agents the leaves are washed for a few minutes in solutions of oxalates or citrates, no crystals appear in the vacuoles. Thus it is apparent that the calcium that enters the vacuoles comes from the outer periphery of the cell.

3. The fact that following stimulation the protoplasm in the cell cortex loses some of its power to bind calcium would in large measure account for the action potential. This follows from the fact that the cortex behaves as a calcium electrode. For a discus-sion of the relation of the action potential to calcium release, see Chapter 4.

4. Calcium release would also increase the permeability of


the cell The semipermeability of the plasma membrane and of the cortex, which may well constitute a part of the plasma mem-brane, depends on the presence of calcium. An increase in the calcium content of the outer membranes of the cell lowers permeability, a decrease in the calcium content increases per-meability. These are well-known facts (for a discussion of them, see Heilbrunn, 1952b).

5. The calcium that enters the cell interior as a result of stimu-lation causes a clotting of the protoplasm there. If cells are im-mersed in solutions containing excess calcium, as the calcium ions very slowly enter the cell, they cause a decrease in the proto-plasmic viscosity of the interior. This has been shown for the cells of the alga Spirogyra, for root hairs of Trianea, for sea urchin eggs, for ameba, and stentor. For literature references and for additional discussion, see Heilbrunn ( 1928 ) ; also Heilbrunn and Daugherty ( 1931 ). On the other hand, if calcium enters the cell interior more rapidly or in more appreciable amounts, instead of causing a decrease in viscosity, it has quite the opposite effect. This is due to the fact that calcium in sufficient amounts can initiate a clotting reaction in protoplasm, a reaction similar to the reaction which occurs when a cell is torn or broken, the sur-face precipitation reaction. In earlier chapters of this book many pages were devoted to this clotting reaction of protoplasm, for it is believed to be a reaction of primary importance for the life of the cell.

When an ameba is stimulated, the entering calcium causes first a transient decrease in the viscosity of the interior protoplasm; this decrease in the viscosity of the interior protoplasm is soon followed by a sharp rise in the viscosity of the protoplasm. Figure 61 shows the sequence of viscosity changes after exposure to ultraviolet radiation from a mercury vapor lamp. An ex-posure to the rays from the lamp for 5 sec. had no effect; a 10-sec. exposure caused a drop in viscosity which was not followed by an increase; longer exposures caused a drop followed by an in-crease. The shape of the curve for these longer exposures can readily be understood if we assume that a very little calcium entering the cell causes the interior protoplasm to become more


fluid; with the entrance of more calcium, the clotting reaction is initiated.

Not only does ultraviolet stimulation produce changes of this sort, other types of stimulation act in the same way; thus an exactly comparable series of viscosity changes is caused by me-

13 12

11

10

& 9

ΐ · $ 7

a 5

« 4

3

2

1

0,

IS seconds . · ·*** 30sec oncts

..···'* y * y s /Osecone/s

V / •S set onals

/

\ V

30 60 90 120 180 240 300 Time a f te r Exposure in Seconds

360

F I G . 6 1 . T h e viscosity of the interior protoplasm of Amoeba dubia

fol lowing exposure to ultraviolet radiation. T h e four curves show the

viscosity changes after four different lengths of exposure to the

radiation (Hei lbrunn and Daugherty , 1 9 3 3 ) .

chanical stimulation. This follows from the work of Angerer ( 1936 ). He shook amebas and then after frequent time intervals he measured the viscosity of the protoplasm in their interior. The results of such an experiment are shown in Fig. 62. The three curves show the results of shaking at a rate of 10.6 jolts/sec for 15, 30, and 45 sec.

In muscle fibers also, when calcium enters the interior of the fiber as a result of stimulation, this entrance of calcium is un-doubtedly followed by a clotting reaction and a shortening of the fiber. Certainly the injection of a small amount of calcium into a muscle fiber can induce a marked shortening (see Chapter 7). And just as in the ameba, the entrance of the first trace of calcium causes a drop in viscosity, which is then followed by a


sharp increase in viscosity as more calcium enters; so too in mus-cle when it is stimulated, the first observable change is a relaxa-tion of the muscle, and only after this preliminary relaxation does the muscle shorten. The occurrence of this preliminary relaxation, originally called Rauh's nose after its discoverer, and

I ι ι ι ι ι ι ι 1 1 1 1 0 30 60 90 120 180 240 300 360 420 480 540

Seconds between jolting and centrifuging

F I G . 6 2 . T h e relative viscosity of the interior protoplasm of Amoeba

dubia fo l lowing exposure to shaking for various t imes. T h e viscosity

measurements were m a d e wi th the centrifuge method and the measure-

ments were m a d e at the t imes shown b y the abscissas (Angerer, 1 9 3 6 ) .

subsequently given the more dignified name of "latency relaxa-tion," is discussed in Chapter 7.

In egg cells also, when they are exposed to stimulating agents that cause them to divide, the calcium that is released from the cortex causes a sharp increase in the viscosity of the protoplasm in the interior of the cell; this protoplasmic clotting produces the mitotic spindle. The colloidal changes that occur when marine egg cells are stimulated to divide are discussed in some detail in Chapter 10. These changes are similar to those that occur in the ameba, and presumably also in muscle fibers.

6. Protoplasmic clotting can in itself cause a sharp increase in oxidative processes. This is probably due to the fact that the clotting of protoplasm, and perhaps also the clotting of blood,


involves an oxidation of SH groups to S-S groups. The relation-ship between protoplasmic clotting and oxidation was discussed in Chapter 7. According to Hultin ( 1950 ), if calcium is added to homogenates of sea urchin eggs, the clotting that ensues is ac-companied by a decided increase in the uptake of oxygen. More work needs to be done on the relation between protoplasmic clotting and oxidation, but the information we now have indicates that the clotting process does increase oxidations. It is as though the clotting reaction threw the oxidative processes into high gear.

Hence on the basis of the theory outlined above, we can have a rather complete interpretation of what happens when an ir-ritable tissue responds to a stimulus. It is of the nature of stimu-lating agents to free calcium from protein, the calcium that is set free can activate proteolytic, clotting enzymes which clot the protoplasm. Such a clotting reaction can make a muscle shorten. The theory also has an explanation for the action potential, for the increase in cell permeability that commonly accompanies a response, and for the increase in metabolism that is a conse-quence of the increased activity of protoplasm.

Let us now consider the other side of the coin—the action of anesthetics. If our theory of stimulation is correct, it should offer a basis for an understanding of the action of anesthetics. Some-how, somewhere, an anesthetic should interfere with one or another of the train of processes that follow the initiation of a response. But it must not be assumed that all types of anesthetics interfere with the course of the series of response reactions in exactly the same way. Some interfere at one point in the chain of reactions, others block at another point.

First let us consider the fat solvent anesthetics. How do they act? Do they interfere with the release of calcium from the cortex; do they prevent the liquefaction of the cortex? They most certainly do not. Indeed fat solvents have the same effect on the cortex that stimulating agents have. Like stimulating agents, they cause a liquefaction of the cortex of the ameba. This was clearly demonstrated for various fat solvents by Daugherty ( 1937 ). She found that ether, as well as ethyl, propyl,


butyl, and amyl alcohols, all cause a sharp decrease in the vis-cosity of the cortex of Amoeba proteus. Similarly Wilson and Heilbrunn (1952) found that dilute solutions of ether cause a liquefaction of the cortex of the Chaetopterus egg. In the presence of 2 per cent ether, it took only 27 per cent as much centrifugal force to dislodge granules from the cortex, as it did for normal eggs not exposed to ether. Butyl alcohol and amyl alcohol acted in similar fashion. If fat solvent anesthetics liquefy the cortex, it might be expected that they would cause a release of calcium. As far as the evidence goes they do actually cause such a release. Thus in 1951 Berwick showed that ether and chloroform caused a release of calcium from frog muscle fibers. (Cocaine has a similar effect.) This is in accord with older evidence cited by Berwick which indicates that ether and other anesthetics cause the tissues to liberate calcium into the blood. Thus we must conclude that fat solvent anesthetics do not in any way interfere with the very first stage of response, with the liquefaction of the cortex, and the freeing of bound calcium. As a matter of fact, they act in the same way as stimulating agents in causing a liquefaction of the cortex and a release of calcium; and, as we shall see later, this is no doubt the reason that anes-thetics can so often excite rather than depress.

If fat solvent anesthetics do not prevent the release of calci-um, what do they do to prevent response? The answer is clear. Fat solvent anesthetics in anesthetic concentration keep the pro-toplasm in the interior of a cell fluid and they do not permit a gelation or clotting reaction to occur. There is a fair amount of evidence in support of this statement, evidence based on trust-worthy methods of determining protoplasmic viscosity. Because the point at issue is an important one, the evidence will be presented in some detail.

Most of the early work on the effect of ether and other fat solvents on protoplasmic viscosity was done on plant cells. In 1914 the German botanist Heilbronn studied the effect of ether on starch-containing cells of oat seedlings. He found that dilute solutions caused the viscosity to decrease, whereas more con-centrated solutions had the opposite effect. Some years later


(1922) he obtained similar results for slime mold protoplasm. Kessler (1935) centrifuged leaves of the plant Sempervivum ghucum and for this material he found that chloroform vapor caused a drop in protoplasmic viscosity. Weber (1921) exposed cells of the alga Spirogyra to 1-2 per cent ether (by volume) and then studied the displacement of the spiral chromatophores under the influence of centrifugal force. In the etherized cells the chloroplasts were displaced more readily. With higher con-centrations of ether (3 per cent by volume), the chloroplasts were less readily displaced. This would indicate that dilute solutions decrease the viscosity and more concentrated solutions have the opposite effect. However, the chloroplasts may, to some extent at least, be anchored in the cortex so that the results give no certain indication of how the viscosity of the protoplasm in the interior is affected.

In the ameba also, fat solvents can cause a decrease in the viscosity of the interior protoplasm. Brinley (1928) studied the rate of Brownian movement in amebas exposed to solutions of ether, chloroform, and alcohol, and he concluded that all of these fat solvents caused a decrease in the protoplasmic viscosity. A similar study was made by Frederikse (1933). This work also indicated that fat solvents caused a decrease in the viscosity of the interior protoplasm of amebas. Frederikse used aqueous solutions of chloroform and chloretone. Daugherty (1937) in some careful studies of protoplasmic viscosity with the centrifuge method found that 2% ether could cause a 50% decrease in the viscosity of the interior protoplasm of Amoeba dubia. Butyl and amyl alcohol had similar effects. Moreover, these fat solvents prevent the clotting caused by ultraviolet radiation. However, Daugherty showed that more dilute solutions of fat solvents could produce effects on the interior protoplasm which were quite the opposite of those produced by somewhat more con-centrated solutions. These apparently contradictory results of Daugherty will be considered later when we come to discuss the stimulating action of dilute solutions of anesthetics.

On the whole, the work with plant cells and with ameba shows clearly enough that ether and other fat solvents may have very


marked effects on the colloidal state of the protoplasm. There can be no doubt of this. But generally speaking, plant proto-plasm and the protoplasm of ameba is not best for the study of anesthesia, for in most cases it is difficult to decide what con-stitutes an anesthetic concentration. Unfortunately the types of protoplasm which show most clearly the results of stimulation are not amenable for colloidal study. There is, however, one type of protoplasmic system that responds to stimulation and that can be anesthetized by fat solvents. A marine egg cell, such as a sea urchin egg, can be stimulated to divide by the same sorts of stimuli as those which excite a nerve or make a muscle contract, and it can be anesthetized by the same types of anes-thetics that act on muscle and nerve. And in marine egg cells it is possible to study the colloidal state of the proptoplasm and how it changes. For that reason we will pay particular attention to the results that have been obtained on marine egg material.

Heilbrunn (1917, 1920a, b, 1925) studied the effect of ether and other fat solvents on the viscosity of the protoplasm of the interior of sea urchin eggs, especially in relation to the anesthetic action of these substances. In a preliminary report (1917) he wrote: "If the normal gelatinization of the cytoplasm is pre-vented, then the spindle never forms, the egg remains quiescent and does not divide. Even after gelatinization has begun, it may be reversed. Ether, chloroform, acetone, paraldehyde, propyl alcohol, isoamyl alcohol, ethyl butyrate, ethyl acetate, ethyl nitrate, acetonitrile, nitromethane, chloral hydrate, phenyl and ethyl urethanes, all prevent or reverse cytoplasmic gelatinization. In the study of the action of these anesthetics, the experimental procedure was usually as follows : soon after fertilization, the eggs were placed in a graded series of concentrations of the anes-thetics. Then the viscosity of the various sets of eggs was de-termined. If the series of concentrations was well chosen, it was found that the highest concentrations produced coagulation, the lowest concentrations no effect, and the intermediate con-centrations a liquefaction or reversal of gelatinization. As my studies progressed, I was soon able to predict the fate of the eggs. In the lower concentrations with no marked effect on the egg


viscosity, the eggs would go on to divide, in the intermediate concentrations which produced liquefaction, development would be interrupted, but removal from the anesthetic (after a few hours), would be followed by a resumption of development. On the other hand, coagulation produced by the higher concentra-tions was generally irreversible, although in a few cases the eggs thus coagulated were able to undergo a few cell-divisions on being returned to sea-water." For details of these experiments, see Heilbrunn (1920a).

In later experiments Heilbrunn ( 1925 ) made a more quantita-tive study of the effect of ether on the interior protoplasm of sea urchin eggs. Table II shows the liquefying effect of ether, the percentages of ether are volume percents, the viscosity is in arbitrary units. A solution of 2.5 per cent ether is anesthetic for sea urchin eggs.

T A B L E II

Liquefying Effect of Ether

Viscosity

Per cent Exposure etherized Viscosity

ether minutes eggs control eggs

2.5 11 10 2 5

2.5 4 15 25

2.5 8 15 2 5

2.5 10 15 2 8

3 15 15 3 5

3 8.5 2 0 4 0

3 3 15 3 0

These results show that in 2.5-3 per cent ether, the viscosity of the protoplasm drops to about half of what it is in sea water. But it must not be supposed that in these relatively high con-centrations of ether the viscosity of the protoplasm remains constant for long periods of time. In the 3 per cent solution the viscosity becomes progressively lower and lower until a minimum is reached and then a sudden coagulation occurs. This takes place at about half an hour (at 22° C ) .

The effect of ether in causing liquefaction is even more pronounced on fertilized eggs. In these eggs the ether causes a


reversal of the mitotic gelation, and the viscosity may drop to one-sixth or less of what it was in the untreated eggs.

The studies on sea urchin eggs show clearly that ether and other fat solvents in anesthetic concentration make the interior protoplasm more fluid and prevent the gelation that is a result of stimulation. The work on the sea urchin eggs was done with an objective, quantitative method, and the results can scarcely be questioned. Unfortunately most of the other pub-lished work on the effect of anesthetics on the colloidal properties of protoplasm is not very satisfactory. Visual observation of a microdissection needle is not an adequate objective method of viscosity determination and in the past it has given undependable and incorrect results. The rate of flow of streaming plant proto-plasm is a function not only of the viscosity but also of the motive force, so that the rate of flow can not with any certainty be taken as a measure of viscosity. Moreover, stimulation of streaming plant protoplasm by mechanical shock, by ultraviolet radiation, by an electric shock, or by any of the other usual methods of stimulation causes stoppage of flow, and this is properly to be regarded as stimulation, not anesthesia. At any rate this is the usual semantic convention, as was pointed out in the last chapter. Hence observations on shocked protoplasm should not be taken as having any relation to anesthesia. And yet, even in recent years, this has been done. Moreover, some authors have made Brownian movement studies on cells that are not anesthetized but dead. Thus, for example, Marinesco ( 1912 ) studied the rate of Brownian movement in isolated nerve ganglion cells, which if they were not killed as a result of the isolation, were certainly killed by the fluid in which Marinesco observed them. This was 16.7% alcohol in distilled water! And yet Marinesco's observa-tions have been quoted in the literature as evidence that anes-thetics cause a coagulation of protoplasm. There have been other similar observations, almost equally as bad.

If ether can keep protoplasm in the cell interior fluid and can prevent the clotting reaction which is believed to result from the entrance of calcium released from the cortex by stimulation, then it might be reasoned that ether (and other fat solvents)


would tend to inhibit the action that calcium has in the surface precipitation reaction. Actually this is the case. In Chapter 5 it was pointed out that a 1 per cent ether solution completely prevented the surface precipitation reaction in the protozoan Stentor, and this is illustrated in Fig. 24. Butyl alcohol, amyl alcohol, acetone, and ethyl urethane act in the same way. How-ever, in the case of the sea urchin egg, solutions of ether in sea water do not prevent the surface precipitation reaction when the eggs are crushed in them. This is due to the fact that there is too much calcium in sea water; this calcium overrides the inhibit-ing effect of the ether. But if the experiment is done in solutions which contain only 3 χ 10"4 molar calcium chloride, then ether will prevent the s.p.r. which normally occurs in the presence of this small amount of calcium. Within the intact cell, presumably the free calcium is present only in very low concentration, so that there also ether can act to prevent the s.p.r.

If fat solvent anesthetics prevent protoplasmic clotting, might it not be possible that they would also prevent blood clotting? If this were true then we might by suitable experimentation be able better to understand how the fat solvents act. The question was investigated by Lundholm and Ehrenberg ( 1952 ). The older literature on the subject is rather contradictory. Bernard (1875, p. 167 ) found that if a 5% opium solution was injected slowly into the jugular vein of a dog, when the animal died its blood was noncoagulable. Babasaki (1932) found that chloral hydrate, luminal, and morphine delay the coagulation of the blood. But apparently under various conditions some anesthetics may either increase or decrease the coagulation time of the blood. At any rate, this is the opinion of Adriani (1946) who unfortunately does not give references to the literature. Presumably the effect of anesthetics on blood clotting depends on the conditions under which clotting was studied (just as the effect of ether on the s.p.r. in sea urchin eggs depends on the concentration of calcium in the medium ). This is exactly what Lundholm and Ehrenberg found. Their work shows clearly that in anesthetic concentration, local as well as general anesthetics prolong the clotting time of blood, when they are added to a blood plasma-thromboplastin


mixture. It is the prothrombin activation that is inhibited, not the fibrin formation. One of Lundholm and Ehrenberg's figures is reproduced in Fig. 63. They believe that the action of the various fat solvents is on the lipid fraction of the thromboplastin. The greater the lipid solubility of the anesthetic, the stronger is its action on the thromboplastin.

F I G . 6 3 . T h e action of n-

alkanols on the coagulation t ime

of horse blood. T h e curve

marked wi th · is that for η butyl

alcohol ( 1-butanol ) that marked

with ο is for normal propyl

alcohol (1-propanol) and that

marked wi th triangles is for

ethyl alcohol ( e t h a n o l ) .

Time, min.

The information that we have about fat solvents thus clearly explains their anesthetic action in terms of their known effects on the colloidal systems of the protoplasm. The fat solvents release calcium from the cortex and then they prevent the clot-ting action that this release of calcium would otherwise cause in the cell interior. Both the action on the cortex and that on the interior is due to an effect on lipoprotein.

Consider now the anesthetic action of the magnesium ion. In spite of the fact that this action has been long known and is very well established, and in spite of the fact that the magnesium ion acts on all animals from coelenterates to man and on muscular as well as nervous tissue, most of those who have attempted to explain the action of anesthetics have given little or no attention to magnesium anesthesia. For this neglect of magnesium, there are two reasons. In the first place, practical books on anesthesia devote very little attention to magnesium, for magnesium is such a universal anesthetic that it tends to prevent the activity of


mammalian breathing muscles. Hence although it has occasion-ally been used in surgical operations on human beings, it is not an anesthetic to be recommended. However, it would be thought that from a theoretical standpoint, those who attempted to interpret the action of anesthetics would be forced to consider magnesium. And yet in Harris' book on "The Mode of Action of Anaesthetics" published in 1951 magnesium anesthesia is not even mentioned. Harris is a practical anesthetist and it is easy to understand his lack of interest in a substance which is a better anesthetic for sea anemones and snails than it is for men. But to anyone interested in what anesthetics do to cells and proto-plasm, magnesium should have a prime importance if only for the fact that it is not a fat solvent and can therefore not be fitted into the Meyer-Overton theory. Nor can its action be explained by any other one of the older theories of anesthesia. But because a phenomenon is not explained by a particular theory is no reason to ignore it; indeed, for this very reason it should be especially interesting. Can magnesium anesthesia be explained in terms of the colloidal theory that we have been presenting in this chapter? Indeed it can.

Magnesium must act at or near the surface of the cell; at least this is usually true. For after exposure to a sufficient quantity of magnesium ion, the onset of anesthesia is rapid. Immerse a sea anemone in sea water to which a small amount of magnesium salt has been added (in isotonic concentration), and the anemone within a few seconds will cease to respond to ordinary stimuli. What happens presumably is that the magnesium ion exchanges for the calcium ion originally present in the cortex of the cells. But why should this replacement of calcium by magnesium tend to anesthetize the protoplasm? The answer is simple. Magne-sium and calcium can both cause a clotting reaction in proto-plasm; both of them can initiate the surface precipitation reaction in sea urchin eggs. But the magnesium ion is far less potent than calcium in causing an s.p.r. Thus according to Heilbrunn ( 1934a), it takes a concentration of approximately 3 χ 10"2 molar magne-sium chloride to produce an s.p.r., whereas as previously noted, a concentration of 3 χ 10"4 molar calcium chloride is sufficient.


In other words, calcium is about a hundred times as potent as magnesium in causing a clotting reaction in the protoplasm. Hence if the calcium cortex of the cell is converted into a magne-sium cortex, it would require the release of a hundred times as much cation in order to get a response. Actually the cortex of the cell almost certainly is not merely a calcium proteinate, but a mixture of calcium and magnesium ( and other cation ) proteinates. But this does not weaken the force of the argument, for if the strongly acting calcium ion is largely replaced by the weaker magnesium ion, then the irritability would be decreased and anesthesia would result. In all probability the Ca-Mg ratio in the cell cortex in large measure determines the threshold of stimulation. Here then is a simple interpretation of magnesium anesthesia, an interpretation that is certainly in accord with the known facts.

Moreover, it is easy to understand why cold can act as an anes-thetic, for although sudden cold by releasing calcium can act as a stimulating agent, cold can prevent the s.p.r. and presumably also prevents protoplasmic clotting (Heilbrunn, 1927). Whether cold acts as a stimulating agent or as an anesthetic undoubtedly depends on how it is applied. Possibly heat just below the lethal temperature acts in somewhat the same manner as cold, but as yet there is no information as to whether this is true or not. However, in view of the fact that heat acts on lipids and lipo-proteins in much the same way that ether does, it is logical to interpret its anesthetic action as being similar to that of ether.

There is left to be considered only the action of anticlotting agents such as heparin. In the chapter on muscular contraction it was pointed out that heparin solutions can cause a relaxation of smooth muscle. Thus heparin and similar substances can doubtless act as anesthetics. This is certainly true for dividing cells. In Chapter 11 it was shown that such substances could inhibit the mitotic gelation and prevent cell division. If heparin and related substances can depress irritability, we are in a posi-tion to interpret the lowered irritability of hibernating animals and of animals in shock. That there is clear evidence for the presence of excess heparin in the blood of hibernating animals


was shown in Chapter 12; and there and also in Chapter 9 there is a discussion of the evidence for the presence of excess heparin in the blood of shocked animals. The fact that heparin can depress irritability, and if it is able to enter cells can act as an anesthetic, is in line with the thesis developed in some of the early chapters of this book. There it was urged that the proto-plasm in the interior of a cell is in a state of balance between the factors that tend to cause clotting and those that tend to prevent such clotting. Various types of anesthetic agents, hep-arin, fat solvents, cold, all prevent the clotting reaction. The magnesium ion, by replacing calcium in the cell cortex, blocks the release of calcium into the cell interior, and in this way it also prevents a clotting of the interior protoplasm.

There are two points still to be considered in this chapter. The first of these is an interpretation of the fact that in cells which can readily be stimulated to divide, the process of division can be prevented not only by substances that inhibit the clotting of protoplasm but also by those that favor or intensify the clotting process. Thus ether keeps the protoplasm fluid and stops the cell from dividing; whereas hypertonic solutions intensify the clotting reaction and yet they also stop the cell from dividing. Should we agree that anesthesia may be due either to a preven-tion of clotting or to an intensification of it? I think not. Actually the question is largely a semantic one. Clearly, hypertonic solu-tions act as stimuli; they induce a mitotic gelation and start the cell on the road to division, but division cannot proceed for the protoplasm is maintained in a state of gelation. Thus hyper-tonic solutions act like those agents which make muscle shorten but do not permit relaxation. A muscle in a shortened state is not capable of response. But in the case of those agents which produce contracture or prolonged shortening, no one has at-tempted to regard the inexcitability that accompanies such pro-longed shortening as anesthesia. Accordingly, if we are to be consistent, it is probably not wise to regard the action of hyper-tonic solutions on dividing egg cells as an example of anesthesia. This is contrarv to the opinion I held many years ago ( Heilbrunn, 1920b).


Finally there is the question of the stimulating action of anes-thetics. This has most often been noted for the fat solvent anesthetics. A dilute solution of ether or alcohol can increase the excitability of nerve or muscle, or can even cause response in nerve or muscle. In the light of what we now know about the colloidal properties of the outer and inner parts of a cell, we are in a position to interpret this strange fact. There can be no doubt but that fat solvent anesthetics act like stimulating agents in causing a liquefaction of the cell cortex and a release of calcium into the cell interior. Thus in their initial effects, fat solvent anesthetics act just like stimulating agents. The reason they are anesthetics is that they prevent the clotting reaction that follows the entrance of calcium into the cell interior. If now we immerse a cell or a tissue in a relatively dilute solution of a fat solvent, the fat solvent because of its tendency to lower interfacial tension and perhaps also because of its ability to dissolve in lipid, would tend to be absorbed or concentrated in the membranous structures at the outer region of the cell and be present in relatively high concentration there. In the cortex it would cause a release of calcium. But not enough of the fat solvent would enter the cell to prevent the clotting reaction caused by the entering calcium. Hence the cell would tend to be stimulated rather than anesthetized. Or we could assume that a fat solvent would release calcium from the cortex but not enough to cause a response. This would facilitate the action of a stimulus, so that the fat solvent ( in dilute concentration ) might act to increase the irritability, that is to say, lower the threshold of an irritable tissue.

If this were true, then we might expect that in some instances at least, relatively dilute solutions of fat solvent anesthetics would tend to increase the viscosity of the protoplasm in the interior of a cell, whereas higher concentrations would have exactly the opposite effect. This is exactly what was found by Daugherty in her studies on ameba. Some of her data were published in 1951 by Heilbrunn in a review of the colloid chemistry of anes-thesia. This review appeared as part of a French symposium, and because the symposium is perhaps not readily available to


many readers and because also the facts are of considerable importance, two of the tables from the symposium article are presented again here. They represent data somewhat more ex-tensive than the data originally published by Daugherty in 1937.

T A B L E III

The Effect of Various Concentrations of Ether on the Viscosity of the Interior

Protoplasm of Amoeba dubia

Concentration T ime of Viscosity

vo lume Normal exposure after

per cent viscosity minutes exposure

1% 7 10 3

30 15

1% 7 6 5

2 6 2 0

36 20

1% 7 7 4

13 10

3 0 2 0

1.5% 6 4 2

26 25

2% 8 2 .5-5 4

18-28 4

Tables III and IV show the effect of varying concentrations of ether and of normal butyl alcohol (1-butanol) on the viscosity of the interior protoplasm of Amoeba dubia. The measurements of viscosity were made by the centrifuge method. The units of viscosity are arbitrary ones; they represent the number of seconds required, at a centrifugal force of 128 times gravity, to move the cytoplasmic inclusions into one half of the cell. In the tables the horizontal lines separate individual experiments. Both in the case of ether and of butanol, if a strong enough concentration of the fat solvent is used, the protoplasm becomes more fluid, and this drop in viscosity may be 50 per cent or more. On the other hand, in the presence of more dilute concentrations, the viscosity may rise to three or four times its original value or even more.


Hence in these experiments, ether and butyl alcohol act just as we would expect them to act if dilute concentrations were to have a stimulating effect and more concentrated solutions were anesthetic. In general, the amebas exposed to these solutions of fat solvents recover, or at least most of them do. Sometimes they stop moving, at other times they continue to move slowly in the presence of the anesthetic. The movement of the ameba is

T A B L E IV

The Effect of Various Concentrations of η Butyl Ahohol on the Viscosity of the

Interior Protoplasm of Amoeba dubia

Concentration T ime of Viscosity

vo lume Normal exposure after

per cent viscosity minutes exposure

0.5% 6 26 4 5

1% 5 6 5

18 10

56 40

1.25% 8 8 3

17 2

not a process that is readily stopped by anesthetics; the move-ment depends on a number of factors. According to most theories, the cortex at one part of the ameba exerts more pressure than the cortex at another weaker part. The latter becomes the anterior end. Stimuli of the usual sort, such as, for example, ultraviolet radiation or an electric shock, generally tend to stop the ameba. This is because they liquefy the cortex all over and they also gel the interior. All in all, it is not easy to decide just what to call anesthesia in an ameba. Our only point is that, in so far as the cortex and the interior protoplasm are concerned, fat solvents act on the colloidal properties of these parts of the cell in accord with theoretical expectation.

The theory presented in this chapter offers an interpretation both of stimulation and of anesthesia in terms of the colloidal properties of protoplasm. The theory can explain why it is that the ordinary types of stimuli act as they do; it explains the action


not only of fat solvent anesthetics, but of the magnesium ion as well. It offers an interpretation of why some agents (such as cold) can have both a stimulating and an anesthetic action; and finally, and this is most important, it offers a clear explanation of why fat solvent anesthetics can, in dilute concentration, in-crease excitability rather than depress it.

But there is one aspect of stimulation that has as yet not been mentioned, and it is a very important aspect. Whenever an excitable tissue is stimulated with ordinary types of stimulating agents, as soon as the response is completed, there is a return to the original resting state and this return to the original state is often a very rapid one. There must be some factor in the stimulus itself, or at least some factor in the train of consequences that follows the application of the stimulus, that brings back the protoplasm to a condition that will make possible response to a second stimulus. What the nature of this factor is, or what the nature of the change or changes are that restore the excitable tissue to a condition in which it can be excited again, is a question that has rarely been faced by physiologists, at least not from a broad theoretical standpoint. In the next chapter this question will be discussed and a possible interpretation offered.

14.

In 1929 and again in 1932, the distinguished mammalian physi-ologist Walter B. Cannon, strongly impressed with the regulatory arrangements of higher animals, proposed the word "homeostasis" as a convenient term for these arrangements. In 1929 he wrote: "The coordinated physiological reactions which maintain most of the steady states in the body are so complex, and are so pecu-liar to the living organism, that it has been suggested (Cannon, 1926) that a specific designation for these states be employed— homeostasis" Cannon was well aware that the idea of a self-regulatory power in living organisms was not a new one. Thus in 1932 he wrote that "The ability of living beings to maintain their own constancy has long impressed biologists" and he quotes earlier authors who emphasized the stability of living organisms.

To Cannon, thinking in terms of mammalian physiology, the constancy of the blood was the prime factor in homeostasis, and in his book on "The Wisdom of the Body," published in 1932, and again in a revised edition in 1939, most of the chapters deal with various aspects of blood stability. On page 387 of the 1939 edition, he wrote: "In the main, stable states for all parts of the organism are achieved by keeping uniform the natural surround-ings of these parts, their internal environment or fluid matrix." And he goes on to say that the course of evolution of higher organisms has been characterized by a gradually increasing con-trol of the functions of the blood and its constancy. "The central problem of understanding the nature of the remarkable stability of our bodies, therefore, is that of knowing how the uniformity of the fluid matrix is preserved." Later, on page 306, he states that "the single-cell organism . . . has no means of controlling its environment and must submit wholly to what the environ-

263

CELLULAR HOMEOSTASIS


ment imposes upon it." Most modern biologists would not agree to this last statement.

For it is of the nature of all living things to be able to adapt themselves so that they can survive under untoward conditions, and this ability is often very highly developed even in single-celled organisms or in plants. If the environment changes—if it becomes warmer or colder—almost all organisms can change so that they can withstand more heat or more cold. This power of acclimatization varies greatly; some organisms acclimatize much more readily than others, but all of them can, to some extent at least, become somewhat more tolerant of the extremes of heat or cold. So too in varying degrees many aquatic animals can adjust to more or less salinity in their environment. The power of adjustment to the environment is often well developed in protozoa. And yeast cells and bacteria sometimes show a remarkable ability to cope with environmental change. Thus a yeast cell, which is normally incapable of ultilizing a foodstuff not normally present in its environment, can develop an enzyme to cope with the strange substance. These acclimatization changes ordinarily do not occur rapidly—acclimatization to tem-perature or to salinity may take days or months, or even years.

There are also much more rapid types of adaptation or adjust-ment. Sense organs adjust to excessive stimulation and become relatively insensitive to it. A nerve can become insensitive to an electric stimulus, if the stimulus increases very slowly in intensity.

Perhaps these phenomena of acclimatization and adaptation do not constitute examples of homeostasis in the sense in which Cannon defined the term, although it might be hard to separate them from true homeostasis. Perhaps a new word is necessary, a word that would apply not merely to higher animals but to all organisms. We need a word that would include all regulatory processes of all organisms, and then perhaps we need additional words to distinguish various manifestations of such regulatory processes.

Possibly the word "biostasis" could be the general term. It could include all the phenomena in which living organisms resist

14. CELLULAR HOMEOSTASIS 265

changes in their environment, in which they tend to preserve their constancy. Acclimatization would be one aspect of it. The immunity that animals develop against bacterial toxins might be another. Then there is the ability of an organism to regain its size and shape when parts of it are destroyed. This is what is known as regeneration. A shrub can be cut to its roots and it will grow back to its original size. A flatworm or an earthworm can have its head or its tail cut off and it will regenerate the lost structures. What clearer example of "the ability of living beings to maintain their own constancy"?

There is nothing essentially new in all this—nothing more than a restatement or a rearrangement of long known facts. But what is not generally realized is that homeostatic or biostatic phenom-ena are a characteristic not only of organisms but of cells. Thus there is a cellular homeostasis; or in view of the fact that Cannon denied the existence of homeostasis in single cells, a cellular biostasis. The basic reaction involved in the homeostatic or bio-static reaction of cells is the surface precipitation reaction. Cut or tear a cell and it seals itself. Thus the cell is able to protect itself against the forces of the environment that would tend to destroy it. To some extent the surface precipitation reaction has made it possible for life to survive on this planet, for if there were no such reaction, with every minor injury the fluid proto-plasm of cells would drain away. And more than that, out of the surface precipitation reaction, living cells have developed a way of responding to stimuli. The colloidal changes that follow stimu-lation are changes like those that occur in the surface precipita-tion reaction. This is the thesis that has been maintained in earlier chapters, it is a thesis that readily explains why excessive stimulation and injury are indistinguishable.

Moreover, reactions which are essentially similar to the surface precipitation reaction are responsible for some of the most in-teresting examples of the homeostatic behavior of higher organ-isms. When blood clots, the reactions involved are of the same type as those which occur in the s.p.r. This was clearly shown in Chapters 5 and 6. And there is perhaps no better example of homeostatic behavior than the way in which the clotting time


of the blood varies under different conditions. Cannon realized that when animals lose blood as a result of hemorrhage, their blood clots more readily. But he was apparently unaware of various other homeostatic mechanisms associated with blood clot-ting. As is well known, citrate prevents the clotting of blood in vitro, for it keeps the calcium of the blood in an unionized state. Therefore when physicians began to transfuse citrated blood into patients, they expected that the citrate injected into the blood stream would increase the time required for coagulation of the patient's blood. They were surprised to find that if small amounts of citrate are injected into animals and into patients, the coagula-tion time of the blood instead of increasing, decreases sharply. This was described first by Weil (1915) and it has been abun-dantly confirmed. The citrate may be injected intravenously, intramuscularly, or subcutaneously, and the effect on the clotting time has been described as "tremendous." Thus Neuhof and Hirshfeld ( 1922 ) found in the case of one patient that the clot-ting time decreased from 10 to 2-3 min. after injection of citrate; and Higgins and Fisher (1924) reported another case in which the clotting time decreased from 11 to 2 min. Perhaps oxalate can act in the same way. At the 1939 meeting of the American Physiological Society in Toronto, Steinberg and Brown (1939) reported that oxalate could speed coagulation if small amounts were injected into the blood stream. This created quite a furor, for the physiologists at the meeting were apparently unaware of the clinical evidence that citrate when injected would greatly speed blood clotting. However, this action of oxalate is open to doubt (Foster, 1940), and further work needs to be done.

Not only does citrate, a clotting inhibitor, hasten the clotting of the blood when injected into the blood stream, but small amounts of the clotting enzyme, thrombin, when injected into the circulation, greatly prolonged the clotting time of the blood ( Tagnon, 1945 ). Trypsin and chymotrypsin, which like thrombin cause blood to clot, act in the same way as thrombin when in-jected into the blood stream, and they also prolong the coagula-tion time of the blood (Rocha e Silva and Dragstedt, 1941; Tag-non, 1945; Tagnon, Weinglass, and Goodpastor, 1945). There


are two ways of interpreting this phenomenon. Tagnon believes that the effect is due to the decrease of fibrinogen and pro-thrombin in the blood, a decrease which might be due to the proteolytic action of the enzymes; on the other hand, Rocha e Silva and Dragstedt believe that trypsin releases heparin and that this causes an increase in the clotting time. Both types of mechanism are possible. And as we shall see later, both types of mechanism can be used as models to interpret phenomena that occur in protoplasm. Also the clotting inhibitor heparin, when injected into the blood will, after a time, instead of preventing blood coagulation, make the blood clot more rapidly. Thus ac-cording to Barnard (1948), "The administration of heparin to a subject with normal hemopoietic function is followed, after the usual period of diminished blood coagulability, by a hypercoagu-lability state. This is familiar to physicians as the heparin re-bound, . . . . Heparin hypercoagulability may also be demon-strated in the rabbit and the dog; in the latter animal the writer has seen coagulation times of 20-30 sec, 4 hrs. after administra-tion of coagulation-abolishing doses of heparin." Barnard goes on to state that during heparin rebound, the thromboplastin concentration of the blood is markedly increased. This thrombo-plastin presumably comes from tissue cells.

As a matter of fact, the homeostatic behavior of blood in respect to the time required for coagulation is undoubtedly due to the contributions the tissue cells make in substances that favor or tend to inhibit clotting. And it is these substances doubtless that are factors in the homeostasis or biostasis of the protoplasm.

The idea that there is a homeostasis of the cell and its proto-plasm was clearly stated in 1952 and again in 1953 (Heilbrunn, 1952b, 1953). Actually there must be some sort of a regulatory change to bring back a cell to its resting state after it has been stimulated, for after a cell has responded to a stimulus, it can soon, sometimes very soon, respond to a second stimulus. A complete theory of stimulation must have some way of account-ing for the ability of protoplasm to reverse the effect of stimula-tion. But how can we interpret this phenomenon in terms of what we know about the protoplasmic colloid and how it is affected by stimulating agents?


There are two points of view that might be held. Thus we could say that protoplasm has a natural resiliency that would tend to make it rebound from any effect imposed on it. This is another way of saying that it is homeostatic or biostatic. But why? What is the nature of the protoplasm to make it act in this way? Or secondly, we might urge that in the very process of response itself, there is some factor that would make it neces-sary for protoplasm to recover from a response. These two points of view are not mutually exclusive. We can, if we please, make use of both of them. What we need are concrete facts on which to base an interpretation. And at the present time there isn't much in the way of useful information.

The fact that from a colloidal standpoint the behavior of proto-plasm is so much like that of blood should be of help to us in understanding the homeostatic behavior of protoplasm. It could perhaps be pointed out that protoplasm, like blood, has buffers which tend to preserve its constancy of hydrogen ion concentra-tion, so that the stability of the hydrogen ion concentration in blood is matched by a similar stability in protoplasm, and for that matter ( to a lesser extent ) in sea water. But this is a minor matter. What concerns us most is the power of the blood to react against factors which tend to upset its colloidal equilibrium. Earlier in the chapter it was pointed out that when an anticlot-ting substance like citrate was added to the blood ( in the bodv ), the blood responded by becoming more readily coagulable; and when clotting enzymes were added (in the body), the blood became less coagulable or even noncoagulable. It is this latter fact that interests us most. In the case of blood, two possible interpretations have been proposed. It has been suggested that the clotting enzymes are capable not only of producing clotting, but also of digesting the substances necessary for the formation of the clot. (Actually these enzymes can also digest the clot after it has been formed). And also it has been claimed that the proteolytic clotting enzymes release heparin into the blood. We should like to believe both of these interpretations, for both of them can be applied to protoplasm.

The first type of interpretation would in large measure help


to explain the colloidal behavior of the protoplasm of the im-mature Chaetopterus egg when the egg is stimulated to begin its maturation divisions. This behavior was discussed at some length in Chapter 10. When the Chaetopterus egg leaves the ovary, there is, according to Goldstein (1953), a liquefaction of the cell cortex and presumably a release of calcium into the cell interior. As a result, there is an activation of a proteolytic en-zyme system. The situation is then comparable to what happens when a proteolytic (clotting) enzyme is injected into the blood stream of a higher animal. In the immature Chaetopterus egg, as Heilbrunn and Wilson (1955b) showed, the protoplasm re-sponds as one might expect. First there is a definite but very transient clotting; this is then followed by a liquefaction. This is illustrated in Fig. 52 in Chapter 10. The transient clotting is an effect due to the clotting action of the proteolytic enzyme system demonstrated by Goldstein. But this enzyme is also capable of exerting a liquefying action, so that the protoplasm first becomes more viscous and then less viscous. This is com-parable to what Tagnon believes happens to the blood of a dog when thrombin or trypsin is injected into the circulation. A proteolytic clotting enzyme can both initiate a clotting reaction and dissolve a clot after it has formed. But for the Chaetopterus egg this is not the complete explanation. For the protease that is activated by the release of calcium into the cell interior does not continue to be active. Indeed Goldstein found that some few minutes after the immature egg had been stimulated, protease activity was lost. Thus in the protoplasm following stimulation there must be some machinery for the inactivation of a protease after it has been activated. This corresponds to what happens in blood following the injection of trypsin, accord-ing to Rocha e Silva and Dragstedt.

It is not only in the Chaetopterus egg that this sort of a system operates. Lundblad (1952) describes a calcium activated pro-tease in sea urchin eggs, and the activity of this protease, almost negligible before the eggs are fertilized, increases as much as 30 or 40 times after fertilization. (These studies were made on cell homogenates.) Then some 40 min. after


fertilization, the protease activity decreases again, and it may become only one-fifth or one-sixth of its peak value. Thus in sea urchin eggs, in the latter half of the mitotic cycle that follows fertilization, the proteolytic activity of the protoplasm fades. This is indicated also by an old observation made by Heilbrunn in 1927. He was extracting from sea urchin eggs a substance which would cause a surface precipitation reaction in the absence of calcium; this he called "ovothrombin." He then decided to find out whether this ovothrombin "can be extracted from fertilized eggs. . . . At various times after fertilization, eggs were shaken with splintered glass. In some of the extracts thus pre-pared, ovothrombin could be demonstrated, in others not. In one experiment extracts prepared 5 and 10 min. after fertiliza-tion showed ovothrombin, those prepared 20, 25, 35, 40, min. after fertilization failed to show ovothrombin. In this experi-ment the temperature was 23-24° C. the time between fertiliza-tion and cleavage 44 min." The conclusion reached was that "it is not improbable that some sort of an antithrombin substance is produced in the egg during the course of the mitotic process." This conclusion is definitely supported by Lundblad's much more extensive and more careful studies.

In muscle also, stimulation apparently results in the production in the protoplasm of substances which can inhibit the action of clotting enzymes. This follows from the work of Most, already discussed in the chapter on muscular contraction. Most found that when a frog muscle is stimulated to fatigue, it liberates to the surrounding medium a substance which prevents the clot-ting of blood in the same manner that heparin does, and may actually be heparin.

Admittedly the data are fragmentary, but the evidence that there is indicates clearly enough that when a cell responds, the activation of a proteolytic and clotting enzyme in the interior protoplasm is followed by the appearance of substances which inhibit the action of such an enzyme. If this is true, then we can go farther and say that the very nature of the response reaction would necessitate such an effect. For if it is true, as we have tried to show in Chapter 6, that heparin and heparin-like sub-stances are generally present in protoplasm, but that they are


often bound to protein, then if a proteolytic clotting enzyme were activated as a result of stimulation, such an enzyme would not only clot the protoplasm, it would also, by virtue of its proteolytic action, free heparin from binding with protein.

Here then is the type of machinery we have been looking for. The very nature of the response mechanism requires that sub-stances are liberated that will block excessive response, prevent a second response, and perhaps also tend to reverse the colloidal changes that the response invokes. We thus have a system which would explain the cyclic colloidal changes that occur in protoplasm and, indeed, must occur if the protoplasmic colloid is to function as a machine capable of doing work. Whether, and to what extent, this interpretation of cyclic colloidal change will help us to understand rhythmic processes such as occur in the heart beat is a question for the future to decide. However, the fact that solutions of heparin stop the frog heart in diastole ( see Chapter 7 ) indicates a possibility in this direction.

If, following a stimulus, an excitable cell is to return to its resting state, this would involve a cycle of colloidal change not only in the cell interior but in the cortex as well. As we have seen, the first effect of a stimulus is to liquefy the cortex; follow-ing such liquefaction, the cortex regains its rigidity. Such a return of rigidity has been followed by Angerer and Wilbur for the ameba cortex. They found that after an electric stimulation either with direct or with alternating current, the relatively fluid cortex almost immediately begins to regain its rigidity. This is shown in Fig. 64.

When the cortex regains its rigidity, this is presumably due to the return of calcium to it. Does the returning calcium come from the cell interior or from the environment? Probably it comes from both. A stimulus that frees cations from the cortex would, after it ceases, leave the cortex relatively avid for cations, and some of the cations taken up by such a cortex would almost certainly come from outside the cell. That they do come from outside the cell is indicated by the fact that stimulation causes a release of potassium from the nerve cell fiber and an uptake of sodium (see Chapter 4) . Hodgkin has assumed that this ex-change involves all of the protoplasm, but it seems more proba-


ble that rapid ion exchange primarily involves the cortex rather than all of the protoplasm. This certainly is a commonly held opinion among students of permeability (see, for example, Mazia, 1940). The fact that following stimulation it is sodium rather than potassium that is taken up by the cell is an indication that the cations that are being replaced in the cortex come mostly

\80\-

from outside the cell, for the outer environment is rich in sodium. Perhaps it is as a result of successive stimulations that cations from the outside of a cell come to enter the interior, or at any rate this may be one aspect of the entrance of cations.

In this chapter it has been shown that from a colloidal stand-point it is possible to understand how a cell can return to a resting state after it has responded to a stimulus. The cortex recovers the calcium it has lost and the protoplasm in the cell interior reverts to its more fluid condition.

F I G . 6 4 . T h e ordinates show relative viscosity in terms of seconds of

centrifugal treatment necessary to displace the granules in the cortex. T h e

abscissas give the t ime in minutes , starting from the moment the electric

current was turned on. (Angerer and W i l b u r ) .

15.

If this book shows nothing else, it shows that there is now definite knowledge about the protoplasmic colloid, knowledge that provides a basis for an understanding of the mechanisms underlying vital activity. This knowledge is primarily of theoreti-cal interest, but eventually it will help to solve problems of prac-tical importance in the fields of human physiology, pharmacology, and pathology.

If we want to know what makes a leg or arm muscle contract, what makes the heart beat, what excites a nerve fiber, or makes a cell divide, we must seek explanations in terms of the material that is in all living cells. This material, chemically so much the same in all types of living organisms, also, by and large, shows a remarkable uniformity in its physical behavior.

In various branches of biology, progress has depended largely on the choice of proper material for study and experimentation. Some types of cells—some types of organisms—can be studied more profitably than others, and by taking advantage of these cells and organisms, great advances have been made that might otherwise never have been realized. What would our knowledge of heredity be like if we had had to depend only on what we could learn about inheritance in mammals? Or our knowledge of embryology? And yet in the all important field of physiology, clinically minded investigators have in large measure concen-trated their attention on the most complicated types of organisms and tissues in the hope that they might arrive as rapidly as pos-sible at information that would help alleviate human suffering.

Surely in attempting to solve a difficult riddle—and what riddle is more complicated than that of living mechanism—it is wise to seek if possible the simplest expression of what it is we attempt

273

CONCLUSION


to explain. The behavior of living cells, infinitely variable as it is, yet follows a definite pattern and this basic uniformity be-comes more and more evident as our knowledge increases.

In the search for a proper understanding of the vital machin-ery, it does not suffice to know the chemical constitution of organisms, tissues, or cells. The chemical analysis of an auto-mobile or a radio would tell us very little as to the ways these machines operate. Because living organisms are internal com-bustion engines, it is interesting to know what types of fuel they require and what happens to the fuel when it is burned. In the search for such information, biochemists have made tre-mendous progress, and we now have a large and important body of information about the chemical changes that occur in extracts of living cells and also considerable information about the changes that occur in the cells themselves. But this information, extensive and impressive as it is, has not shown us how the living machine operates. The oxidative changes that occur in a muscle fiber do not in themselves offer any real explanation of the action of the muscle engine. And the attempts of chemists to explain stimulation and anesthesia have had a broad appeal only because chemists (and many physiologists also) are often unaware of the phenomena they seek to explain.

One difficulty with the purely chemical attempt to explain living mechanism is that the theories that have been proposed are much too simple. Anesthetics or narcotics depress oxidation —stimulation presumably has the opposite effect. But if this is so, why do anesthetics in dilute concentration have a stimulating effect? To disregard facts such as these can make explanations easier to come by, but certainly not correct explanations. For true progress has never been made by disregarding phenomena which are in contradiction to proposed theories.

The study of the colloid chemistry of protoplasm has prog-ressed a long way since I published my book on the subject in 1928. And yet even in 1928 many important truths had begun to emerge and subsequent work has only served to substantiate much of what was known twenty-seven years ago. On the other hand, there is also much new and significant information.

15. CONCLUSION 275

Here in very brief summary is what we know now: 1. In cells generally the major part of the protoplasm is fluid.

At any rate, the hyaline part of the protoplasm is fluid. Sus-pended in the hyaline protoplasm there are granules of one sort or another. If these granules are very numerous, the viscosity of the entire protoplasmic suspension may be rather high. Such dense suspensions may show a dilatancy similar to that of con-centrated starch suspensions, that is to say the viscosity may become progressively greater when more and more shearing force is applied. (In such dilatant suspensions, the viscosity of the dispersion medium is of necessity low ).

2. Typically (and perhaps always) the outer part of the proto-plasm consists of a rigid cortex. This cortex may constitute a rather delicate membrane containing a single layer of granules, or it may be decidedly thicker than this. The cortex is an ex-tremely important part of the cell.

3. The rigidity of the cortex depends on the fact that it contains calcium bound to protein ( and also to lipid ). Chemical and physical agents which are able to free the calcium in the cortex from its bound state cause a liquefaction of the cortex.

4. The fact that the cortex can bind calcium ions and can also release them makes it a calcium electrode. This calcium electrode is an important factor in the electrochemical behavior of the cell. But it is not the only factor—in the cell interior (at least of some cells) chloride ions are bound, so that in addition to the calcium electrode the cell also has a chloride electrode. Both of these electrodes contribute to the bioelectric potentials that the cell is capable of producing.

5. The colloidal behavior of protoplasm is not like that of a pure protein or of any ordinary combination of protein and lipid. When a cell is torn or broken, it seals itself by a reaction called the surface precipitation reaction. This reaction is similar to blood clotting, for it does not occur in the absence of calcium, and the calcium ion is necessary because it activates an enzyme system which acts both as a protease and as a clotting enzyme. The surface precipitation reaction, or s.p.r., is the basic colloidal reaction of protoplasm, and many of the effects produced by


chemical or physical agents on protoplasm can be interpreted in terms of this reaction. If a cell is broken gently, the s.p.r. results merely in the formation of a new precipitation membrane at the outer boundary of the emerging droplet of protoplasm, but if the cell is broken more harshly, the reaction spreads so as to involve a sizable fraction of the cell or perhaps all of the cell. In an ordinary s.p.r., the contents of the cell become exposed to the calcium of the medium. The same type of reaction can occur if calcium is injected into the cell interior or if calcium is liberated from the cortex and enters the interior. Because of the similarity of the s.p.r. to blood clotting, it is proper to speak of the reaction that occurs in the cell interior as a protoplasmic clotting.

6. Protoplasm is always in a state of equilibrium between the factors that induce clotting and those that tend to prevent clot-ting. Cells generally contain thrombin or thromboplastin or similar substances; they also contain heparin or heparin-like sub-stances. Both the clotting factors (thrombin, thromboplastin, etc.) and the anticlotting factors (heparin) can be liberated from cells and can have an influence on other cells of an organism.

7. When a cell is exposed to stimuli, such as heat, cold, mechanical impact, electric shock, ultraviolet radiation, etc., the cortex is liquefied and calcium is released from the cortex into the cell interior. It is of the very nature of stimuli to produce this effect. Thus, for example, ultraviolet radiation is known to break the carboxyl bond that links calcium to protein, and an electric current would tend to separate calcium ion from calcium proteinate.

8. The freeing of calcium from the cortex would tend to make the outer region of the cell negative to the interior. This would be a factor in the initiation of an action potential. Freeing of calcium from the outer region of the cell would also increase the cell permeability.

9. As the calcium enters the cell interior, it activates an en-zyme which is both a protease and a clotting enzyme. This causes the protoplasm or at least some constituent of the proto-plasm to undergo a clotting reaction. In a muscle fiber the result of such a clotting reaction is the shortening of the fiber; in a

15. CONCLUSION 277

marine egg cell, the clotting reaction results in the formation of the mitotic spindle.

10. The protoplasmic clotting reaction involves a change of SH groups to S-S groups. This oxidative change starts off a chain of oxidative reactions, and accordingly one common result of stimulation and response is an increase in the rate of metabolism.

11. The calcium-activated proteolytic enzyme not only causes a clotting reaction, it also tends to digest away the protein that is bound to heparin, or at least to break the bond between protein and heparin. As a result, heparin or some substances like it is liberated, and this heparin inhibits further activity of the enzyme. There is thus a cellular homeostasis, or biostasis, in that the very nature of the response reaction or series of reactions in itself causes an inhibition of excessive reaction (and perhaps also a reversal of the colloidal changes induced by the response ).

12. Fat solvent anesthetics liquefy the cortex and free calcium just as stimulating agents do, but they prevent the clotting reac-tion that would otherwise follow the release of calcium. Under certain conditions, usually in relatively low concentrations, the fat solvents liberate calcium but are not able to prevent the clotting of the protoplasm that follows such a liberation. This is the reason that fat solvent anesthetics in low concentration can act to increase rather than decrease excitability.

13. When cells are exposed to an excess of the magnesium ion, the calcium of the cortex is to a large extent replaced by magnes-ium. The result is a cortex in which the Mg/Ca ratio is increased. Hence when the cells are stimulated, it is largely magnesium ion rather than calcium ion that is freed and enters the cell interior. The magnesium ion is far less potent in causing protoplasmic clot-ting than is the calcium ion. Thus in the sea urchin egg it takes a hundred times as much magnesium to produce a surface preci-pitation reaction as it does calcium. This is why stimulation of nerve or muscle in the presence of excess magnesium fails to elicit a response.

14. Heparin and heparin-like substances can also have an anesthetic action. They cause a relaxation of smooth and heart


muscle and they probably play a part in depressing excitability in hibernating animals and animals in shock.

15. When cells are stimulated or injured, they may give off to the surrounding medium both substances like thromboplastin which promote clotting and substances like heparin which pre-vent clotting (both of blood and of protoplasm). Thus cells injured severely by burns, by mechanical crushing, by anoxia, by roentgen radiation, etc., can give off substances which have an effect on cells and tissues throughout the organism. On the basis of these facts a theory of shock can be formulated, a theory which would explain why substances like thrombin, trypsin and pep-tone can cause shock.

The above summary should not be regarded as anything more than the statement of a set of conclusions arrived at on the basis of a considerable amount of data. These data are presented in some detail in the earlier chapters of this book. Moreover, evidence in support of our conclusions is constantly growing.

Our story has been a long and a complex one. It has taken us into various aspects of physiology and, indeed, we have invaded the fields of muscle physiology, nerve physiology, cell division, cytochemistry, pathology, and pharmacology, and we have even ventured into the practical field of medical science. Whether the excursions into any or all of these fields have been profitable or not, only the future can tell. Doubtless mistakes in interpreta-tion have been made. In this era of scientific complexity and specialization, an era in which there are so many different branches and subdivisions of science, it is hard for any one person to understand the facts and the points of view of widely diver-gent disciplines. Concentration in very specialized parts of scien-tific inquiry is the order of the day; and yet in spite of this, or perhaps because of it, there seems to be a need for a broad outlook, for a theory or theories that would interpret the varied activities of the living substance in terms of a consistent general plan, a plan that is in keeping with and not in contradiction to our knowledge of the physical and colloidal properties of the living material. If, as we believe, these properties are funda-mentally similar in almost all types of living cells and organisms,

15. CONCLUSION 279

then there should certainly be a fundamental basis for interpret-ing vital activity, whether it be in a muscle cell, a nerve cell, or in a dividing egg cell.

In attempting to find out what this general plan is, I have tried to view all the facts available to me and to present them as simply and clearly as possible. At the risk of failing to be impres-sive, I have avoided mathematical formulations and abstruse dis-cussions of physical chemistry. This has been done in the hope that more biologists and more physiologists, and possibly also some of those concerned with clinical problems will become interested in the colloid chemistry of protoplasm. There are so many important facts still to be learned and so few workers in the field. And at the present time, with new techniques and new apparatus available, it should be possible to make more rapid progress than was possible in the past. But apparatus alone, no matter how complicated or how expensive, is not a substitute for thought and imagination. And the mere gathering of data, no matter how precise they may be, is not necessarily of great value. What is needed is a clear appreciation and understanding of the problems involved. Experiments should be planned, not merely on the basis of apparatus available, but primarily in terms of an attempt to interpret one or another of the mysterious phenomena involved in vital activity.

With properly trained workers, men trained not only in tech-niques but in ways of thought as well, it should be possible to make rapid progress, so that perhaps before many years pass, what has been presented in this book may constitute but a small fraction of our knowledge concerning the dynamics of living protoplasm.

BIBLIOGRAPHY

Abelous, J. E. 1894. Toxicité du sang et des muscles des animaux fatigués. Arch. Physiol, norm, pathol. 6 [5 ] : 433-439.

Abrahams, D. G., and Howarth, S. 1950. A peripheral action of heparin. Brit. Heart J. 12: 429.

Adriani, J. 1946. "Chemistry of Anesthesia." C. C Thomas, Spring-field, 111.

Ahlquist, R. P. 1950. The action of various drugs on the arterial blood flow of the pregnant, canine uterus. / . Am. Pharm. Assoc. 39: 370-373.

Alcock, Ν. H. 1906. The action of anesthetics on living tissues. I. The action on isolated nerve. Ρ roc. Roy. Soc. B77: 267-283.

Alday-Redonnet, T. 1920. Sensibilisierung des Trendelenburgschen Froschpräparates zur Adrenalinmessung. Biochem. Ζ. 110: 306-318.

Allen, J. G., and Egner, W. 1948. The reaction caused by the intra-venous injection of salmine sulfate in dogs. Federation Proc. 7: 1-2.

Allen, J. G., and Jacobson, L. O. 1947. Hyperheparinemia, cause of hemorrhagic syndrome associated with total body exposure to ioniz-ing radiation. Science 105: 388-389.

Allen J. G , Moulder, P. V., and Enerson, D. M. 1951. Pathogenesis and treatment of the postirradiation syndrome. /. Am. Med. Assoc. 145: 704-711.

Allen, J. G , Sanderson, M., Milham, M., Kirschon, Α., and Jacobson, L. O. 1948. Hyperheparinemia (? ) . An anticoagulant in the blood of dogs with hemorrhagic tendency after total body exposure to roentgen rays. /. Exptl. Med. 87: 71-86.

Ambache, N., and Lippold, O. C. J. 1949. Brachycardia of central origin produced by injection of tetanus toxin into the vagus nerve. /. Physiol. (London) 108: 186-196.

Angerer, C. A. 1936. The effects of mechanical agitation on the relative viscosity of amoeba protoplasm. J. Cellular Comp. Physiol. 8: 329-345.

Angerer, C. Α., and Wilbur, Κ. M. 1943. The action of various types of electric fields on the relative viscosity of the plasmagel of Amoeba proteus. Physiol. Zoo/. 16: 84-92.

Anslow, G. Α., Foster, M. L. and Klingler, C. 1933. The absorption spectra of glycine solutions and their interpretation. /. Biol. Chem. 103: 81-92.

280

BIBLIOGRAPHY 281

Ashkenaz, Ε. W. 1938a. The release of calcium from muscle on stimulation by ultraviolet radiation. /. Cellular Comp. Physiol. 12: 139-147.

Ashkenaz, E. W. 1938b. Anomalous behavior of isolated muscle fibers toward certain chemical stimuli. Proc. Soc. Exptl. Biol. Med. 38: 719-720.

Astbury, W. T. 1939. X-ray studies of the structure of compounds of biological interest. Ann. Rev. Biochem. 8: 113-132.

Astbury, W. T. 1947. On the structure of biological fibers and the problem of muscle. Proc. Roy. Soc. B134: 303-328.

Austin, J. H., Sunderman, F. W., and Camack, J. G. 1927. Studies in serum electrolytes. II. The electrolyte composition and the pH of serum of a poikilothermous animal at different temperatures. /. Biol. Chem. 72 : 677-685.

Autenrieth, H. F. 1833. "Ueber das Gift der Fische." Osiander, Tübingen.

Baas-Becking, L. G. M., Sande Bakhuyzen, H. v.d., and Hotelling, H. 1928. The physical state of protoplasm. Verhandl. Koninkl. Akad. Wetenschap. Amsterdam, Afdeel. Natuurk. (Tweede Sectie) 25(5), 1-53.

Babasaki, Y. 1932. Pharmakologische Beiträge zur Kenntnis der Blutgerinnung. III. Über den Einfluss der verschiedenen Narkotika auf die Blutgerinnung sowie auf die koagulative Bestandteile im Blut. Folia Pharmacol. Japon. Opera Orig. 14: 14-19.

Bainbridge, F. Α., and Trevan, J. W. 1917. In a report on shock and some allied conditions, issued by the Medical Research Com-mittee. Brit. Med. J. i: 381-383.

Balazs, Α., and Holmgren, H. 1949. Effect of sulfomucopolysac-charides on growth of tumor tissue. Proc. Soc. Exptl. Biol. Med. 72: 142-145.

Bärlund, Η. 1938. Einfluss des Athyläthers auf die Permeabilität der Chara-Zellen. Protoplasma 30: 70-78.

Barnard, R. D. 1948. Similarity to heparin of the clotting inhibitor in acute leucemia and the significance of hyperheparinemia in estrapenic cholinergic states. Science 107: 571-572.

Barone, V. G. 1932. Azione dell'acetilcolina suireccitabilitâ elettrica dei vaghi. Arch. Fisiol. 30: 590-609.

Barr, G. 1931. "A Monograph of Viscometry." Oxford, New York, 1931.

Baumberger, J. P. 1941. Some evidence in support of a sulfhydryl mechanism of blood clotting. Am. J. Physiol. 133: P206-P207.

Bayliss, W. M. 1915. "Principles of General Physiology," 1st ed. Longmans, Green, London.


Bayliss, VV. M. 1924. "Principles of General Physiology." 4th ed. Longmans, Green, London.

Beams, H. W. and Evans, T. C. 1940. Some effects of colchicine upon the first cleavage in Arbacia punctulata. Biol. Bull 79: 188-198.

Bear, R. S., Schmitt, F. Ο., and Young, J. Z. 1937. The ultrastruc-ture of nerve axoplasm. Proc. Roy. Soc. Β123: 505-519.

Belar, K. 1927. Beiträge zur Kenntnis des Mechanismus der indirek-ten Kernteilung. Naturwissenschaften 15: 725-734.

Beraldo, W. T. 1950. Formation of bradykinin in anaphylactic and peptone shock. Am. J. Physiol 163: 283-289.

Bernard, C. 1875. "Leçons sur les anesthésiques et sur l'asphyxie." Balliere, Paris.

Bernard, C. 1878-1879. "Leçons sur les phénomènes de la vie com-muns aux animaux et aux végétaux," 2 vols. Balliere, Paris.

Bernstein, J. 1915. Experimentelles und Kritisches zur Theorie der Muskelkontraktion. Pflügers Arch. ges. Physiol. 162: 1-53.

Berwick, M. C. 1951. The effect of anesthetics on calcium release. /. Cellular Comp. Physiol. 38: 95-107.

Beznàk, A. von. 1931. Über die Erhöhung des Calciumgehaltes des Blutserums bei Strychninvergiftung. Naunyn-Schmiedebergs Arch, exptl Pathol u. Pharmakol. 160: 397-400.

Bezold, A. von. 1861a. Über einige Zeitverhältnisse welche bei der direkten elektrischen Erregung des Muskels in's Spiel kommen. Monatsher. Kön. Preuss. Akad. Wiss. Berlin 1860, 760-765.

Bezold, A. von. 1861b. Über die zeitlichen Verhältnisse, welche bei der elektrischen Erregung der Nerven in's Spiel kommen. Monatsher. Kön. Preuss. Akad. Wiss. Berlin 1860, 736-743.

Biedermann, W. 1879. Beiträge zur allgemeinen Nerven-und Muskelphysiologie. III. Über die polaren Wirkungen des elek-trischen Stromes im entnervten Muskel. Sitzber. Akad. Wiss. Wien. Math, naturw. Kl Abt. III 79: 289-320.

Biedermann, W. 1881. Beiträge zur allgemeinen Nerven-und Muskelphysiologie. VI. Über rhythmische, durch chemische Rei-zung bedingte Contractionen quergestreifter Muskeln. Sitzber. Akad. Wiss. Wien. Math, naturw. Kl. Abt. III 82: 257-275.

Biedermann, W. 1909. Vergleichende Physiologie der irritablen Substanzen. Ergeh. Physiol 8: 26-211.

Blinks, L. R. 1949. The source of the bioelectric potentials in large plant cells. Proc. Natl. Acad. Set. (U.S.) 35: 566-575.

Blumenfeldt, E. 1925. Über den Einfluss von Kalium- und Calci-umsalzen auf die Erregbarkeit des Froschnerven. Biochem. Ζ. 156: 236-248.

Bocher, C. 1952. The effect of an extract of starfish gonads and heparin upon cells grown in tissue culture. Master's thesis, Univer-sity of Pennsylvania, Philadelphia.

BIBLIOGRAPHY 283

Bois Reymond, Ε. du 1874. Experimentalkritik der Entladungs-hypothese über die Wirkung von Nerv auf Muskel. Monatsher. Kön. Preuss Akad. Wiss. Berlin 1874: 519-560.

Botta, Β. 1931. RicerchesuUa carica e sul trasporto elettrico della figura cromatica della cellule in mitosi in culture di cuore embrio-nale di polio (nota preventiva). Arch, exptl. Zellforsch. Gewebe-zücht. 12: 455-464.

Bouckaert, J. J. 1927. L'ion calcium, condition d'excitabilité des nerfs vaso-dilatateurs de même que du pneumogastrique. /. physiol. (Paris) 25: 507-517.

Braun, H. 1924. "Local Anesthesia: Its Scientific Basis and Practical Uses." 2nd Amer. ed. (translated from the 6th German ed. by M. L. Harris). Lea and Febiger, Philadelphia.

Brink, F., and Bronk, D. W. 1937. Rhythmic activity of single nerve fibers induced by low calcium. Proc. Soc. Exptl. Biol. Med. 37: 94-95.

Brink, F., and Posternak, J. M. 1948. Thermodynamic analysis of the relative effectiveness of narcotics. /. Cellular Comp. Physiol. 32: 211-233.

Brinley, F. J. 1928. The effect of chemicals on viscosity of proto-plasm of amoeba as indicated by brownian movement. Protoplasma 4: 177-191.

Brooks, S. C, and Brooks, M. M. 1941. "The Permeability of Living Cells." Borntraeger, Berlin.

Brown, R. H. J. 1940. The protoplasmic viscosity of paramecium. /. Exptl. Biol. 17: 317-324.

Bryant, S. H., and Tobias, J. H. 1952. Studies on optical properties of active nerve. Federation Proc. 11: 19.

Bucciante, L. 1927. Qualche rilievo suH'attivita ameboide delle cellule in mitosi. Protoplasma 2: 80-88.

Buchthal, F., Deutsch, Α., and Knappeis, G. G. 1944. Release of contraction and changes in birefringence caused by adenosine tri-phosphate in isolated cross striated muscle fibers. Acta Physiol. Scand. 8: 271-287.

Buchthal, F., Deutsch, Α., and Knappeis, G. G. 1946. Further in-vestigations on the effect of adenosine triphosphate and related phosphorus compounds on isolated striated muscle fibres. Acta Physiol. Scand. 11: 325-334.

Buchthal, F., Knappeis, G. G., and Sjöstrand, T. 1939. Optisches Verhalten der quergestreiften Muskelfaser im natürlichen Licht. Skand. Arch. Physiol. 82: 225-257.

Bülbring, E. 1931. Über die Beziehungen zwischen Epithelkörper-chen, Calciumstoffwechsel und Knochenwachstum. Naunyn-Schmie-debergs Arch, exptl. Pathol, u. Pharmakol. 162: 209-248.


Bullough, W. S. 1948. The effects of experimentally induced rest and exercise on the epidermal mitotic activity of the adult male mouse, Mus musculus L. Proc. Roy. Soc. B135: 233-242.

Bullough, W. S., and Green, H. N. 1949. Shock and mitotic activity in mice. Nature 164: 795-796.

Burgen, A. S. V., and Terroux, K. G 1953. On the negative ino-tropic effect in the cat's auricle. / . Physiol. (London) 120: 449-464.

Burns, C. M. 1933. The calcium content of muscle. Biochem. J. 27: 22-32.

Biitschli, O. 1894. "Investigations on Microscopic Foams and on Protoplasm. Black, London.

Cameron, G. R. 1952. "Pathology of the Cell." C. C Thomas, Spring-field, 111.

Cannon, W. B. 1939. "The Wisdom of the Body." Norton, New York.

Carter, J. R., and Warner, E. D. 1954. Evaluation of disulfide bonds and sulfhydryl groups in the blood clotting mechanism. Am. J. Physiol 179: 549-556.

Cecil, R. L., and Loeb, R. F. 1951. "A Textbook of Medicine," 8th ed. Saunders, Philadelphia.

Chaet, A. B. 1951. The physiological action of heparin solutions. Biol. Bull. 101: 206.

Chaet, A. B. 1955. Heat death in Phascolosoma. Physiol Zool In press.

Chambers, R., and Kao, C. 1950. Micro-injection of CaCl 2 into the giant nerve axon of the squid. Biol. Bull 99: 344.

Chambers, R., and Kao, C. 1951. The physical state of the axoplasm in situ in the nerve of the squid mantle. Biol. Bull. 101: 206.

Chambers, R., and Kao, C. 1952. The effect of electrolytes on the physical state of the nerve axon of the squid and of stentor, a protozoön. Exptl. Cell. Research 3: 564-573.

Chambers, R., and Pollack, H. 1927. Micrurgical studies in cell physiology. IV. Colorimetric determination of the nuclear and cytoplasmic pH in the starfish egg. / . Gen. Physiol. 10: 739-755.

Chargaff, E., and Bendich, A. 1943. On the coagulation of fibrino-gen. /. Biol. Chem. 149: 93-110.

Charles, A. F., and Scott, D. A. 1933. Studies on heparin. II. Hep-arin in various tissues. /. Biol Chem. 102: 431-435.

Churney, L. 1936. The quantitative determination of mitotic elon-gation. Biol Bull. 70: 400-407.

Churney, L. 1940. The physico-chemical properties of the nucleus. Univ. of Pennsylvania Bicentennial Conference, pp. 113-128. Phila-delphia.

BIBLIOGRAPHY 285

Churney, L., and Klein, H. M. 1937. The electrical charge on nu-clear constituents (salivary gland cells of Sciara coprophila). Biol. Bull 72: 384-388.

Cicardo, V. H., and Beninson, D. 1952. El bloqueo neuromuscular por descalcificantes. Rev. soc. argentina biol 28: 174-180.

Clark, A. J., Percival, G. H., and Stewart, C. P. 1928. Action of calcium ions on the frogs heart. /. Physiol. (London) 66: 346-355.

Clark, J. H. 1923. The effect of ultraviolet light on the condition of calcium in the blood. Am. } . Hyg. 3: 481.

Cloetta, M., Fischer, H., and van der Loeff, M. R. 1934. Die Bio-chimie von Schlaf und Erregung, mit besonderer Berücksichtigung der Bedeutung der Kationen. Naunyn-Schmiedeberg's Arch, exptl. Pathol, u. Pharmakol. 174: 589-675.

Cole, Κ. S. 1932. Surface forces of the Arbacia egg. /. Cellular Comp. Physiol. 1: 1-9.

Cole, Κ. S., and Curtis, H. J. 1939. Electric impedance of the squid giant axon during activity. /. Gen. Physiol. 22: 649-670.

Cole, K. S., and Michaelis, Ε. M. 1932. Surface forces of fertilized Arbacia eggs. /. Cellular Comp. Physiol. 2: 121-126.

Conklin, E. G. 1902. Karyokinesis and cytokinesis in the maturation, fertilization and cleavage of Crepidula and other Gasteropoda. /. Acad. Natl. Set. {Philadelphia) [2] 12: 1-121.

Conklin, E. G. 1912. Experimental studies on nuclear and cell divi-sion in the eggs of Crepidula. /. Acad. Natl. Set. (Philadelphia) [2] 15: 503-591.

Coombs, H. C, Searle, D. S., and Pike, F. H. 1934. The changes in the concentration of inorganic calcium and phosphorus during con-vulsions of experimental origin, in cats, before and after thyropara-thyroidectomy, with and without bromide therapy. Am. J. Psychiat. [n. s.] 13: 761-797.

Coppée, G. 1946. Le rôle des ions calcium dans la transmission neuro-musculaire. Arch, intern. Physiol, 54: 323-336.

Costello, D. P. 1932. The surface precipitation reaction in marine eggs. Protoplasma 17: 239-257.

Costello, D. P. 1939. The volumes occupied by the formed cyto-plasmic components in marine eggs. Physiol Zool. 12: 13-21.

Couillard, P. 1953. Antimitotic substance in the ovary of the com-mon puffer Sphaeroides maculatus. Biol. Bull. 105: 372.

Couillard, P. 1954. The effect of tetrodotoxin and related substances on cell division. Biol. Bull. 107: 308.

Cowan, S. L. 1940. The action of eserine-like compounds upon frog's nerve-muscle preparations, and conditions in which a single shock can evoke an augmented muscular response. Proc. Roy. Soc. B129: 356-391.


Crane, Ε. E. 1950. Bioelectric potentials, their maintenance and function. Progr. Biophys. and Biophys. Chem. 1: 85-136.

Cremer, M. 1909. Die allgemeine Physiologie der Nerven. In "Handbuch der Physiologie des Menschen" (Nagel, ed.), Vol. 4, pp. 792-992. Vieweg, Braunschweig.

Cullis, W. C, and Lucas, C. L. T. 1936. Action of acetylcholine on the aneural chick heart. /. Physiol. (London) 86: 53P-55P.

Cunningham, E. 1910. On the velocity of steady fall of spherical particles through a fluid medium. Proc. Roy. Soc. A83: 357-365.

Curtis, H. J., and Cole, K. S. 1942. Membrane resting and action potentials from the squid giant axon. /. Cellular Comp. Physiol 19: 135-144.

Dahlgren, U. 1915. Structure and polarity of the electric motor nerve-cell in torpedoes. Carnegie Inst. Wash. Puhl No. 212: 213-256.

Dale, H. H., Feldberg, W., and Vogt, M. 1936. Release of acetyl-choline at voluntary motor nerve endings. /. Physiol (London) 86: 353-380.

Dan, K. 1933. Electrokinetic studies of marine ova. I. Arbacia punctulata. /. Cellular Comp. Physiol. 3: 477-492.

Dan, K. 1934. Electrokinetic studies of marine ova. II. Cumingia tellinoides, Asterias forbesii, Echinarachnius parma, Nereis limbata and Cerebratulus lacteus. Biol. Bull 66: 247-256.

Daugherty, K. 1937. The action of anesthetics on amoeba proto-plasm. Physiol Zool 10: 473-483.

Davidman, Α., and Dolley, D. H. 1921. Cloudy swelling a process of stimulation. /. Med. Research 42: 515-536.

Davis, H. A. 1949. "Shock and Allied Forms of Failure of the Circulation." Grune, New York.

Davson, H., and Danielli, J. F. 1952. "The Permeability of Natural Membranes," 2nd ed. Cambridge, New York.

De, P. 1928. The rate of action of drugs and ions on frog's heart. /. Pharmacol. Exptl. Therap. 33: 115-128.

Debie, P., Lafontaine, Α., and Willot, W. 1946. Influence des régions brûlées sur l'état général chez la grenouille. Arch, intern. pharmacodynamic 73: 287-293.

Delezenne, C. 1897. Recherches sur la coagulation du sang chez les oiseaux. I. Arch, physiol. norm, et pathol. [5] 9: 333-346.

Delezenne, C. 1905. Sur le role des sels dans i'activation du suc pancréatique. Spécificité du calcium. Compt. rend. soc. biol 59: 478-480.

BIBLIOGRAPHY 287

Dixon, H. H., Davenport, Η. Α., and Ranson, S. W. 1929. The calci-um content of muscular tissue during parathyroid tetany. J. Biol. Chem. 83: 737-739.

Dreyer, Ν. B. 1925. Zur Herzwirkung des Magnesiums. Naunyn-Schmiedebergs Arch, exptl Pathol, u. Pharmakol. 105: 54-57.

Dujardin, F. 1835. Recherches sur les organismes inférieurs. III. Sur les prétendus estomacs des animalcules Infusoires et sur une substance appelée Sarcode. Ann. sei. not. Zool [2] 4: 364-377.

Dujardin, F. 1838. Mémoire sur l'organisation des Infusoires. Ann. sei. nat. Zool. [2] 10: 230-315.

Dujardin, F. 1841. "Histoire naturelle des zoophytes." Roret, Paris. Dustin, P. 1947. Suppl. Lloydia 10: 106-114.

Eccles, J. C. 1953. "The Neurophysiological Basis of Mind." Ox-ford, New York.

Edsall, J. T., and Lever, W. F. 1951. Effect of ions and neutral molecules on fibrin clotting. J. Biol. Chem. 191: 735-756.

Eigsti, O. J. 1947. Colchicine bibliography. Lloydia 10: 65-105. Eilers, H. 1941. Die Viskosität von Emulsionen hochviskoser Stoffe

als Funktion der Konzentration. Kolloid-Z. 97: 313-321. Engelberg, H., and Masseil, T. B. 1953. Heparin in the treatment

of advanced peripheral atherosclerosis. Am. J. Med. Set. 225: 14-19. Engelmann, T. W. 1867. Über den Ort der Reizung in den Muskel-

faser bei Schliessung und Öffnung eines constanten electrischen Stromes. Jen. Z. Naturw. 3: 445-447.

Engelmann, T. W. 1868. Über Reizung der Muskelfaser durch den constanten Strom. Jen. Z. Naturw. 4: 295-306.

Engelmann, T. W. 1870. Beiträge zur allgemeinen Muskel- und Nervenphysiologie. Pflügers Arch. ges. Physiol. 3: 247-326.

Engelmann, T. W. 1893. "Über den Ursprung der Muskelkraft," 2nd ed. Engelmann, Leipzig.

Erlanger, J., and Gasser, H. S. 1919. Studies in secondary traumatic shock. III. Circulatory failure due to adrenalin. Am. J. Physiol 49: 345-376.

Essner, E. S. 1954. The breakdown of isolated yolk granules by cations. Protoplasma 43: 79-89.

Falkenhagen, H., and Leist, M. 1952. Eine Bemerkung zu den Arbeiten von G. B. Bonino und Mitarbeitern über die Konzentra-tionsabhängigkeit der Dielektrizitätskonstanten. Z. physik. Chem. 199: 370-375.

Fatt, P. 1950. The electromotive action of acetylcholine at the motor end-plate. J. Physiol (London) 111:408-422.


Fatt, P., and Katz, B. 1953. The electrical properties of crustacean muscle fibers. /. Physiol (London) 120: 171-204.

Federow, B. G. 1935. Essai de l'étude intravitale des cellules nerveuses et des connexions interneuronales dans le système nerveux autonome. Trav. Lab. recherche biol Univ. Madrid 30: 403-434.

Fenn, W. O. 1945. Contractility. In Höber's "Physical Chemistry of Cells and Tissues" (R. Höber, ed.), pp. 445-522. Blakiston, Phila-delphia.

Fenn, W. O., Cobb, D. M., Manery, J. F., and Bloor, W. R. 1937. Electrolyte changes in cat muscle during stimulation. Am. /. Physiol. 121: 595-608.

Ferguson, J. 1939. The use of chemical potentials as indices of toxicity. Proc. Roy. Soc. B127: 387-404.

Ferguson, J. 1951. Relations between thermodynamic indices of narcotics. In Mécanisme de la Narcose. Colloq. interna. Centre natl. recherche sei. (Paris) No. 26, 25-39.

Fessard, A. 1936. "Recherches sur l'activité rythmique des nerfs isolés." Hermann, Paris.

Fetter, D. 1926. Determination of the protoplasmic viscosity of Paramecium by the centrifuge method. /. Exptl. Zool. 44: 279-283.

Fischer, Albert. 1930. Regeneration. Versuche an Gewebekulturen in vitro. Virchows Arch, pathol. Anat. u. Physiol. 279 : 94-136.

Fischer, Albert. 1934. Über die Identität des Muskel- und Blut-thrombins. Biochem. Z. 270: 275-280.

Fischer, Albert. 1936. Über die Wirkung des Heparins auf das Wachstum von Gewebezellen in Vitro. Protoplasma 26: 344-350.

Fischer, Alfred. 1899. "Fixirung, Färbung und Bau des Proto-plasmas." Fischer, Jena.

Flaig, J. V. 1947. Viscosity changes in axoplasm under Stimulation. /. Neurophysiol. 10: 211-221.

Fleckenstein, Α., Janke, J., Davies, R. E., and Krebs, Η. A. 1954. Contraction of muscle without fission of adenosine triphosphate or creatine phosphate. Nature 174: 1081-1083.

Flory, P. J. 1953. "Principles of Polymer Chemistry." Cornell Univ. Press, Ithaca.

Folkow, B. 1949. The vasodilator action of adenosine triphosphate. Acta Physiol Scand. 17: 311-316.

Foster, R. Η. K. 1940. On the role of oxalic acid in blood clotting. Proc. Soc. Exptl. Biol. Med. 44: 136-139.

Frederikse, A. M. 1933. Viskositätsänderungen des Protoplasmas während der Narkose. Protoplasma 18:194-207.

Freund, H. 1920. Über die pharmakologischen Wirkungen des defibrinierten Blutes. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 86: 266-280.

BIBLIOGRAPHY 289

Freundlich, H., and Röder, H. L. 1938. Dilatancy and its relation to thixotropy. Trans. Faraday Soc. 34: 308-316.

Fröhlich, F. W. 1929. Nervenreize. In Bethe's "Handbuch der normalen und pathologischen Physiologie" (Bethe, ed.), vol. 9, pp. 177-211. Springer, Berlin.

Frommhagen, L. H., Fahrenbach, M. J., Brockman, J. Α., and Stokstad, E. L. R. 1953. Heparin-like anticoagulants from mollusca. Proc. Soc. Exptl. Biol. Med. 82: 280-283.

Fuchs, H. J., and Falkenhausen, M. von. 1931. Über die Beziehung von Glykolyse zur Blutgerinnung. Z. ges. exptl. Med. 79: 23-28.

Fühner, H. 1904. Pharmakologische Studien an Seeigeleiern. Der Wirkungsgrad der Alkohole. Naunyn-Schmiedeberg's Arch, exptl. Pathol, u. Pharmakol. 52: 69-82.

Fürth, R. 1930. Über die Messung der Viskosität sehr kleiner Flüssigkeitsmengen mit Hilfe der Brownschen Bewegung. Z. Physik 60: 313-316.

Gahringer, J. E. 1926. The sensitization of pigeons to foreign pro-teins. /. Immunol. 12: 477-488.

Gellhorn, Ε. 1929. "Das Permeabiltätsproblem." Springer, Berlin. Gerber, C. F., and Blanchard, E. W. 1945. The effect of certain

substances on clotting time in vitro. Am. J. Physiol. 144: 447-456. Ghosh, J. J., and Quastel, J. H. 1954. Narcotics and brain respira-

tion. Nature 174: 28-31. Gilbert, N. C. and Nalefski, L. A. 1949. The effect of heparin and

dicumarol in increasing the coronary flow volume. /. Lab. Clin. Med. 34: 797-805.

Gley, E., and Bouckaert, J. J. 1927. Diminution d'excitabilité des nerfs vaso-dilatateurs, a la suite de la diminution des ions calcium du sang. Compt. rend. soc. biol. 96: 770-772.

Goerner, A. 1931. The influence of anticlotting agents on trans-plantation and growth of tumor tissue. /. Lab. Clin. Med. 16: 369-372.

Goldstein, L. 1953. A study of the mechanism of activation and nuclear breakdown in the Chaetopterus egg. Biol. Bull. 105: 87-102.

Graham, T. 1861. Liquid diffusion applied to analysis. Phil. Trans. Roy. Soc. 151: 183-224.

Gramenizki, M. I. 1934. Anatomisch-histologische mikroskopische Untersuchungen am schlagenden Froschherzen. Ζ. Zellforsch, u. mikrosrop. Anat. 21: 580-595.

Greenstein, J. P. 1947. "Biochemistry of Cancer," 1st ed. Academic Press, New York.

Greenstein, J. P. 1954. "Biochemistry of Cancer," 2nd ed., Academic Press, New York.


Gross, P. R. 1952. A study of colloidal changes in sea urchin egg homogenates. Biol. Bull 103: 293.

Gross, P. R. 1954a. On the mechanism of the yolk-lysis reaction. Protoplasma 43: 416-428.

Gross, P. R. 1954b. Alterations in the proteins of sea urchin egg homogenates treated with calcium. Biol Bull 107: 364-385.

Guest, M. M., Ware, A. G., and Seegers, W. H. 1947. A quantita-tive study of antifibrinolysin activity during pteroylglutamic acid deficiency. Am. J. Physiol 150: 661-669.

Gwathmey, J. T. 1924. "Anesthesia," 2nd ed. Macmillan, New York.

Haeckel, E. 1857. Ueber die Gewebe des Flusskrebses. Arch. Anat. Physiol u. wiss. Med, 1857: 469-568.

Hahnert, W. F. 1932. A quantitative study of reactions to electricity in Amoeba proteus. Physiol Zool 5: 491-526.

Haist, R. E., and Hamilton, J. I. 1944. Reversibility of carbohydrate and other changes in rats shocked by a clamping technique. / . Physiol (London) 102:471-483.

Harding, D. 1951. Initiation of cell division in the Arbacia egg by injury substances. Physiol Zool 24: 54-69.

Hardy, W. B. 1895. On some histological features and physiological properties of the post-oesophageal nerve cord of the C r u s t a c e a . Phil

Trans. Roy. Soc. B185: 83-117. Hardy, W. B. 1913. Note on differences in electrical potential

within the living cell. J. Physiol (London) 47: 108-111. Harkins, H. N. 1942. "The Treatment of Burns." C. C Thomas,

Springfield, 111. Harris, D. L. 1939. An experimental study of the pigment granules

of the Arbacia egg. Biol Bull 77: 310. Harris, D. L. 1943. The osmotic properties of cytoplasmic granules

of the sea urchin egg. Biol Bull. 85: 179-192. Harris, J. E. 1935. Studies on living protoplasm. I. Streaming

movements in the protoplasm of the egg of Sabellaria alveolata (L . ) . / . Exptl. Biol 12: 65-79.

Harris, T. A. B. 1951. "Mode of Action of Anaesthetics." Williams & Wilkins, Baltimore.

Hartmann, Ε., and Kühnau, J. 1930. Bestehen Beziehungen der Glykolyse zu der Blutgerinnung? Z. ges. exptl. Med. 73: 720-737.

Hartwell, J. L. 1951. Survey of compounds which have been tested for carcinogenic activity. Public Health Service (U.S.) Publ No. 149, 2nd ed.

Hartwell, J. L., Shear, M. J., and Adams, J. R. 1943. Chemical treatment of tumors. VII. Nature of the hemorrhage-producing fraction from Serratia marcescens (Bacillus prodigiosus) culture filtrate. / . Natl. Cancer. Inst. 4: 107-122.

BIBLIOGRAPHY 291

Harvey, A. M. 1939. The peripheral action of tetanus toxin. /. Physiol (London) 96:348-365.

Heilbronn, A. 1914. Zustand des Plasmas und Reizbarkeit. Ein Beitrag zur Physiologie der lebenden Substanz. Jahrb. wiss. Botan. 54: 357-390.

Heilbronn, A. 1922. Eine neue Methode zur Bestimmung der Vis-kosität lebender Protoplasten. Jahrb. wiss. Botan. 61: 284-338.

Heilbrunn, L. V. 1915a. Studies in artificial parthenogenesis. II. Physical changes in the egg of Arbacia. Biol. Bull. 29: 149-203.

Heilbrunn, L. V. 1915b. The measurement of oxidation in the sea-urchin egg. Science 42: 615-616.

Heilbrunn, L. V. 1917. An experimental study of cell division. Anat. Record 11: 487-489.

Heilbrunn, L. V. 1920a. An experimental study of cell-division. /. Exptl. Zool. 30: 211-237.

Heilbrunn, L. V. 1920b. The physical effect of anesthetics upon living protoplasm. Biol Bull 39: 307-315.

Heilbrunn, L. V. 1921. Protoplasmic viscosity changes during mitosis. /. Exptl. Zool. 34: 417-447.

Heilbrunn, L. V. 1924. The colloid chemistry of protoplasm. IV. The heat coagulation of protoplasm. Am. J. Physiol. 69: 190-199.

Heilbrunn, L. V. 1925. The action of ether on protoplasm. Biol. Bull. 49: 461-476.

Heilbrunn, L. V. 1926a. The absolute viscosity of protoplasm. /. Exptl. Zool. 44: 255-278.

Heilbrunn, L. V. 1926b. The centrifuge method of determining protoplasmic viscosity. /. Exptl Zool. 43: 313-320.

Heilbrunn, L. V. 1926c. The physical structure of the protoplasm of sea-urchin eggs. Am. Naturalist 60: 143-156.

Heilbrunn, L. V. 1927. The colloid chemistry of protoplasm. V. A preliminary study of the surface precipitation reaction of living cells. Arch, exptl. Zellforsch. Gewebeziicht. 4: 246-263.

Heilbrunn, L. V. 1928. "The Colloid Chemistry of Protoplasm." Borntraeger, Berlin.

Heilbrunn, L. V. 1929a. Protoplasmic viscosity of amoeba at dif-ferent temperatures. Protoplasma 8: 58-64.

Heilbrunn, L. V. 1929b. The absolute viscosity of amoeba proto-plasm. Protoplasma 8: 65-69.

Heilbrunn, L. V. 1929c. Protoplasma 6: 519-520. Heilbrunn, L. V. 1930. The action of various salts on the first stage

of the surface precipitation reaction in Arbacia egg protoplasm. Protoplasma 11: 558-573.

Heilbrunn, L. V. 1934a. The effect of anesthetics on the surface precipitation reaction. Biol. Bull. 66: 264-275.


Heilbrunn, L. V. 1934b. The action of anaesthetics on living proto-plasm. Proc. Am. Phil. Soc. 74: 159-165.

Heilbrunn, L. V. 1937. "An Outline of General Physiology," 1st ed. Saunders, Philadelphia.

Heilbrunn, L. V. 1940. The action of calcium on muscle protoplasm. Physiol. Zool. 13: 88-94.

Heilbrunn, L. V. 1950. Viscosity measurements. In "Biophysical Research Methods" (F. M. Uber, ed.), chapter 4, New York.

Heilbrunn, L. V. 1951. The colloid chemistry of narcosis. In Mécanisme de la Narcose. Colloq. intern, centre natl. recherche

sei. (Paris) No. 26, 163-177. Heilbrunn, L. V. 1952a. The physiology of cell division. In "Modem

Trends in Physiology and Biochemistry" (E. S. G Barron, ed.), pp. 123-134. Academic Press, New York.

Heilbrunn, L. V. 1952b. "An Outline of General Physiology," 3rd ed. Saunders, Philadelphia.

Heilbrunn, L. V. 1953. Cellular homeostasis. Abstr. Communs. 19th

Intern. Physiol. Congr. Montreal, pp. 448-449. Heilbrunn, L. V., and Ashkenaz, E. W. 1941. The irritability of

frog muscle in relation to the sodium ion. Physiol. Zool. 14: 281-295. Heilbrunn, L. V., Chaet, A. B., Dunn, Α., and Wilson, W. L. 1954.

Antimitotic substances from ovaries. Biol. Bull. 106: 158-168. Heilbrunn, L. V., and Daugherty, K. 1931. The action of the

chlorides of sodium, potassium, calcium, and magnesium on the protoplasm of Amoeba dubia. Physiol. Zool. 4: 635-651.

Heilbrunn, L. V., and Daugherty, K. 1932. The action of sodium, potassium, calcium, and magnesium ions on the plasmagel of Amoeba proteus. Physiol. Zool. 5: 254-274.

Heilbrunn, L. V., and Daugherty, K. 1933. The action of ultraviolet rays on amoeba protoplasm. Protoplasma 18: 596-619.

Heilbrunn, L. V., and Daugherty, K. 1939. The electric charge of protoplasmic colloids. Physiol. Zool. 12: 1-12.

Heilbrunn, L. V., Halaban, Α., and Wilson, W. L. 1952. An attempt to influence the survival time of cancerous mice. Biol. Bull. 103: 282.

Heilbrunn, L. V., and Hamilton, P. 1942. The presence of chloride in muscle fibers. Physiol. Zool. 15: 363-374.

Heilbrunn, L. V., Harris, D. L., LeFevre, P. G, Wilson, W. L., and Woodward, A. A. 1946. Heat death, heat injury, and toxic factor. Physiol. Zool. 19: 404-429.

Heilbrunn, L. V., and Mazia, D. 1936. The action of radiations on living protoplasm. In "Biological Effects of Radiation" (Duggar, ed.), vol. 1, pp. 625-676. McGraw-Hill, New York.

Heilbrunn, L. V., and Wiercinski, F. J. 1947. The action of various cations on muscle protoplasm. /. Cellular Comp. Physiol. 29: 15-32.

BIBLIOGRAPHY 293

Heilbrunn, L. V., and Wilbur, Κ. M. 1937. Stimulation and nuclear breakdown in the Nereis egg. Biol. Bull 73: 557-564.

Heilbrunn, L. V., and Wilson, W. L. 1948. Protoplasmic viscosity changes during mitosis in the egg of Chaetopterus. Biol Bull 95: 57-68.

Heilbrunn, L. V., and Wilson, W. L. 1949. The effect of heparin on cell division. Proc. Soc. Exptl Biol Med. 70: 179-182.

Heilbrunn, L. V., and Wilson, W. L. 1950a. Effect of bacterial polysaccharide on cell division. Science 112: 56-57.

Heilbrunn, L. V., and Wilson, W. L. 1950b. The prevention of cell division by anticlotting agents. Protoplasma 39: 389-399.

Heilbrunn, L. V., and Wilson, W. L. 1954. Changes in the proto-plasm during maturation. Biol. Bull. 107: 312-313.

Heilbrunn, L. V., and Wilson, W. L. 1955a. The initiation of cell division in the Chaetopterus egg. Protoplasma 44: 377-384.

Heilbrunn, L. V., and Wilson, W. L. 1955b. Changes in the proto-plasm during maturation. Biol. Bull 109: in press.

Heilbrunn, L. V., and Wilson, W. L. 1955c. The action of fatigue substances on cell division. Pubbl. staz. zool. Napoli in press.

Heilbrunn, L. V., Wilson, W. L., and Harding, D. 1951. The action of tissue extracts on cell division. /. Natl. Cancer Inst. 11: 1287-1298.

Heilbrunn, L. V., Wilson, W. L., and Lippman, M. 1953. Anti-mitotic substances from ovaries. Biol. Bull. 105: 375.

Helmholtz, Arminius (Hermann). 1842. De fabrica systematis nervosi evertebratorum. Diss., Berlin.

Hering, Ε. 1879. Beiträge zur allgemeinen Nerven- und Muskel-physiologie. II. Über die Methoden zur Untersuchung der polaren Wirkungen des elektrischen Stromes im quergestreiften Muskel. Sitzber. Akad. Wiss. Wien. Math, naturw. Kl. Abt. III 79 : 237-262.

Hering, E. 1882. Beiträge zur allgemeinen Nerven- und Muskel-physiologie. IX. tiber Nervenreizung durch den Nervenstrom. Sitzber. Akad. Wiss. Wien. Math, naturw. Kl. Abt. III 85: 237-275.

Hermann, S. 1932. Die Beeinflussbarkeit der Zustandsform des Calciums im Organismus durch Adrenalin. Naunyn-Schmiedebergs Arch, exptl. Pathol, u. Pharmakol. 167: 82-84.

Hermans, P. Η. 1949. Gels. In "Colloid Science" (H. R. Kruyt, ed.), vol. 2, pp. 483-651. Elsevier, New York.

Heubner, W., and Rona, P. 1923. Über die Kalkgehalt der Organe bei kalkbehandelten Katzen. Biochem. Z. 135: 248-281.

Heuking, G. von, and Szent-Györgyi, A. 1923. Über die Wirkung des defibrinierten Blutes auf das isolierte Säugetierherz. Pflügers Arch. ges. Physiol 197: 516-517.

Hevesy, G. 1945. On the effect of roentgen rays on cellular division. Revs. Mod. Phys. 17: 102-111.


Higgins, S. G,. and Fisher, D. 1924. Effects of the intramuscular injection of sodium citrate upon bleeding. Ann. Surg. 80: 268-271.

Hill, Α. V. 1936. Excitation and accommodation in nerve. Proc.

Roy. Soc. B119: 305-355. Hill, Α. V. 1948. On the time required for diffusion and its relation

to processes in muscle. Proc. Roy. Soc. B135: 446-453. Hill, Α. V. 1949a. The onset of contraction. Proc. Roy. Soc. B136:

242-254. Hill, Α. V. 1949b. The abrupt transition from rest to activity in

muscle. Proc. Roy. Soc. B136: 399-420. Hill, D. K. 1950a. The effect of stimulation on the opacity of a

crustacean nerve trunk and its relation to fibre diameter. /. Physiol.

(London) 111: 283-303. Hill, D. K. 1950b. The volume change resulting from stimulation

of a giant nerve fiber. /. Physiol. (London) 111: 304-327. Hill, D. K., and Keynes, R. D. 1949. Opacity changes in stimulated

nerve. /. Physiol. (London) 108: 278-281. Höber, R., and Strohe, H. 1929. Uber den Einfluss von Salzen auf

die elektronischen Ströme, die Erregbarkeit und das Ruhepotential des Nerven. Pflügers Arch. ges. Physiol. 222: 71-88.

Hodge, C. F. 1892. A microscopical study of changes due to func-tional activity in nerve cells. /. Morphol. 7: 95-168.

Hodgkin, A. L. 1951. The ionic basis of electrical activity in nerve and muscle. Biol. Revs. 26: 339-409.

Hodgkin, A. L., and Huxley, A. F. 1939. Action potentials recorded from inside a nerve fibre. Nature 144: 710-711.

Hodgkin, A. L., and Katz, B. 1949a. The effect of sodium ions on the electrical activity of the giant axon of the squid. /. Physiol.

(London) 108: 37-77. Hodgkin, A. L., and Katz, B. 1949b. The effect of calcium on the

axoplasm of giant nerve fibers. /. Exptl. Biol. 26: 292-294. Höfler, Κ., and Weber, F. 1926. Die Wirkung der Äthernarkose auf

die Harnstoffpermeabilität von Pflanzenzellen. Jahrb. wiss. Botan.

65: 643-737. Hollingsworth, J. 1941. Activation of Cumingia and Arbacia eggs

by bivalent cations. Biol. Bull. 81: 261-276. Holloman, A. L. 1947. Reactions of patients to injections of S.

marcescens in eight cases of malignant disease. In "Approaches to Tumor Chemotherapy," pp. 273-276. Am. Assoc. Advancement of Sei., Washington, D. C.

Holmes, Β. E. 1949. The indirect effect of X rays on the synthesis of nucleic acid in vivo. Brit. J. Radiol, [n. s.] 22: 487-491.

Homburger, F., and Fishman, W. H. (eds.). 1953. "The Physio-pathology of Cancer." Hoeber, New York.

BIBLIOGRAPHY 295

Hooker, D. R. 1911. The chemical regulation of vascular tone as studied upon the perfused blood vessels of the frog. Am. J. Physiol. 28: 361-367.

Hopkins, A. L. 1948. Autotomy as a test for toxic factor. Federation Proc. 7: 57.

Hopkins, A. L. 1955. Effects of lyophilization on the contractile mechanism of muscle. Federation Proc. 14: 75-76.

Hopkins, H. 1935. Chemical studies in the epileptic syndrome. II. Nocturnal and diurnal rhythm and blood chemistry. Am. J. Psychiat. 92: 75-88.

Houssay, Β. Α., and Molinelli, E. A. 1928. Excitabilité des fibres adrénalino-sécrétoires du nerf grand splanchnique. Fréquences seuil et optimum de stimulus. Rôle de l'ion calcium. Compt. rend. soc. Hol. 99: 172-174.

Howard, E. 1932. The structure of protoplasm as indicated by a study of the apparent viscosity of sea-urchin eggs at various shear-ing forces. J. Cellular Comp. Physiol. 1: 355-369.

Hughes, A. 1952. "The Mitotic Cycle; The Cytoplasm and Nucleus During Interphase and Mitosis." Academic Press, New York.

Hultin, T. 1949. The effect of calcium on respiration and acid formation in homogenates of sea-urchin eggs. Arkiv Kernt Mineral. Geol. 2 6 A ( 2 7 ) : 1-10.

Hultin, T. 1950a. On the oxygen uptake of Paracentrotus lividus egg homogenates after the addition of calcium. Exptl. Cell Research 1: 159-168.

Hultin, T. 1950b. On the acid formation, breakdown of cytoplasmic inclusions, and increased viscosity of Paracentrotus egg homogenates after the addition of calcium. Exptl. Cell Research 1: 272-283.

Humboldt, F. A. von. 1797. Versuche über die gereizte Muskel-und Nervenfaser nebst Vermuthungen über den chemischen Process des Lebens in der Thier- und Pflanzenwelt. 2 vols. Rottmann, Berlin.

Hunt, R. 1915. A physiological test for cholin and some of its applications. / . Pharmacol. Exptl. Therap. 7: 301-337.

Hürthle, Κ. 1909. Über die Struktur der quergestreiften Muskel-fasern von Hydrophilus im ruhenden und tätigen Zustand. Pflügers Arch. f. ges. Phi/siol. 126: 1-164.

Ikawa, M., Koepfli, J. B., Mudd, S. G., and Niemann, C. 1952. An agent from Ε. coli causing hemorrhage and regression of an ex-perimental mouse tumor. I. Isolation and properties. /. Natl. Cancer Inst. 13: 157-166.

Immers, J., and Vasseur, E. 1949. Comparative studies on the coagulation process with heparin and sea-urchin fertilizin. Ex-perientia 5: 124-125.


Inoué, S. 1951. A method for measuring small retardations of struc-tures in living cells. Exptl. Cell. Research 2: 513-517.

Inoué, S. 1953. Polarization optical studies of the mitotic spindle. I. The demonstration of spindle fibers in living cells. Chromosoma 5: 487-500.

Inoué, S., and Dan, K. 1951. Birefringence of the dividing cell. / . Morphol. 89: 423-456.

Jacobs, M. H. 1922. The influence of ammonium salts on cell reac-tion. /. Gen. Physiol. 5: 181-188.

Jacobs, M. H., and Parpart, A. K. 1937. The influence of certain alcohols on the permeability of the erythrocyte. Biol. Bull. 73: 380-381.

Jahn, D. 1924. Über den Einfluss von Ionen auf die Erregbarkeit des Nerven durch Momentan- und Zeitreize. Pflügers Arch. ges. Physiol. 206: 66-80.

Janeway, T. C , Richardson, Η. Β., and Park, Ε. Α. 1918. Experi-ments on the vasoconstrictor action of blood serum. Arch. Intern. Med. 21: 565-603.

Jaques, L. B., and Waters, Ε. T. 1941. The identity and origin of the anticoagulant of anaphylactic shock in the dog. / . Physiol (London) 99: 454-466.

Jeener, R. 1947. Sur le rôle des groupes thiol dans la coagulation du plasma sanguin. Experientia 3: 243-244.

Johnson, F. H., Eyring, H., and Polissar, M. J. 1954. "The Kinetic Basis of Molecular Biology." Wiley, New York.

Jorpes, J. E. 1946. "Heparin in the Treatment of Thrombosis: An Account of Its Chemistry, Physiology, and Application in Medicine," 2nd ed., Oxford, New York.

Jowett, M., and Quastel, J. H. 1937. The effects of ether on brain oxidations. Biochem. J. 31: 1101-1112.

Just, Ε. E. 1931. Die Rolle des kortikalen Cytoplasmas bei vitalen Erscheinungen. Naturwissenschaften 19: 953-962, 980-984, 998-1001.

Just, Ε. E. 1939. "The Biology of the Cell Surface." Blakiston, Philadelphia.

Katz, B. 1939. "Electric Excitation of Nerve." Oxford, New York. Kayser, C. 1948. Le sommeil. J. physiol. (Paris) 41: 1A-60A. Kelly, J. W. 1950. The localization of a metachromatic substance

in the Chaetopterus egg. Protoplasma 39: 386-388. Kelly, J. W. 1951. Effects of x-ray and trypsin on the metachromasy

of heparin-basic protein combinations. Biol. Bull. 101: 223. Kelly, J. W. 1954. Metachromasy in the eggs of fifteen lower

animals. Protoplasma 43: 329-346.

BIBLIOGRAPHY 297

Kelly, J. W. 1955. Suppression of metachromasy by basic proteins. Arch. Biochem. and Biophys. 55: 130-137.

Kessler, W. 1935. Über die inneren Ursachen der Kälteresistenz der Pflanzen. Planta 24: 312-352.

Keynes, R. D. 1951a. The leakage of radioactive potassium from stimulated nerve. /. Physiol. (London) 113: 99-114.

Keynes, R. D. 1951b. The ionic movements during nervous activity. /. Physiol. (London) 114: 119-150.

Knisely, M. H., Eliot, T. S., and Bloch, Ε. Η. 1946. Sludged blood in traumatic shock. I. Microscopic observations of the precipitation and agglutination of blood flowing through vessels in crushed tis-sues. Arch. Surg. 51: 220-236.

Koshtojanz, C. S. 1944. An analysis of the mode of action of acetyl-choline as chemical agent of nervous excitation. Compt. rend. Acad. Sei. U.R.S.S. 43: 357-359.

Kraus, F., and Fuchs, H. J. 1929. Über das Koagulin des Muskels. II. Z. ges. exptl. Med. 68: 245-257.

Kraus, F., Fuchs, H. J., and Merländer, R. 1932. Über das Koagulin des Muskels. III. Z. ges. exptl. Med. 79: 59-75.

Kreisler, L. 1952. Effect of heparin on the growth of a transplant-able lymphosarcoma in mice. Science 115: 145-146.

Krieger, K. R. 1880. Ueber das Centrainervensystem des Flusskreb-ses. Z. wiss. Zool. 33: 527-594.

Kruyt, H. R. (ed.). 1949, 1952. "Colloid Science." Vols. 1 and 2. Elsevier, New York.

Kuffler, S. W. 1944. The effect of calcium on the neuromuscular junction. /. Neurophysiol. 7: 17-26.

Kuffler, S. W. 1948. Physiology of neuro-muscular junctions: elec-trical aspects. Federation Proc. 7: 437-446.

Kuffler, S. W. 1952. Transmission processes at nerve-muscle junc-tions. In "Modern Trends in Physiology and Biochemistry" ( E. S. G. Barron, ed.), pp. 277-290. Academic Press, New York.

Kiihnau, J., and Morgenstern, V. 1934. Glutathion, Schwermetalle und Blutgerinnung. Z. physiol. Chem. 227: 145-168.

Kühne, W. 1863. Eine lebende Nematode in einer lebenden Muskel-faser beobachtet. Virchow's Arch, pathol. Anat. u. Physiol. 26: 222-224.

Kühne, W. 1864. "Untersuchungen über das Protoplasma und die Contractilität." Leipzig.

Kühne, W. 1888. Secundäre Erregung vom Muskel zum Muskel. Z. Biol. 24: 383-422.

Labhardt, E. 1927. Fortgesetzte Untersuchungen über Muskeler-müdung. Z. Biol. 86: 27-38.


Lambers, K. 1951. Über Organ Veränderungen bei chronischer Col-chicinvergiftung. Virchow's Arch, pathol. Anat. u. Physiol. 321: 88-100.

Lânczos, A. 1935. Freiwerden von Calcium durch Reizung der Herznerven. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 177: 752-754.

Lânczos, Α. 1936. Freiwerden von Calcium bei Reizung des Sym-pathicus. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 180: 313-318.

Lapicque, L. 1934. Preface. In "L'Excitation Electrique des Tissus" (Monnier, ed.), pp. vii-xiii. Presses Univ. de France, Paris.

Lawaczek, H. 1928. Ueber das Verhalten des Kalziums unter Adren-alin. Deut. Arch. klin. Med. 160: 309-322.

Lehotzky, P. von. 1935. Die Wirkung des elektrischen Stromes auf lebende Zellen und Gewebe. Arch, exptl. Zellforsch. Gewebezucht. 18: 3-12.

Lewis, W. H. 1919. Degeneration granules and vacuoles in the fibroblasts of chick embryos cultivated in vitro. Bull. Johns Hopkins Hosp. 30: 81-91.

Lillie, F. R. 1906. Observations and experiments concerning the elementary phenomena of embryonic development in Chaetopterus. /. Exptl. Zool. 3: 153-268.

Lillie, F. R. 1908. Polarity and bilaterality of the annelid egg. Ex-periments with centrifugal force. Biol. Bull. 16: 54-79.

Lillie, F. R. 1919. "Problems of Fertilization." U. of Chicago Press, Chicago.

Lillie, R. S. 1914. The action of various anaesthetics in suppressing cell-division in sea-urchin eggs. /. Biol. Chem. 17: 121-140.

Lillie, R. S. 1916. The theory of anesthesia. Biol. Bull. 30: 311-366. Lillie, R. S. 1923. "Protoplasmic Action and Nervous Action." U.

of Chicago Press, Chicago. Lillie, R. S. 1941. Further experiments on artificial parthenogenesis

in starfish eggs, with a review. Physiol. Zool. 14: 239-267. Lippman, M. 1955. The effect of heparin and related substances

on ascites tumors in mice. Master's Thesis, Univ. of Pennsylvania. Lissâk, K. 1934. Beiträge zur Frage der Beziehung zwischen Tetanie

und Tetanus. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 176: 425-428.

Locke, F. S. 1894. Notiz über den Einfluss physiologischer Koch-salzlösung auf die elektrische Erregbarkeit von Muskel und Nerv. Centralbl. Physiol. 8: 166-167.

BIBLIOGRAPHY 299

Loeb, J. 1901. On an apparently new form of muscular irritability (contact irritability?) produced by solutions of salts (preferably sodium salts) whose anions are liable to form insoluble calcium compounds. Am. /. Physiol. 5: 362-373.

Loeb, J. 1909. "Die chemische Entwicklungserregung des tierischen Eies." Springer, Berlin.

Loeb, J. 1913. "Artificial Parthenogenesis and Fertilization." Univ. of Chicago Press, Chicago.

Loeb, L. 1903. On the coagulation of the blood of some arthropods and on the influence of pressure and traction on the protoplasm of the blood cells of arthropods. Biol. Bull 4: 301-318.

Loeb, L. 1905. Untersuchungen über Blutgerinnung. VI. Beitr. Chem. Physiol, u. Pathol. 6: 260-286.

Loeb, L. 1906. Untersuchungen über Blutgerinnung. VII. Beitr. Chem. Physiol, u. Pathol 8: 67-94.

Loewi, O. 1945. Aspects of the transmission of the nervous impulse. /. Mt. Sinai Hosp., Ν. Ύ. 12: 803-816, 851-865.

Lohmann, K. 1934. Über die enzymatische Aufspaltung der Krea-tinphosphorsäure, zugleich ein Beitrag zum Chemismus der Muskel-kontraktion. Biochem. Z. 271: 264-277.

Lohmann, K. 1937. Chemische Vorgänge bei der Muskelkontrak-tion. Angew. Chem. 50: 97-100.

Loicq, R. 1937. Recherches sur les effects de la colchicine sur la coagulation du sang. Arch, intern, méd. exptl 12: 371-396.

Lorand, L. 1952. Fibrino-peptide. Biochem. J. 52: 200-203. Lorand, L. 1954. Interaction of thrombin and fibrinogen. Physiol.

Revs. 34: 742-752. Lorand, L., and Middlebrook, W. R. 1952. The action of thrombin

on fibrinogen. Biochem. J. 52: 196-199. Lorente de No, R. 1947. "A Study of Nerve Physiology," Vols. 131

and 132. Studies from the Rockefeller Institute for Medical Re-search, New York.

Lorente de No, R. 1949. On the effect of certain quarternary am-monium ions upon frog nerve. J. Cellular Comp. Physiol. 33 Suppl.: 1-231.

Love, R., and Frommhagen, L. H. 1953. Histochemical studies on the clam Mactra solidissima. Proc. Soc. Exptl. Biol. Med. 83: 838-844.

Lucké, Β., Parpart, A. K., and Ricca, R. A. 1941. Failure of choleic acids of carcinogenic hydrocarbons to alter permeability of marine eggs and of mammalian erythrocytes. Cancer Research 1: 709-713.

Lullies, H. 1930. Über die Polarisation in Geweben. III. Die Polarisation im Nerven. II. Pflügers Arch. ges. Physiol. 225: 87-97.


Lumière, A. 1929. "Le cancer: Maladie des cicatrices." Masson, Paris.

Lundblad, G. 1952. Proteolytic activity in sea urchin gametes. II. Activity of extracts and homogenates of the egg subjected to dif-ferent treatments. Arkiv Kemi 4: 537-565.

Lundblad, G 1954. Proteolytic activity in sea urchin gametes. IV. Further investigations of the proteolytic enzymes of the egg. Arkiv Kemi 7: 127-157.

Lundblad, G, and Lundblad, I. 1954. Proteolytic activity in sea urchin gametes. III. A study of the proteolytic enzymes of the egg. Arkiv Kemi 6: 387-415.

Lundholm, E., and Ehrenberg, L. 1952. The effects of anesthetics on blood coagulation in vitro. Acta Chem. Scand. 6: 341-351.

Lundsgaard, E. 1930. Untersuchungen über Muskelkontraktionen ohne Milchsäurebildung. Biochem. Z. 217: 162-177.

Lustig, B., Ernst, T., and Reuss, E. 1937. Die Zusammensetzung des Blutes von Helix pomatia bei Sommer- und Wintertieren. Biochem. Z. 290: 95-98.

Lyons, R. N. 1945. Thiol-vitamin Κ mechanism in the clotting of fibrinogen. Australian J. Exptl. Biol. Med. Sei. 23: 131-140.

McBain, J. W. 1950. "Colloid Science." Reinhold, New York. McClendon, J. F. 1910. On the dynamics of cell division. I. The

electric charge on colloids in living cells in the root tips of plants. Arch. Entwicklungsmech. Organ. 31: 80-90.

McClendon, J. F. 1913. The laws of surface tension and their ap-plicability to living cells and cell division. Arch. Entwicklungsmech. Organ. 37: 233-247.

McLean, F. C, and Hastings, A. B. 1934. A biological method for the estimation of calcium ion concentration. /. Biol. Chem. 107: 337-350.

McNamara, B., Krop, S., and McKay, E. A. 1948. The effect of calcium on the cardiovascular stimulation produced by acetylcholine. /. Pharmacol. Exptl. Therap. 92: 153-161.

Marinesco, G 1912. Forschungen über den kolloiden Bau der Nervenzellen und ihre erfahrungsgemässen Veränderungen. Kolloid-Z. 11: 209-225.

Mast, S. O. 1931. The nature of the action of electricity in produc-ing response and injury in Amoeba proteus (Leidy) and the effect of electricity on the viscosity of protoplasm. Z. vergleich. Physiol. 15: 309-328.

Mathews, A. P. 1936. "Principles of Biochemistry." Wood, Balti-more.

BIBLIOGRAPHY 301

Matis, P., and Scheele, J. 1953. Zur Vasoaktivität der Anticoagu-lantia. Wien. Hin. Wochschr. 65: 102-103.

Mazia, D. 1937. The release of calcium in Arbacia eggs on fertiliza-tion. /. Cellular Comp. Physiol. 10: 291-304.

Mazia, D. 1940. The binding of ions by the cell surface. Cold Spring Harbor Symposia Quant. Biol. 8: 195-203.

Mazia, D. 1954. SH and growth. In "Glutathione: A Symposium" (S. Colowick et al, eds.), pp. 209-228. Academic Press, New York.

Mazia, D., and Clark, J. M. 1936. Free calcium in the action of stimulating agents on Elodea cells. Biol Bull. 71: 306-323.

Mazia, D., and Dan, K. 1952. The isolation and biochemical charac-terization of the mitotic apparatus of dividing cells. Proc. Natl. Acad. Sei. (U.S.) 38: 826-838.

Meitzer, S. J. 1908. The nature of shock. Arch. Intern. Med. 1: 571-588.

Meyer, Κ. H. 1951a. "Natural and Synthetic High Polymers," 2nd ed. (translated from the German by L. E. R. Picken) Interscience, New York.

Meyer, K. H. 1951b. Les bases expérimentales de la théorie lipoidi-que de la narcose. Mécanisme de la Narcose. Colloq. intern, centre not. recherche sei. (Paris) No. 26, 17-23.

Meyerhof, O. 1930. "Die chemischen Vorgänge im Muskel und ihr Zusammenhang mit Arbeitsleistung und Wärmebildung." Springer, Berlin.

Meyerhof, O. 1947. The main chemical phases of the recovery of muscle. Ann. Ν. Ύ. Acad. Sei. 47: 815-834.

Mihâlyi, Ε., and Lorând, L. 1948. The mechanism of the clotting of fibrinogen with formaldehyde and quinone. Interactions between formaldehyde and thrombin. Acta Physiol. Acad. Set. Hung. 1: 218-237.

Miko, J. von, and Pala, T. 1927. Über Blutveränderungen infolge Strychninwirkung. Naunyn-Schmiedebergs Arch, exptl. Pathol u. Pharmakol 119: 273-284.

Mills, C. A. 1921. The action of tissue extracts in the coagulation of blood. /. Biol. Chem. 46: 167-192.

Mitchison, J. M. 1952. Cell membranes and cell division. Symposia Soc. Exptl Biol. 6: 105-127.

Mitchison, J. M. 1953. Microdissection experiments on sea-urchin eggs at cleavage. /. Exptl. Biol 30: 515-524.

Mommaerts, W. F. H. M. 1954. Is adenosine triphosphate broken down during a single muscle twitch? Nature 174: 1083-1084.

Monnier, A. M. (ed.). 1934. "Excitation électrique des tissus." Hermann, Paris.


Monnier, A. M. 1947. Nouvelles recherches sur la résonance des tissus excitables. II. Influence de la température et des décalcifiants sur le déclenchement de l'activité périodique des nerfs. Arch, intern, physiol 54: 348-371.

Monnier, A. M., and Coppée, G. 1939. Nouvelles recherches sur la résonance des tissus excitables. I. Relation entre la rythmicité de la réponse nerveuse et la résonance. Arch, intern, physiol. 48: 129-180.

Moon, V. H. 1942. "Shock. Its Dynamics, Occurrence and Manage-ment." Lea and Febiger, Philadelphia.

Moreau, L. 1948. Shock due to head injury in the frog. Am. J. Physiol, 155: 92-97.

Moreau, L., Balistocky, M., and Heilbrunn, L. V. 1948. Shock due to electrical injury in frogs. Am. J. Physiol. 154: 38-44.

Morgan, T. H. 1898. The effect of salt solutions on unfertilized eggs of Arbacia. Science [n. s.] 7: 222-223.

Morgan, T. H. 1899. The action of salt-solutions on the unfertilized and fertilized eggs of Arbacia, and of other animals. Arch. Entwick-lungsmech. Organ. 8: 448-539.

Morgan, T. H. 1927. "Experimental Embryology." Columbia U. P., New York.

Morgenstern, V. 1933. Die Ermüdbarkeit "tonischer" und "nicht tonischer" Froschmuskeln und deren Beeinflussung durch Ringer-lösung mit verschiedenem Ionengehalt. Pflügers Arch. ges. Physiol. 232: 174-186.

Moser, F. 1939. Studies on a cortical layer response to stimulating agents in the Arbacia egg. I. Response to insemination. /. Exptl. Zool. 80: 423-445.

Mosso, A. 1890. Le sang des animaux fatigués, alors même qu'il est privé du C0 2 , fait augmenter la frequence de la respiration et la pression du sang si l'on opère sa transfusion à un autre animal. Trans. Intern. Med. Congr., Berlin, 2, part 2: 13-14.

Most, S. N. 1950. Studies on fatigue substances and clotting factors. Thesis, University of Pennsylvania, Philadelphia.

Most, S. 1951. Effect of Shear's polysaccharide on plasma clotting. Nature 168: 342-343.

Moussatché, H., and Cruz, W. O. 1952. In vitro release by the anti-platelet serum of a substance from the platelets active on the iso-lated gut of the guinea-pig. Arch, intern, pharmacodynamic 91: 224-229.

Müller, J. 1838. "Elements of Physiology," Vols. 1 and 2. London. Mylon, E., Winternitz, M. C, Katzenstein, R., and de Sütö-Nagy, G. J.

1942. Studies on mechanisms involved in shock. Am. J. Physiol. 137: 280-298.

BIBLIOGRAPHY 303

Nauts, H. C, and Coley, B. L. 1947. A review of the treatment of malignant tumors by Coley bacterial toxins. In "Approaches to Tumor Chemotherapy," pp. 217-235. Amer. Assoc. Advance Sei., Washington, D. C.

Nauts, H. C, Fowler, G. Α., and Bogatko, F. H. 1953. A review of the influence of bacterial infection and of bacterial products (Coley's toxins) on malignant tumors in man. Acta Med. Scand. 145, Suppl. 276.

Nauts, H. C, Swift, W. E., and Coley, B. L. 1946. The treatment of malignant tumors by bacterial toxins as developed by the late William B. Coley, M.D., reviewed in the light of modern research. Cancer Research 6: 205-216.

Nekrassow, P. Α., and Nekrassowa, Ν. V. 1936. Die Wirkung von Serum auf den ermüdeten Muskel. Fiziol. Zhur. U.S.S.R. 21: 519-531.

Neuhof, H., and Hirshfeld, S. 1922. The intramuscular administra-tion of sodium citrate. A new method for the control of bleeding. Ann. Surg. 76: 1-8.

Nolf, P., and Adant, M. 1954. La defibrination "in vivo" par injec-tion de thrombine chez le chien normal ou hépatectomisé. Le Sang 25: 193-261.

Nysten, P. H. 1811. "Recherches de physiologie et de chemie patho-logique." Brosson, Paris.

Oakey, R. 1947. Reactions of patients to injection of S. marcescens polysaccharide in nine further cases of malignant disease. In "Ap-proaches to Tumor Chemotherapy," pp. 277-278. Amer. Assoc. Advance. Sei. Washington, D. C.

Opie, E. L. 1948. An osmotic system within the cytoplasm of cells. /. Exptl. Med. 87: 425-444.

Osterhout, W. J. V. 1931. Physiological studies of single plant cells. Biol. Revs. 6: 369-411.

Osterhout, W. J. V. 1933. Some aspects of cell physiology. Ann. Intern. Med. 7: 396-400.

Osterhout, W. J. V., and Hill, S. 1933. Anesthesia produced by distilled water. /. Gen. Physiol. 17: 87-98.

Osterhout, W. J. V., and Hill, S. E. 1935. Nature of the action current in Nitella. III. Some additional features. /. Gen. Physiol. 18: 499-514.

Overbeek, J. T. G. 1952. Rheology of lyophobic systems. In "Col-loid Science" (H. R. Kruyt, ed.), vol. 1, pp. 342-368. Elsevier, New York.

Overton, E. 1902. Beiträge zur allgemeinen Muskel- und Nerven-physiologie. II. Ueber die Unentbehrlichkeit von Natrium (oder Lithium- ) Ionen für den Contractionsact des Muskels. Pflügers Arch, ges. Physiol. 92: 346-386.


Palmer, W. L. 1951. Diseases of the stomach. In "Textbook of Medicine" (R. L. Cecil and R. F. Loeb, eds.) , pp. 682-717. Saunders, Philadelphia.

Partos, A. 1932. Gesetzmässiger Zusammenhang zwischen Blut-zuckergehalt und Blutgerinnungszeit. Über Gerinnungstheorie von Stuber und Lang. Pflügers Arch. ges. Physiol. 229. 336-343.

Pauli, W. 1912. Ueber den Zusammenhang von elektrischen, mech-anischen und chemischen Vorgängen im Muskel (Kolloidchemie der Muskelkontraktion). Kolloidchem. Beih. 3: 361-384.

Pekarek, J. 1930a. Absolute Viskositätsmessung mit Hilfe der Brownschen Molekularbewegung. I. Prinzip der Methode, Voraus-setzungen, Fehlerquellen der Messungen. Protoplasma 10: 510-532.

Pekarek, J. 1930b. Absolute Viskositätsmessung mit Hilfe der Brownschen Molekularbewegung. Viskositätsbestimmung des Zell-saftes der Epidermiszellen von Allium cepa und des Amoeben-Protoplasmas. Protoplasma 11: 19-48.

Pekarek, J. 1931. Absolute Viskositätsmessungen mit Hilfe der Brownschen Molekularbewegung. Protoplasma 13: 637-665.

Pekarek, J. 1932. Absolute Viskositätsmessungen mit Hilfe der Brownschen Molekularbewegung. IV. Plasmaviskositätsmessungen an Rhizoiden von Chara fragilis Desv. Protoplasma 17: 1-24.

Pekarek, J. 1933a. Absolute Viskositätsmessungen mit Hilfe der Brownschen Molekularbewegung. V. Plasmolyse und Zellsaftvis-kosität. Protoplasma 18: 1-53.

Pekarek, J. 1933b. Absolute Viskositätsmessungen mit Hilfe der Brownschen Molekularbewegung. VI. Der Einfluss der Temperatur auf die Zellsaftviskosität. Protoplasma 20: 251-278.

Pentimalli, F. 1909. Influenza della corrente elettrica sulla dinamica del processo cariocinetico. Arch. Entwicklungsmech. Organ. 28: 260-276.

Pentimalli, F. 1912. Sulla carica elettrica della sostanza nucleare cromatica. Arch. Entwicklungsmech. Organ. 34: 444-451.

Permeschko. 1879. Ueber die Theilung der thierischen Zellen. Arch. mikroskop. Anat. 16: 437-457.

Pflüger, E. 1859. Über die Veränderung der Erregbarkeit des Nerven durch einen constanten elektrischen Strom vor. Monatsher. Kön Preuss. Akacl. Wiss. Berlin 1858: 198-205.

Piulachs, 1945. "Shock traumâtico." Salvat Barcelona. Proctor, Ν. Κ. 1952. The effects of calcium on isolated arthropod

muscle fibers. Biol. Bull. 103: 421-432. Punt, A. 1942. L'influence de la température et des electrolytes sur

la contracture acétylcholinique. Arch, néerl. physiol. 26: 212-221. Purr, A. 1935. The activation phenomena of papain and cathepsin.

Biochem. J. 29: 13-20.

BIBLIOGRAPHY 305

Quastel, J. H. 1951. Biochemical basis of narcosis. Mécanisme de la Narcose. Colloq. intern, centre natl. recherche sei. (Paris) No. 26, 105-116.

Ranke, J. 1863. Untersuchung über die chemischen Bedingungen der Ermüdung des Muskels. Arch. Anat. Physiol. u. wiss. Med. 1863: 422-450.

Ranke, J. 1864. Untersuchung über die chemischen Bedingungen der Ermüdung des Muskels. II. Arch. Anat. Physiol. u. wiss. Med. 1864: 320-348.

Rapkine, L. 1931. Sur les processus chimiques au cours de la divi-sion cellulaire. Ann. physiol. physicochim. biol. 7: 382-418.

Rashevsky, N. 1948. "Mathematical Biophysics." Univ. of Chicago, Chicago.

Rashkis, H. A. 1952. Systemic stress as an inhibitor of experimental tumors in Swiss mice. Science 116: 169-171.

Rathgeb, P., and Sylvén, Β. 1954a. Fractionation studies on the tumor-necrotizing agent from Serratia marcescens (Shear's poly-saccharide). /. Natl. Cancer Inst. 14: 1099-1108.

Rathgeb, P., and Sylvén, Β. 1954b. Chemical studies on the tumor-necrotizing agent from Serratia marcescens ( Shear's polysaccharide ). /. Natl. Cancer Inst. 14: 1109-1117.

Rauh, F. 1922. Die Latenzzeit des Muskelelementes. Ζ. Biol. 76: 25-48.

Reid, C, and Bick, M. 1942. Pharmacologically active substances in serum. Australian J. Exptl. Biol. Med. Set. 20: 33-46.

Rex-Kiss, B., and Lissâk, K. 1940. Ca- und Κ—Gehalt der Nerven bei lokalem Tetanus. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 196: 542-544.

Rieser, P. 1949a. The protoplasmic viscosity of muscle. Proto-plasma 39: 95-98.

Rieser, P. 1949b. The protoplasmic viscosity of muscle and nerve. Biol. Bull. 97: 245-246.

Rieser, P. 1955. The effects of roentgen irradiation and other types of injurious agents on capillaries. Proc. Soc. Exptl. Biol. Med. 89: 39-41.

Riker, W. F. 1953. Excitatory and anti-curare properties of acetyl-choline and related quarternary compounds at the neuromuscular junction. Pharmacol. Revs. 5: 1-86.

Robbie, W. A. 1949. Respiration of the tissues of some invertebrates and its inhibition by cyanide. /. Gen. Physiol. 32: 655-670.

Rocha e Silva, M., and Dragstedt, C. A. 1941. Liberation of heparin by trypsin. Proc. Soc. Exptl. Biol. Med. 48: 152-155.


Röder, H. L. 1939. Rheology of suspensions. A study of dilatancy and thixotropy. Thesis, Utrecht, Amsterdam.

Roeder, F. 1930. Über Elektrolytgehalt und elementare Zusammen-setzung der Froschnerven in dem Verlauf der Leitungsstrecke und ihre Veränderungen mit der Reizung. Biochem. Z. 218: 404-411.

Rosso, C. 1934. Variazioni delle frazioni del calcio ematico in seguito alla iperormonizzazione adrenalinica. Clin, pediatr. 16: 229-238.

Runnström, J. 1930. Zur Kenntnis der Stoffwechselvorgänge bei der Entwicklungserregung des Seeigeleies. Biochem. Z. 258: 257-279.

Rusch, H. P., and Kline, Β. Ε. 1944. The effect of exercise on the growth of a mouse tumor. Cancer Research 4: 116-118.

Sandow, A. 1947. Latency relaxation and a theory of muscular mechano-chemical coupling. Ann. Ν. Ύ. Acad. Set. 47: 895-929.

Schaefer, H., and Göpfert, H. 1937. Aktionsstrom und optisches Verhalten des Froschmuskels in ihrer zeitlichen Beziehung zur Zuckung. Pflügers Arch. ges. Physiol. 238: 684-708.

Schmidt, A. 1892. "Zur Blutlehre." Vogel, Leipzig. Schmidt, A. 1895. "Weitere Beiträge Zur Blutlehre." Bergmann,

Wiesbaden. Schmidt, W. J. 1937. "Die Doppelbrechung von Karyoplasma,

Zytoplasma und Metaplasma." Borntraeger, Berlin. Schmidt, W. J. 1939. Doppelbrechung der Kernspindel und Zug-

fasertheorie der Chromosomenbewegung. Chromosoma 1: 253-264. Schräder, F. 1953. "Mitosis," 2nd ed. Columbia Univ. Press, New

York. Scott Blair, G. W. 1938. "An Introduction to Industrial Rheology."

Blakiston, Philadelphia. Scott Blair, G. W. 1949. "A Survey of General and Applied

Rheology," 2nd ed. Pitman, London. Shaver, J. 1953. Studies on the initiation of cleavage in the frog

egg. /. Exptl. Zool. 122: 169-192. Shear, M. J., and Turner, F. C. 1943. Chemical treatment of tumors.

V. Isolation of the hemorrhage-producing fraction from Serratia marcescens (Bacillus prodigiosus) culture filtrate. /. Natl. Cancer Inst. 4: 81-97.

Shenk, W. D. 1950. The chloride content of frog muscle. Arch. Biochem. 25: 168-170.

Shenk, W. D. 1954. Tissue chloride. Arch. Biochem. and Biophys. 49: 138-148.

Simha, R. 1949. Effect of concentration on the viscosity of dilute solutions. /. Research Natl. Bur. Standards 42: 409-418.

BIBLIOGRAPHY 307

Simha, R. 1950. The concentration dependence of viscosities in dilute solutions. /. Colloid Sei. 5: 386-392.

Simha, R. 1952. A treatment of the viscosity of concentrated sus-pensions. /. Appl. Phys. 23: 1020-1024.

Simonson, E. 1935. Der heutige Stand der Theorie der Ermüdung. Ergeh. Physiol. 37: 299-365.

Smith, F. M, Miller, G. H., and Graber, V. C. 1926. The action of adrenalin and acetyl-cholin on the coronary arteries of the rabbit. Am. J. Physiol. 77: 1-7.

Smitten, N. A. 1946. Vital studies of the neuroplasm. Am. Rev. Soviet Med. 3: 414-425.

Sodeman, W. A. 1950. "Pathologic Physiology/' Saunders, Phila-delphia.

Spek, J. 1918. Oberflächenspannungsdifferenzen als eine Ursache der Zellteilung. Arch. Entwicklungsmech. Organ. 44: 5-113.

Spiegel-Adolf, M., Henny, G. C, and Ashkenaz, E. W. 1944. X-ray diffraction studies on frog muscles. /. Gen. Physiol. 28: 151-178.

Staffelt, M. G. 1949. Effect of heteroauxin and colchicine on proto-plasmic viscosity. Exptl. Cell Research 1 Suppl.: 63-78.

Staudinger, H., and Staudinger, M. 1954. "Die makromoleculare Chemie und ihre Bedeutung für die Protoplasmaforschung." (In volume 1 of Protoplasmatologia ). Vienna.

Steinberg, Α., and Brown, W. R. 1939. A new concept regarding the mechanism of clotting and the control of hemorrhage. Am. J. Physiol. 126: P638.

Stiles, W. 1923. The indicator method for the determination of coefficients of diffusion in gels, with special reference to the diffu-sion of chlorides. Proc. Roy. Soc. A103: 260-275.

Stoner, H. B., and Green, H. N. 1947. Bodily reactions to trauma. The influence of ischaemia on the clotting factor of muscle. Brit. J. Exptl. Pathol. 28: 127-141.

Storti, E., Vaccari, F., and Scardovi, G 1954. Die vasomotorische Wirkung von Heparin bei Normalen und bei an Reynaudscher Krankheit leidenden Patienten. Experientia 10: 225-227.

Strangeways, T. S. P. 1922. Observations on the changes seen in living cells during growth and division. Proc. Roy. Soc. B94: 137-141.

Straub, W. 1912. Bedeutung der Zellmembran. Verhandl. Ges. Deut. Naturforsch, u. Arzte 84: 194-214.

Stuber, B., and Lang, K. 1926. Untersuchungen zur Lehre von der Blutgerinnung. XV. Über die Beziehungen des Blutzuckerabaues zur Blutgerinnung. Biochem. Z. 179: 70-85.

Stuber, B., and Lang, K. 1930. "Die Physiologie und Pathologie der Blutgerinnung." Urban, Schwarzenberg, Berlin.


Stuber, B., and Lang, K. 1931. Über die Beziehungen der Glykolyse zur Blutgerinnung. Ζ. ges. exptl. Med. 79: 802-805.

Stuber, B., and Lang, K. 1932. Über Blutglykolyse and Blutge-rinnung. Pflügers Arch. ges. Physiol. 230: 154.

Sugai, K. 1944. Studies on protease. VI. The effect of the addition of various salts on the tryptic activity. / . Biochem. (Japan) 36: 91-100.

Suntzeft, V., and Carruthers, C. 1943. The effect of methylcholan-threne upon epidermal sodium and calcium. Cancer Research 3: 431-433.

Suntzeff, V., and Carruthers, C. 1944. Potassium and calcium in epidermal carcinogenesis induced by methylcholanthrene. /. Biol. Chem. 153 : 521-527.

Suomalainen, P. 1938. Magnesium and calcium content of hedgehog serum during hibernation. Nature 141: 471.

Suomalainen, P. 1939. Magnesium and calcium content of human serum during diurnal sleep. Suomen Kemistilehti B12: 8.

Suomalainen, P., and Lehto, E. 1952. Prolongation of clotting time in hibernation. Experientia 8: 65.

Svihla, Α., Bowman, H., and Pearson, R. 1952. Prolongation of blood clotting time in the dormant hamster. Science 115: 272.

Svihla, Α., Bowman, H. R., and Ritenour, R. 1951. Prolongation of clotting time in dormant estivating mammals. Science 114: 298-299.

Swann, M. M. 1951a. Protoplasmic structure and mitosis. I. The birefringence of the metaphase spindle and asters of the living sea-urchin egg. /. Exptl Biol 28: 417-433.

Swann, M. M. 1951b. Protoplasmic structure and mitosis. II. The nature and cause of birefringence changes in the sea-urchin egg at anaphase. /. Exptl Biol 28: 434-444.

Swann, M. M., and Mitchison, J. M. 1953. Cleavage of sea-urchin eggs in colchicine. / . Exptl Biol 30: 506-514.

Szent-Györgyi, A. 1940. On protoplasmic structure and functions. (A survey). Enzymologia 9: 98-110.

Szent-Györgyi, A. 1945. Studies on muscle. Acta Physiol Scand. 9: Suppl. 2 5 /

Szent-Györgyi, A. 1947. "Chemistry of Muscular Contraction," 1st ed. Academic Press, New York.

Szent-Györgyi, A. 1948. "Nature of Life: A Study on Muscle." Academic Press, New York.

Szent-Györgyi, A. 1949. Free-energy relations and contraction of actomyosin. Biol Bull 96: 140-161.

Szent-Györgyi, A. 1951. "Chemistry of Muscular Contraction," 2nd ed. Academic Press, New York.

BIBLIOGRAPHY 309

Szent-Györgyi, Α. 1953. "Chemical Physiology of Contraction in Body and Heart Muscle." Academic Press, New York.

Tagnon, H. J. 1945. The nature of the mechanism of the shock produced by the injection of trypsin and thrombin. /. Clin. Invest. 24: 1-10.

Tagnon, H. J., Weinglass, A. R., and Goodpastor. 1945. The nature and mechanism of shock produced by the intravenous injection of chymotrypsin. Am. J. Physiol. 143: 644-655.

Terry, R. L. 1950. The surface precipitation reaction in the ovarian frog egg. Protoplasma 39: 206-221.

Thoma, R. 1906. Untersuchungen über die wachsartige Umwand-lung der Muskelfasern. Virchows Arch, pathol. Anat. u. Physiol. 186: 64-96.

Thomas, L. J. 1951. A blood anticoagulant from surf clams. Biol. Bull. 101: 230-231.

Thomas, L. J. 1954. The localization of heparin-like blood anti-coagulant substances in the tissues of Spisula solidissima. Biol. Bull. 106: 129-138.

Thompson, W. R. 1900. Die physiologische Wirkung der Protamine und ihre Spaltungsprodukte. Ζ. physiol. Chem. 29: 1-19.

Thornton, F. E. 1935. The action of sodium, potassium, calcium and magnesium ions on the plasmagel of Amoeba proteus at dif-ferent temperatures. Physiol. Zool. 8: 246-254.

Tobias, J. M., and Solomon, S. 1950. Electrically induced polar changes in viscosity in the hyaline protoplasm of Elodea with ob-servations on streaming and plastid charge. /. Cellular Comp. Physiol. 35: 1-9.

Trendelenburg, P., and Goebel, W. 1921. Tetanie nach Entfernung der Epithelkörperchen und Calciummangel im Blute. Naunyn-Schmiedebergs Arch, exptl. Pathol. u. Pharmakol. 89: 171-199.

Umrath, K. 1954. Über das elektrische Potential und den Aktions-strom von Eierstockeiern von Rana esculenta. Protoplasma 43: 385-401.

Van Harreveld, A. 1936. A physiological solution for freshwater crustaceans. Proc. Soc. Exptl. Biol. Med. 34: 428-432.

Vernon, Η. M. 1912. Die Abhängigkeit der Oxydasewirkung von Lipoiden. Biochem. Z. 47: 374-395.

Vernon, Η. M. 1914. Die Abhängigkeit der Oxydasewirkung von Lipoiden. II. Biochem. Z. 60: 202-220.

Verworn, M. 1896. Die polare Erregung der lebendigen Substanz durch den constanten Strom. IV. Pflügers Arch. ges. Physiol. 65: 47-62.


Voelkel, H. 1921. Die Beziehungen des Ruhestromes zur Erregbar-keit. Pflügers Arch. ges. Physiol. 191: 200-210.

Wacker, L. 1929. Zur Kenntnis der Vorgänge bei der Arbeit und Ermüdung des Muskels. Klin. Wochschr. 8: 244-249.

Weber, F. 1921. Zentrifugierungsversuche mit ätherisierten Spiro-gyren. Biochem. Z. 126: 21-32.

Weil, R. 1915. Sodium citrate in the transfusion of blood. /. Am. Med. Assoc. 64: 425-426.

Weimar, V. 1953. Calcium binding in frog muscle brei. Physiol. Zool. 26: 231-242.

Weimar, V. 1955. Peptone shock in frogs. Am. /. Physiol In press. Weise, Ε. 1934. Untersuchungen zur Frage der Verteilung und der

Bildungsart des Calciums im Muskel. Naunyn-Schmiedebergs Arch. exptl. Pathol, u. Pharmakol 176: 367-377.

Wiercinski, F. J., and Cookson, Β. A. 1949. Action of dilute trypsin on muscle protoplasm. Federation Proc. 8: 165.

Wiggers, C. J. 1950. "Physiology of Shock." Harvard Univ. Press, Cambridge.

Wilander, O. 1939. Studien über Heparin. Skand. Arch. Physiol 81: Suppl. 15.

Wilbur, Κ. M. 1940. Effects of colchicine upon viscosity of the Arbacia egg. Proc. Soc. Exptl. Biol. Med. 45: 696-700.

Wilbur, Κ. M. 1941. The stimulating action of citrates and oxalates on the Nereis egg. Physiol. Zool. 14: 84-95.

Willstätter, R., Grassmann, W., and Ambros, O. 1926. Blausäure-Aktivierung und -Hemmung pflanzliche Proteasen. Ζ. physiol. Chem. 151: 286-306.

Wilson, W. L. 1950. The effect of roentgen rays on protoplasmic viscosity changes during mitosis. Protoplasma 39: 305-317.

Wilson, W. L. 1951. The rigidity of the cell cortex during cell divi-sion. /. Cellular Comp. Physiol. 38: 409-415.

Wilson, W. L., and Heilbrunn, L. V. 1952. The protoplasmic cortex in relation to stimulation. Biol. Bull 103: 139-144.

Wilson, W. L., and Heilbrunn, L. V. 1953. The action of cyanide on the protoplasmic colloid. Biol Bull 105: 388.

Wilson, W. L., and Heilbrunn, L. V. 1954. An antimitotic substance from muscle. Biol. Bull. 107: 322-323.

Wilson, W. L., and Schub, Y. 1953. The effect of strong centrifugal force on the development of Chaetopterus eggs. Biol. Bull. 105: 389.

Winterstein, H. 1905. Wärmelähmung und Narkose. Z. allgem. Physiol. 5: 323-350.

Winterstein, H. 1926. "Die Narkose," 2nd ed. Springer, Berlin. Witschi, F. 1927. Fortgesetzte Untersuchungen über Muskeler-

müdung. Z. Biol. 86: 1-26.

BIBLIOGRAPHY 311

Wohlfarth-Bottermann, Κ. Ε. 1954. Cytologische Studien. I. Zur sublichtmikroskopischen Struktur des Cytoplasmas und zum Nach-weis seiner "Partikelpopulationen." Protoplasma 43: 347-381.

Wood, F. D., and Wood, Η. E. 1932. Autotomy in decapod Crusta-cea. /. Exptl Zool 62: 1-55.

Woodward, Α. A. 1948. Protoplasmic clotting in isolated muscle fibers. /. Cellular Comp. Physiol. 31: 359-394.

Woodward, A. A. 1949. The release of radioactive Ca 4 5 from muscle during stimulation. Biol. Bull. 97: 264.

Yamaha, G. 1937. Kataphoretische Versuche an den Pollenmutter-zellen einiger Pflanzen. Cytologia Fujii Jub. Vol 617-626.

Yasutake, T. 1925. Studien über antagonistische Nerven. Nachweis der Mobilisierung von Kalzium im Herzen durch Reizung des Ner-vus accelerans. Z. Biol. 82: 605-610.

Young, J. Z. 1936. The structure of nerve fibers in cephalopods and Crustacea . Proc. Roy. Soc. B121: 319-337.

Young, J. Z. 1938. The functioning of the giant nerve fibres of the squid. /. Exptl Biol. 15: 170-185.

Yudkin, W. H. 1944. Tetrodon poisoning. Bull. Bingham Oceanogr. Collection 9: No. 1.

Yudkin, W. H. 1945. The occurrence of a cardio-inhibitor in the ovaries of the puffer, Sphéroïdes maculatus. /. Cellular Comp. Physiol. 25: 85-95.

Yung, E. 1878. Recherches sur la structure intime et les fonctions du système nerveux central chez les crustacés décapodes. Arch. Zool. exptl. et gén. 7: 401-534.

Zakrzewski, Z. 1932. Untersuchungen über den Einfluss von Hep-arin auf das Wachstum von transplantablen Sarkomen. Bull intern, acad. polon. sei. Classe med. 1932: 239-259.

Zucker, M. J. 1944. A study of the substances in blood serum and platelets which stimulate smooth muscle. Am. J. Physiol. 142: 12-26.

AUTHOR INDEX

A Abelous, 105, 2 8 0

Abrahams, 118, 2 8 0

Adams, 205 , 2 9 0

Adant, 161 , 3 0 3

Adriani, 2 8 0

Ahlquist, 118, 2 8 0

Alcock, 146, 2 8 0

Alday-Redonnet , 109, 2 8 0

Allen, 160, 163, 2 0 3 , 2 8 0

Ambache , 136, 2 8 0

Ambros, 199, 3 1 0

Angerer, 37 , 2 3 9 - 2 4 1 , 246 , 247 , 2 7 1 ,

272 , 2 8 0

Anslow, 178, 239 , 2 8 0

Asher, 105

Ashkenaz, 96, 107, 110, 140, 153, 2 8 1

Astbury, 96, 281

Austin, 239 , 2 8 1

Autenrieth, 2 1 1 , 281

Β

Baas-Becking, 20 , 281

Babasaki, 254 , 281

Bainbridge, 160, 281

Balazs, 202 , 281

Balistocky, 163, 3 0 2

Bàrlund, 2 3 1 , 2 8 1

Barnard, 267 , 2 8 1

Barone, 152, 281

Barr, 11 , 2 8 1

Bartholinus, 227

Baumberger, 120, 281

Bayliss, 65 , 2 8 1 , 2 8 2

Beams, 200 , 2 8 2

Bear, 129, 2 8 2

Belar, 4 2 , 2 8 2

Bendich, 120, 2 8 4

Beninson, 153, 285

Beraldo, 163 , 2 8 2

Bernard, 2 2 1 , 2 3 3 , 2 5 4 , 2 8 2

Bernstein, 53 , 54 , 98 , 2 8 2

Berwick, 239 , 249 , 2 8 2

Beznak, 109, 2 8 2

Bezold, 111-113 , 137, 2 8 2

Bick, 117, 305

Biedermann, 112, 123, 140, 2 8 2

Blanchard, 120, 2 8 9

Blinks, 60 , 2 8 2

Bloor, 124, 2 8 8

Blumenfeldt , 139, 2 8 2

Bocher, 202 , 2 8 2

Bogatko, 205 , 3 0 3

Bois Reymond, 150, 151 , 2 8 3

Botta, 48 , 2 8 3

Bouckaert, 142, 2 8 3

B o w m a n , 228 , 308

Braun, 226 , 227 , 2 8 3

Brink, 139, 224 , 2 8 3

Brinley, 250 , 2 8 3

Brockman, 90 , 2 8 9

Bronk, 139, 2 8 3

Brooks, M. M., 7, 2 8 3

Brooks, S. C , 7, 2 8 3

Brown, R. H. J., 25 , 26 , 2 8 3

Brown, W . R., 266 , 307

Bryant, 144, 2 8 3

Bucciante, 173, 2 8 3

Buchthal , 102, 123 , 2 8 3

Bülbring, 125, 2 8 3

Bullough, 2 1 3 , 2 1 4 , 2 8 4

Burgen, 151 , 2 8 4

Burns, 125, 2 8 4

Bütschli, 76 , 2 8 4

C

Camack, 239 , 2 8 1

Cameron, 77 , 2 8 4

Cannon, 162, 2 6 3 - 2 6 6 , 284

Carruthers, 192, 3 0 8

Carter, 120, 2 8 4

Cecil , 155, 2 8 4

Chaet, 9 1 , 118, 119, 157, 208 , 2 8 4

Chambers, 95 , 131 , 133, 284

Chargaff, 120, 2 8 4

Charles, 89 , 2 8 4

Churney, 4 8 , 172, 284 , 285

Cicardo, 153, 2 8 5

Clark, A. J., 108, 109, 2 8 5

Clark, J. H., 239 , 2 8 5

Clark, J. M., 244 , 285

312

AUTHOR INDEX

Cloetta, 2 2 9 , 2 8 5

Cobb, 124, 2 8 8

Cole, 3 3 , 54 , 143, 144, 2 8 5

Conklin, 4 2 , 170, 174, 2 8 5

Cookson, 116, 3 1 0

Coombs, 110, 2 8 5

Coppée , 139, 153 , 2 8 5

Costel lo, 2 4 , 67 , 2 8 5

Couillard, 9 0 , 2 1 0 , 2 8 5

Cowan , 153, 2 8 5

Crane, 5 2 , 2 8 6

Cremer, 134, 137, 2 8 6

Cruz, 117, 3 0 2

Cullis, 151 , 2 8 6

Cunningham, 13, 2 8 6

Curtis, 5 4 , 143 , 144, 2 8 6

D Dahlgren, 4 8 , 2 8 6

Da le , 151 , 2 8 6

D a n , 4 5 , 168, 174, 2 8 6

Daniel l i , 7, 2 8 6

Daugherty , 39 , 4 9 - 5 1 , 178, 239 , 2 4 2 , 245 ,

246 , 2 4 8 - 2 5 0 , 2 5 9 - 2 6 1 , 286 , 2 9 2

Davenport , 125, 2 8 7

D a v i d m a n , 77 , 2 8 6

Davies , 101 , 2 8 8

Davis , 158, 2 8 6

Davson , 7, 2 8 6

D e , 108, 2 8 6

D e b i e , 156, 2 8 6

D e l e z e n n e , 7 1 , 7 2 , 115, 2 8 6

Deutsch , 102 , 2 8 3

Dixon, 125, 287

Dol l ey , 77 , 2 8 6

Dragstedt , 2 6 6 , 2 6 7 , 2 6 9 , 3 0 5

Dreyer, 102 , 287

Dujardin, 6 3 , 75 , 9 1 , 2 8 7

D u n n , 207 , 2 0 8 , 2 9 2

Dust in , 200 , 2 8 7

Ε Eccles , 55 , 287

Edsall , 120, 2 8 7

Egner , 160, 2 8 0

Ehrenberg, 2 5 4 , 255 , 3 0 0

Eigsti , 2 0 0 , 2 8 7

Eilers, 2 4 , 2 8 7

Einstein, 14, 2 3

Enerson, 2 0 3 , 2 8 0

Engelberg , 118, 287

Enge lmann, 97 , 98 , 112, 2 8 7

Erlanger, 160, 287

Ernst, 228 , 3 0 0

Essner, 7 0 - 7 2 , 2 8 7

Evans , 200 , 2 8 2

Eyring, 2 3 3 , 2 9 6

F

Fahrenbach, 90 , 2 8 9

Falkenhagen, 4 5 , 2 8 7

Falkenhausen, 120, 2 8 9

Fatt , 55 , 152 , 287 , 2 8 8

Federow, 144, 2 8 8

Feldberg , 151 , 2 8 6

Fenn , 124, 125, 2 8 8

Ferguson, 2 2 4 , 2 8 8

Fessard, 139, 2 8 8

Fetter, 24 -26 , 2 8 8

Fischer, Albert, 115, 2 0 1 , 2 8 8

Fischer, Alfred, 130, 2 8 8

Fischer, H., 2 2 9 , 2 8 5

Fisher, 2 6 6 , 2 9 4

Fishman, 192 , 2 9 4

Flaig, 132, 145, 2 8 8

Fleckenstein, 101 , 2 8 8

Fletcher, 104

Flory, 4 , 5, 2 8 8

Foster, M. L. , 178, 2 3 9 , 2 8 0

Foster, R. Η. Κ., 266 , 2 8 8

Fowler , 205 , 3 0 3

Frederikse, 250 , 2 8 8

Freund, 161 , 2 8 8

Freundlich, 27 , 2 8 9

Fröhlich, 134, 2 8 9

Frommhagen , 90 , 2 8 9

Fuchs , 115, 118-120 , 289 , 2 9 7

Fühner, 197, 2 8 9

Fürth, 15, 2 8 9

G

Gahringer, 163 , 2 8 9

Gasser, 160, 2 8 7

Gellhorn, 7, 2 8 9

Gerber, 120, 2 8 9

Ghosh, 2 3 2 , 2 8 9

313

314 AUTHOR INDEX

Gilbert, 118, 2 8 9

Gley, 142, 2 8 9

Goebel , 108, 309

Goemer , 2 0 1 , 2 8 9

Goldstein, 9 2 , 118, 269 , 2 8 9

Goodpastor, 266 , 3 0 9

Göpfert, 123, 3 0 6

Graber, 151, 307

Graham, 4 , 2 8 9

Gramenizki, 144, 2 8 9

Grassman, 199, 3 1 0

Green, 115, 162, 214 , 284 , 307

Greenstein, 191 , 2 8 9

Gross, 70 , 9 2 - 9 5 , 2 9 0

Guest, 2 1 6 , 2 9 0

Gwathmey , 227 , 2 9 0

H

Haeckel , 75 , 131, 132, 290

Hahnert, 50 , 2 9 0

Haist, 156, 2 9 0

Halaban, 214 , 215 , 2 9 2

Hamilton, J. I., 156, 290

Hamilton, P., 58 , 59 , 2 9 2

Harding, 9 1 , 179, 180, 207 , 2 9 0

Hardy, 47 , 48 , 131 , 2 9 0

Harkins, 158, 2 9 0

Harris, D . L., 5, 79 , 84 , 9 1 , 2 9 0

Harris, J. E. , 16, 19, 20 , 2 9 0

Harris, T. A. B., 220 , 2 3 4 , 256 , 2 9 0

Hartmann, 120, 2 9 0

Hartwell , 190, 205 , 2 9 0

Harvey, 136, 2 9 1

Hastings, 108, 3 0 0

Heilbrunn, 15-17, 249 , 250 , 2 9 1

Helmholtz , 44 , 131 , 2 9 3

Henny , 96 , 307

Hering, 112, 149, 2 9 3

Hermann, 154, 2 9 3

Hermans, 35 , 2 9 3

Heubner, 125, 2 9 3

Heuking, 116, 2 9 3

Hevesy , 2 0 3 , 2 9 3

Higgins , 266 , 2 9 4

Hill , Α. V., 97 , 101 , 125-127 , 134, 2 9 4

Hill, D . K., 144, 294

Hill , S. E . , 56 , 219 , 220 , 226 , 3 0 3

Hirshfeld, 266 , 3 0 3

Höber, 139, 230 , 2 3 1 , 294

H o d g e , 78 , 2 9 4

Hodgkin , 54 -56 , 6 1 , 133-135 , 2 7 1 , 2 9 4

Höfler, 2 3 1 , 2 9 4

Holl ingsworth, 178, 294

Hol loman, 206 , 2 9 4

Holmes , 2 0 3 , 294

Holmgren, 2 0 2 , 281

Homburger, 192, 294

Hooker, 109, 295

Hopkins , A. L., 102, 157, 2 9 5

Hopkins, H. , 229 , 295

Hotel l ing, 20 , 2 8 1

Houssay, 142 , 2 9 5

Howard, 2 1 , 22 , 295

Howarth, 118, 2 8 0

Hückel , 4 5

Hughes , 167, 2 9 5

Hult in, 9 3 , 95 , 248 , 2 9 5

Humboldt , 234 , 295

Hunt , 151 , 2 9 5

Hürthle, 102, 295

Huxley, 54 , 294

I

Ikawa, 206 , 2 9 5

Immers, 9 0 , 2 0 4 , 2 9 5

Inoué, 174, 2 9 6

J Jacobs, 162, 2 3 1 , 2 9 6

Jacobson, 163 , 2 0 3 , 2 8 0

Jahn, 139, 2 9 6

Janeway, 117, 2 9 6

Janke, 101 , 2 8 8

Jaques, 163 , 229 , 2 9 6

Jeener, 120, 2 9 6

Johnson, 2 3 3 , 2 9 6

Jowett , 2 3 2 , 2 9 6

Just, 34 , 2 9 6

Κ

Kao, 131 , 133 , 2 8 4

Katz, 54 , 55 , 133 , 134, 137, 294 , 2 9 6

Katzenstein, 161 , 163 , 3 0 2

Kaye, 2 0 1

Kayser, 2 2 9 , 2 9 6

Kelly, 90 , 2 0 4 , 207 , 296 , 2 9 7

Kessler, 250 , 297

Keynes, 143 , 144, 294 , 2 9 7

AUTHOR INDEX 315

Kirschon, 2 0 3 , 2 8 0

Klein, 48 , 285

Kline, 215 , 3 0 6

Klingler, 178, 239 , 2 8 0

Knappeis , 102, 123 , 2 8 3

Knisely, 86 , 297

Koepfli, 2 0 6 , 2 9 5

Koshtojanz, 152, 297

Kraus, 115, 118, 297

Krebs, 101 , 2 8 8

Kreisler, 202 , 297

Krieger, 131 , 2 9 7

Krop, 152, 300

Kruyt, 4 , 2 9 7

Kuffler, 140, 151 , 297

Kühnau, 120, 199, 2 9 7

Kuhne, 28 , 47 , 149, 297

L

Labhardt, 105, 2 9 7

Lafontaine, 156, 2 8 6

Lambers , 200 , 2 9 8

Lanczos , 136, 2 9 8

Lang, 120, 307 , 3 0 8

Lapicque , 134, 2 9 8

Larson, 25 -27

Lawaczek, 154, 2 9 8

Leeuwenhoek , 3 2

LeFevre , 84 , 9 1 , 2 9 2

Lehotzky, 48 , 2 9 8

Lehto , 163 , 2 2 8 , 3 0 8

Leist, 45 , 287

Lever, 120, 2 8 7

Lewis , 78 , 2 9 8

Lill ie, F . R., 33 -45 , 4 1 , 187, 2 9 8

Lillie, R. S., 150, 182, 197, 2 2 1 , 230 , 2 3 1 ,

2 9 8

Lippman, 202 , 2 0 3 , 2 1 1 , 2 9 8

Lippold, 136, 2 8 0

Lissâk, 109 , 136, 2 9 8

Locke, 153 , 2 9 8

L o e b , J., 78 , 79 , 176, 177, 2 9 9

Loeb , L. , 115, 199, 2 9 9

L o e b , R. F. , 155, 2 8 4

Loewi , 150, 2 9 9

Lohmann, 97 , 101 , 106, 2 9 9

Loicq, 2 0 0 , 2 9 9

Lorand, 92 , 120, 133 , 2 9 9

Lorente de No, 55 , 60 , 2 9 9

Love , 90 , 2 9 9

Lowel l , 201

Lucas, 151 , 2 8 6

Lucké, 192, 2 9 9

Lullies, 143, 2 9 9

Lumière, 192, 3 0 0

Lundblad , G., 92 , 269 , 270 , 3 0 0

Lundblad , L, 92 , 3 0 0

Lundholm, 254 , 255 , 300

Lundsgaard, 97 , 3 0 0

Lustig, 228 , 300

Lyons, 120, 3 0 0

M

McBain, 4, 3 0 0

MacCal lum, 5 8

McClendon, 48 , 194, 3 0 0

McKay, 152 , 3 0 0

McLean , 108, 3 0 0

McNamara, 152, 300

Manery, 124, 2 8 8

Marinesco, 2 5 3 , 3 0 0

Massell , 118, 2 8 7

Mast, 50 , 144, 3 0 0

Mathews , 119, 120, 3 0 0

Matis, 118, 301

Mazia, 76 , 167, 185, 192, 2 4 4 , 2 7 2 , 301

Meitzer, 154, 155, 301

Merländer, 118, 2 9 7

Meyer, H. H., 2 2 3 , 2 2 4

Meyer, Κ. H. , 5, 2 2 4 , 3 0 1

Meyerhof, 2 , 101 , 3 0 1

Michaelis , 3 3 , 2 8 5

Middlebrook, 92 , 2 9 9

Mihalyi, 120, 3 0 1

Miko, 109, 3 0 1 Milham, 2 0 3 , 2 8 0

Miller, 151 , 307

Mills, 161 , 3 0 1

Mitchison, 193 , 200 , 3 0 1 , 3 0 8

Molinelli , 142, 2 9 5

Mommaerts , 101 , 3 0 1

Monnier, 139, 140, 3 0 1 , 3 0 2

Moon, 158, 1 6 1 , 3 0 2

Moreau, 163 , 2 2 9 , 230 , 3 0 2

Morgan, 2 1 , 32 , 176, 3 0 2

Morgenstern, 199, 3 0 2

316 AUTHOR INDEX

Moser, 3 3 , 34 , 3 0 2

Mosso, 105, 3 0 2

Most, 118, 205 , 270 , 3 0 2

Moulder, 2 0 3 , 2 8 0

Moussatché, 117, 3 0 2

Mudd, 206 , 295

Müller, 235 , 3 0 2

Mylon, 161 , 163 , 3 0 2

Ν

Nalefski, 118, 2 8 9

Nauts , 205 , 3 0 3

Nekrassow, 118, 3 0 3

Nekrassowa, 118, 3 0 3

Nernst, 137

Neuhof, 266 , 3 0 3

Niemann, 206 , 2 9 5

Nolf, 161 , 3 0 3

Nysten, 122, 3 0 3

Ο Oakey, 206 , 3 0 3

Opie, 5, 3 0 3

Osterhout, 56 , 2 1 9 - 2 2 1 , 226 , 3 0 3

Overbeek, 2 3 , 3 0 3

Overton, 107, 2 2 3 , 2 2 4 , 3 0 3

Ρ

Pala, 109, 301

Palmer, 192, 304

Paré, 2 2 6

Park, 117, 2 9 6

Parpart, 34 , 192, 2 3 1 , 296 , 2 9 9

Partos, 120, 304

Pauli, 123 , 3 0 4

Pearson, 228 , 3 0 8

Pekarek, 15, 22 , 3 0 4

Pentimalli , 48 , 3 0 4

Percival, 108, 109, 2 8 5

Permeschko, 172, 3 0 4

Pflüger, 137, 3 0 4

Pike, 110, 2 8 5

Piulachs, 155, 3 0 4

Polissar, 2 3 3 , 2 9 6

Pollack, 95 , 2 8 4

Posternak, 2 2 4 , 2 8 3

Proctor, 80 , 3 0 4

Punt, 152, 304

Purr, 199, 3 0 4

Q Quastel, 232 , 305

R Ranke, 105, 305

Ranson, 125, 287

Rapkine, 182, 185, 3 0 5

Rashevsky, 137, 3 0 5

Rashkis, 215 , 3 0 5

Rathgeb, 205 , 305

Rauh, 104, 305

Reid, 117, 3 0 5

Reuss, 228 , 300

Rex-Kiss, 136, 305

Ricca, 192, 2 9 9

Richardson, 117, 2 9 6

Rieser, 28 -30 , 34 , 122, 131 , 164, 305

Riker, 151 , 305

Ritenour, 228 , 308

Robbie, 198, 305

Rocha e Silva, 266 , 267 , 269 , 305

Röder, 27 , 3 0 6

Roeder, 136, 3 0 6

Rona, 125, 2 9 3

Rosso, 154, 3 0 6

Runnström, 95 , 3 0 6

Rusch, 215 , 3 0 6

Rutman, 2 1 1

S

Sande Bakhuyzen, 20 , 281

Sanderson, 2 0 3 , 2 8 0

Sandow, 104, 3 0 6

Scardovi, 118, 307

Schaefer, 123, 3 0 6

Scheele , 118, 301

Schmidt, Α., 87 , 115, 3 0 6

Schmidt, W . J., 4 1 , 4 2 , 173, 174, 306

Schmitt, 129, 2 8 2

Schräder, 167, 3 0 6

Schub, 35 , 3 1 0

Scott, 89 , 2 8 4

Scott Blair, 11 , 306

Searle, 110, 2 8 5

Seegers, 216 , 2 9 0

Severino, 227

Shaver, 181, 182, 306

Shear, 205 , 306

Shenk, 58 , 3 0 6

Simha, 2 3 , 306 , 307

Simonson, 105, 307

AUTHOR INDEX 317

Smith, 151 , 307

Smitten, 144, 307

Smoluchowski , 4 5

Sodeman, 155, 307

Solomon, 50 , 3 0 9

Spek, 194, 3 0 7

Spiegel-Adolf, 96 , 307

Stâlfelt, 200 , 307

Staudinger, H. , 5, 307

Staudinger, M., 5, 307

Steinberg, 2 6 6 , 307

Stewart, 108, 109, 285

Stiles, 126, 307

Stokstad, 90 , 2 8 9

Stoner, 115, 162, 307

Storti, 118, 307

Strangeways, 172, 173 , 307

Straub, 108, 307

Strohe, 139, 2 9 4

Stuber, 120, 307 , 3 0 8

Sugai, 199, 308

Sunderman, 2 3 9 , 2 8 1

Suntzeff, 192 , 308

Suomalainen, 163 , 228 , 229 , 308

Si i to-Nagy, 161 , 163 , 3 0 2

Svihla, 228 , 308

Swann, 174, 193, 200 , 3 0 8

Swift, 205 , 3 0 3

Sylvén, 205 , 305

Szent-Györgyi , 98 -102 , 116, 2 9 3 , 308 ,

3 0 9

Τ

Tagnon, 161 , 266 , 269 , 309

Terroux, 151 , 2 8 4

Terry, 69 , 70 , 3 0 9

Thoma, 80 , 309

Thomas, 90 , 3 0 9

Thompson, 160, 309

Thornton, 38 , 238 , 3 0 9

Tobias, 50, 144, 283 , 309

Trendelenberg, 108, 309

Trevan, 160, 2 8 1

Turner, 205 , 306

U

Umrath, 56 , 3 0 9

Van der Loeff, 229 , 2 8 5

Van Harreveld, 142, 3 0 9

Vasseur, 90 , 204 , 2 9 5

Vernon, 232 , 2 3 3 , 3 0 9

Verworn, 50 , 309

Voelkel, 146, 3 1 0

Vogt, 151 , 2 8 6

W

Wacker, 110, 3 1 0

Ware, 216 , 2 9 0

Warner, 120, 2 8 4

Waters, 163, 229 , 2 9 6

Weber , 2 3 1 , 250 , 294 , 3 1 0

Wei l , 266 , 3 1 0

Weimar, 111 , 163 , 239 , 2 4 3 , 3 1 0

Weinglass , 266 , 3 0 9

Weise , 110, 310

Wiercinski, 113, 114, 116, 117, 292 , 310

Wiggers , 154, 158, 3 1 0

Wilander, 229 , 3 1 0

Wilbur, 141 , 187, 200 , 240 , 2 4 1 , 2 7 1 ,

2 7 2 , 280 , 2 9 3 , 3 1 0

Willot , 156, 2 8 6

Willstätter, 199, 3 1 0

Wilson, 16, 35 , 38 , 39 , 4 1 , 84 , 9 1 , 170,

174, 178, 179, 183, 198, 2 0 1 , 2 0 3 , 205,

207 , 208 , 2 1 1 , 2 1 2 , 2 1 4 , 2 4 2 , 249 , 269 ,

2 9 2 , 2 9 3 , 3 1 0

Winternitz, 161 , 163 , 3 0 2

Winterstein, 161 , 163 , 227 , 2 3 1 , 234,

235 , 3 1 0

Witschi , 105, 3 1 0

Wohlfarth-Bottermann, 130, 311

W o o d , F. D . , 157, 311

W o o d , Η. E. , 157, 311

Woodward , 79 , 82 , 84 , 9 1 , 110, 116,

292 , 3 1 1

Y

Yamaha, 48 , 311

Yasutake, 110, 311

Young, 123, 124, 129, 131 , 132, 282 , 311

Yudkin, 210 , 311

Yung, 75 , 131 , 132, 311

Ζ

Zakrzewski, 2 0 1 , 311

Zsigmondy, 4

Zucker, 117, 311 Vaccari, 118, 307

V

SUBJECT INDEX

A Acclimatization, 2 6 4

Acetylchol ine, 150-153 , 2 3 4

Acid, as parthenogenet ic agent, 176, 179

Acid polysaccharide, 2 0 7

Acidity, 60 , 95 , 182

Action current, in conduction, 149, 150

Action potential, 53-57 , 105, 111 , 143 ,

2 3 0 , 2 4 4

Actomyosin, 99 , 100

Adaptation, 2 6 4

Adaptive enzymes , 162, 2 6 4

Adenosine triphosphate, 97 , 9 9 - 1 0 2

Adrenaline, 150, 151 , 153 , 154, 160, 162

Alcohol, 248 , 249 , 2 5 4 , 255 , 260 , 261

stimulation by, 2 3 4 , 2 6 1

Algae, 74 . See also Chara, Nitella,

Valonia, etc.

Alkali, as parthenogenetic agent, 176,

179

Alternating current, 240 , 2 4 1 , 2 7 1 , 2 7 2

Ameba, anodal breakdown, 143, 144, 2 4 0

cortex, 36 , 37 , 2 3 8 - 2 4 2 , 248 , 249 , 2 7 1 ,

2 7 2

effect of calcium on, 39 , 2 4 5

of cold on cortex, 38 , 238 , 2 3 9

of fat solvents, 2 4 8 - 2 5 0 , 259 -261

of heat on cortex, 38 , 238 , 2 3 9

of potassium, 39 , 2 4 2

of stimulation on, 2 3 8 - 2 4 2 , 245 -247

electric charges in protoplasm of, 5 1 ,

5 2

surface precipitation reaction, 67

viscosity, 2 2

Amoeba dubia, 36 , 2 5 0 , 260 , 261

Amoeba proteus, 36 -38 , 67 , 238 -240 , 2 4 9

Ammonia, 150

Anaphylactic shock, 159, 160, 163 , 2 2 9

Anesthesia, 217 , 218 , 220 -236 . See also

Anesthetics.

agents causing it, 2 2 2 - 2 2 9

colloidal theory, 2 3 7 - 2 6 2

definition, 218 , 2 2 0 - 2 2 2 , 225 , 2 2 6

effect on metabol ism, 2 3 1 - 2 3 3

in a strict sense, 221

oxidation theory, 2 3 1 - 2 3 3

permeabil i ty theory, 230 , 2 3 1 , 2 3 6

Anesthetics , 2 2 2 - 2 2 9 . See also Anesthesia.

action of, 2 4 6 - 2 6 2

on nerve protoplasm, 146

as stimulants, 227 , 233 -236 , 259 -261

depolarizing effect, 146

effect on blood clotting, 254 , 255

on cell division, 168, 197

on electric potential , 5 3

on metabol ism, 2 3 1 - 2 3 3

on muscle , 121

on nerve, 145

Anion electrode, 5 8 - 6 0

Arbacia egg . See also Sea urchin egg.

calcium release, 138, 2 4 4

division, 170, 171 , 180, 204

effect of X-rays on, 2 0 3 , 2 0 4

of cyanide on, 198

entrance of calcium, 138

mitotic elongation, 172

parthenogenesis , 179, 180

viscosity, 18, 19, 22 -24

Arenicola larva, 197

Arginine, 97

Artificial parthenogenesis , 175-182

Ascites tumors, 2 0 2 , 2 0 3

Asterias egg , 67 , 68 . See also Starfish

egg.

Asters, 167

Astral rays, 171 , 172, 193, 194

ATP, 97 , 9 9 - 1 0 2

Atropine, 152

Autonomic nerves, 142

Autotomy, 157

Axoplasm, 131-134

Β

Bacterial extracts, 205 , 206 , 208 , 2 0 9

Barium, 7 3 , 102

Bean, 16, 17

Benzanthrenes, 191 , 192

Bernstein theory, 5 3 , 54 , 56 , 2 3 0

Bioluminescence, 2 2 0

Biostasis, 264 , 265

318

SUBJECT INDEX 319

Birds, shock in, 163

Birefringence, 4 1 , 4 2 , 173 , 174, 194

Blood. See also Blood clotting.

calcium, 109, 110

homeostat ic behavior, 2 6 5 - 2 6 7

platelets, 117

pressure, 155

serum, 117, 118, 154, 181

s ludge, 8 6

vessels , 9 1 , 109, 118, 155, 2 0 5

Blood clotting, 8 6 - 9 2

as homeostat ic reaction, 2 6 5 - 2 6 7

comparison wi th s.p.r., 7 2 , 8 2 , 8 3

effect of anesthetics on, 2 5 4 , 2 5 5

of concentrated calcium solutions on,

7 2

of cyanide on, 199

of muscle extracts on, 115

in hibernating animals, 2 2 8

relation to oxidation, 119, 120

similarity to protoplasmic clotting, 9 2 -

94

substances in muscle , 114, 115

Brain, 159

Bromide, 5 8

Brownian movement , 14, 19, 21

Bubbl ing movements , 173

Burn shock, 156, 159

C

Calcium. See also Calcium release.

activation of protease by, 7 1 , 7 2 , 9 2 ,

188, 2 6 9

as parthenogenetic agent, 178

as stimulating agent, 2 2 2 , 2 4 3

diffusion coefficient, 126

diffusion in muscle , 125-127

effect of concentrated solutions of, 7 1 ,

7 2

on cortex, 39 , 4 0

on homogenates , 9 3 - 9 5

on muscle , 113 , 114

on nuclear breakdown, 141 , 187, 188

on protoplasmic viscosity, 133, 189,

245 -247

electrode, 57 , 5 8

importance for nerve conduction, 142

for neuromuscular transmission, 153 ,

154

in muscle , 124, 125

in muscular contraction, 107-111 , 247

l iquefaction of protoplasm by, 133,

188, 189

proteinate, 46 , 56 , 57

radioactive, 110

relation to acetylcholine, 152, 153

to adrenaline, 153

to bioelectric potential , 56 -58 , 61

to nerve excitation, 135 -143

to nuclear membrane breakdown,

141 , 187, 188

to protoplasmic clotting, 92 -94

to s.p.r., 6 5 - 7 3 , 80 -84

Calcium release, b y acetylchol ine, 152

b y anesthetics, 2 4 9

b y carcinogens, 192

b y cold, 2 3 9

b y fat solvents, 2 3 9 , 2 4 9

b y heat, 2 3 9

b y parthenogenet ic agents, 178

b y sodium and potassium, 138

b y st imulating agents , 2 4 3 , 2 4 4

electrical effect of, 57

in musc le , 1 0 9 - 1 1 1 , 124, 125

in nerve, 136

Callicrein, 161

Cancer, 166, 167, 169

carcinogenesis , 175, 190-192

chemotherapy, 196

effect of exercise on, 2 1 4 , 2 1 5

of heparin on, 2 0 1 - 2 0 3

of metabol ic inhibitors on, 2 1 6

of X-rays on, 2 0 3

Capillary leakage, 164

Carcinogenesis , 175, 190-192

Carbon dioxide, effect on electric charge,

49 , 5 1 , 5 2

on electric potential , 6 0

Cat, 78 , 110, 116, 124, 125

Catgut, 97 , 9 8

Cathepsin, 199

Cathode, contraction at, 103 , 111-113

excitation at, 137

320 SUBJECT INDEX

Cell division, 166-216

antimitotic agents, 195-216

morphological changes during, 169-173

physical changes during, 183-185 , 189,

190

suppression, 195-216

Cellular homeostasis, 2 6 3 - 2 7 2

Centrifuge method, 14

Centromere, 167

Centrosomes, 167

Centrospheres, 171

Cerebratulus egg , 67 , 68

Chaetopterus egg , cortex, 33 -35 , 37, 2 3 9

effect of antimitotics on, 2 0 6 - 2 1 3

of cyanide on, 198

of fat solvents on, 249

of heparin on, 91

of muscle extracts on, 212 , 213

of ovarian extracts on, 206 -212

of Shear's polysaccharide on, 205

homeostatic behavior, 269

maturation, 185, 186, 188-190, 269

mitotic spindle, 170, 174

parthenogenesis , 179, 180

protease in, 188, 2 6 9

t ime sequence during division, 168,

170

viscosity, 16, 18

changes during mitosis, 183-185 , 2 0 6

Chara, 5 3

Chick heart, 151

Chicken blood, 120

muscle , 115

Chloral hydrate, 197

Chloretone, 198

Chloride electrode, 58-60

Chloride in cells, 58 , 59

Chloroplasts, 49 , 50

Choline, 55

Chromatin, 47 , 4 8

Chromosomes, 48 , 167, 193

Chymotrypsin, 116, 161, 2 6 6

Citrate, 8 1 , 82 , 87, 141 , 142, 187, 2 6 6

Clam, 9 0

Clam eggs . See Cumingia egg, Spisula

egg.

Clam heart, 208

Cloudy swel l ing, 77

Coal tar, 175

Cocaine, 2 3 4 , 2 4 9

Colchicine, 199, 2 0 0

Cold, anesthetic action, 227 , 257

as parthenogenetic agent, 178, 179

calcium release by, 111, 2 3 9

effect on cortex, 38 , 39

on s.p.r., 7 3 , 257

vacuolization caused by, 76

Colloid, 3 , 4

Compression (of e g g c e l l ) , 3 3

Condenser, 45 , 54

Conduction, 148-150

Convulsions, 110

Cortex, 3 1 , 3 3 - 4 0

cycl ic colloidal change in, 2 7 1 , 2 7 2

effect of fat solvents on, 249 , 2 5 0

of parthenogenetic agents on, 178

of stimulation on, 2 3 9 - 2 4 3

importance, 3 2 , 34

l iquefaction by ultraviolet, 239

muscle fiber, 30 , 3 1 , 126

nerve fiber, 131

relation to calcium, 38 -40

C o w ovary, 2 1 1 , 2 1 2

Crab, injury, 157

muscle , 55 , 64 , 80 , 81

nerve, 74 , 75 , 77 , 142

Crayfish nerve, 77, 142

Creatine, 97

Crepidula egg , 170, 171

Crepidula plana, 171

Crustacean nerve, 144. See also Crab,

Crayfish, Lobster.

Cumingia egg , 18, 2 1 , 67, 183-185

Cunningham correction, 13, 14

Curare, 151

Cyanide, 120, 198, 199

Cyclic colloidal change, 2 7 1 , 272

Cyclosis, 2 2 0

Cytochrome oxidase, 2 3 3

Cytolysis, 78 , 177, 192, 197

D

Degenerat ion, 77 -79

waxy, 8 0

SUBJECT INDEX 321

Deoxypentosenucleoprote in , 167

Depolarization, 5 3 , 60 , 6 1 , 146

of end plate, 152

Dibenzanthrene , 192

Dielectric constant, 44 , 4 5

Diffusion, 125-127 , 149

Diffusion coefficient, 126

Di latancy, 2 5 , 2 6

Dilatation, 9 1 , 118

D o g , 105, 109, 110, 158, 2 0 3 , 2 1 1

D o u b l e layer, 44

Duck, 115

Ε

Echinarachnius egg , 67

Ectoplasm, 3 4

E g g organization, 2 1 , 3 2 , 3 5

Einstein's formulae, 14, 19, 2 3

Elasticity, 3 2 , 3 3

Electric charge, 4 4 - 5 2

Electric current, action on ameba, 2 4 0 ,

2 4 1 , 2 7 1 , 2 7 2

shock caused by, 159

vacuolization caused by, 7 6

Electric potential , 7, 4 4 , 45 , 52 -61

Electric ray, 4 8

Electrokinetic potential , 44 , 4 5

Electron microscopy, 130

Electrophoresis , 4 5 - 5 2

Elodea, 4 9 , 50 , 77 , 200 , 2 4 4

Emuls ions , viscosity of, 2 4

E n d plate, 150, 152

potential , 151

Escherichia coli, 205 , 206 , 208 , 2 0 9

Ether. S e e also Fat solvents, Anesthetics.

as parthenogenet ic agent, 178, 179

convulsions caused by, 2 3 4 , 2 3 5

effect on ameba, 250 , 260 , 2 6 1

on oxidation, 2 3 2

on sea urchin eggs , 2 5 0 - 2 5 3

on s.p.r., 7 3 , 74 , 2 5 4

Excitation, 217 -220 , 2 2 2 , 2 3 0 - 2 3 6

and permeabil i ty, 2 3 0 , 2 3 1


Exercise, effect on cell division, 2 1 3 , 2 1 4

on tumors, 214 , 2 1 5

F Fat solvents, action on protoplasm, 2 4 8 -

255 , 2 5 9 - 2 6 1

as anesthetics, 2 2 3 , 224 , 2 2 6

calc ium release by, 2 3 9

depolarizing effect, 146

effect on bioelectric potential, 146

on cell division, 197

on fermenting enzymes , 2 3 3

on protoplasmic clotting, 2 5 4

on s.p.r., 7 3 , 2 5 4

Fat igue , 104, 105, 118

Fat igue substances, 105, 2 1 2 , 2 1 3

Fermentation, 2 3 3

Fertilization, 3 3

Fertilizin, 2 0 4

Fibrin, 9 2 , 120

Fibrinogen, 120

Fibrinolysin, 2 1 6

Fish ovaries, 208 , 2 1 0 , 211

Fluid loss theory, 156, 157

F o a m , 7 6

Fol ic acid, 2 1 6

Frog

e g g , 5 6 , 6 9 - 7 2 , 180-182 , 201

head injury, 229 , 2 3 0

heart, 9 1 , 102, 108, 109, 118, 119, 136,

144, 2 0 8

insensitivity to histamine, 160

muscle , 79 , 80 , 82 , 8 3 , 103-105 , 107,

110, 111 , 112, 114, 116-118 , 125,

126, 149, 152, 2 1 3 , 2 3 4

nerve, 136, 139

ovary, 2 1 1

shock in, 156

F u g u , 2 1 0

G

Ganglion cells, 78 , 144

Gelation, 4 1 . See also Blood clotting,

Protoplasmic clotting.

mitotic, 183, 184

of nerve cell protoplasm, 144, 145

Gels, 3 1 - 4 2

definition, 35

in glycerinated muscle , 101

muscle , 98

322 SUBJECT INDEX

Germinal vesicle, 141 , 185-188

Glucosamine, 88

Glycerinated muscle , 100-102

Glycolysis, 120

Granules, 4, 15, 2 1 , 3 2

breakdown, 68-74

cortical, 3 3 , 34 . See also Cortex.

electric charge, 50, 51

in mitotic spindle, 170, 171 , 174, 194

relation to s.p.r., 68 -74

specific gravity, 18

transformation to vacuoles , 79

Grasshopper muscle , 8 1 , 8 2

Ground squirrel, 2 2 8

Guinea pig, 157, 161

H

Halicystis, 6 0

Hamster, 228

Haptogen membrane, 6 5

H e a d injury, 159, 229 , 2 3 0

Heart, 9 1 , 102, 108-110 , 118, 119, 151

ganglion cells of, 144

Heat , as anesthetic, 2 2 7

as parthogenetic agent, 176, 178, 179

as stimulating agent, 2 2 2

calcium release by, 111 , 2 3 9

effect on cortex, 38 , 39 , 238 , 2 3 9

prostration, 159

vacuolization caused by, 76

Hedgehog , 228

Helix pomatia, 2 2 8

Hemorrhage, 159, 162, 200 , 205 , 266 .

See also Petechiae.

Heparin, 88-91

as anesthetic, 2 2 7 - 2 2 9 , 257 , 2 5 8

as antimitotic agent, 2 0 1 - 2 0 3

effect on heart, 118, 119

in hibernating animals, 163, 228

in muscle , 89 , 116, 212 , 220 , 2 7 0

in sea urchin eggs , 2 0 4

in shock, 162-164

rebound, 267

relation to muscular contraction, 118,

119

release b y trypsin, 2 6 7

by X-rays, 203 , 204

spectrum, 2 0 8 , 2 0 9

Heparin-like substances, 88-91

effect on dividing cells, 2 0 4 - 2 1 3

from muscle , 116, 2 1 2

in shock, 163

Hexosamine, 8 8

Hibernation, 163, 227 -229 , 257

Histamine, 158, 160, 162

Hoeppler viscosimeter, 12

Homeostasis , 163, 2 6 3 - 2 6 6

cellular, 188, 2 6 3 - 2 7 2

Homogenates , 92 -94 , 111 , 121

Horse, blood, 87 , 255

muscle , 115

Hydrocyanic acid, 120

Hydrogen ion concentration, 49 , 5 1 , 52 ,

9 5

Hydrogen sulfide, 120

Hydroides e g g , 6 7

Hypertonic solutions, 76 , 176, 179, 180,

198, 2 5 8

Hyposulfites, 120

Hypotonic solutions, 76

I

Immature eggs , 4 1 , 189, 196

Injury, 64 , 95 . See also Shock.

as cause of cancer, 193

as stimulus to cell division, 174, 175

head, 169, 229 , 2 3 0

release of chloride by, 58

to nerve protoplasm, 75 , 131 , 132

vacuolization caused by, 76 , 78 , 79

Injury potential , 52 -54

Injury substances, 84 , 90 -92 , 158, 175

as parthenogenetic agents, 179, 180

Insect muscle , 64 , 81

Insulin shock, 161 , 163

Interfacial tension, 3 3 , 194

Intestinal manipulation, 159

Iodoacetic acid, 9 7

Iron rods, 15

Isoelectric point, 46 , 47 , 5 1 , 52 , 58

J Jactitation, 2 3 4

Κ

Kallicrein, 161

Karyokinetische Streckung, 172

Kinetochore, 167

SUBJECT INDEX 323

L heparin in, 89 , 90 , 118, 2 7 0

models of contraction, 9 7 - 1 0 2

opacity, 122-124

relaxation, 103, 118

smooth. See Smooth muscle.

stretched, 126, 127

surface precipitation reaction, 64 , 7 9 -

8 3

transparency, 122 , 123

twitch, 103 , 104

vacuolization, 7 9

viscosity, 2 8 - 3 0

Ν

Narcosis. See Anesthesia.

definition, 2 1 8 , 2 2 0 , 2 2 1

Nelson complex, 2 3 1

Nereis egg , 16, 4 1 , 141 , 186-189

Nerve , 129-149

conduct ion, 148, 149

excitation, 134-147

excited b y anesthetics, 2 3 5

fibrils, 129, 130, 132

s.p.r., 75 , 132

viscosity, 131

Neuromuscular transmission, 150-154

Nitella, action potential , 5 3 , 5 6

anesthet ized b y water, 2 2 6

conduct ion in, 148

excitation, 2 1 9

injury potential , 5 3

N o d e of Ranvier, 149

N o n - N e w t o n i a n fluid, 11

Noradrenaline, 151

Nucleoprotein, 95 , 167

Nucleus , electric charges in, 47 , 4 8

membrane breakdown, 141 , 186-188

Ο Oat, 89

Onion, 47 , 77

Oil drop, 28 -30 , 131 , 194

Opacity, gangl ion cells, 144

muscle , 122-124

nerve, 144, 145

Opium, 2 5 4

Osmosis , 6, 7

Ostwald viscosimeter, 11 , 12

Lact ic acid, 97 , 98 , 105, 120, 150

Latency relaxation, 104, 2 4 7

Ligature, 156, 159, 162

Lily, 174

Lipid, as antimitotic constituent, 205 ,

2 0 6

Lobster, blood, 115, 199

muscle , 115, 116, 212 , 2 1 3

nerve, 30 , 131

Lockjaw, 109, 110

Lol igo muscle , 123 , 124

M Magnes ium, as anesthetic, 224 -226 , 230 ,

2 3 1 , 2 5 5 - 2 5 7

as parthenogenet ic agent, 177

effect on muscle , 113

electrode, 58

in b lood during s leep, 2 2 9

in hibernating animals, 2 2 8

relation to s.p.r., 7 3 , 74 , 2 5 6

release, 57, 109

Marine eggs , 45 , 78 , 175. See also

Arbacia egg, Chaetopterus egg,

etc.

Mast cells, 88 -90

Mechanical stimulation, 239 , 240

Metachromatic reaction, 89 , 90 , 207

Meyer-Overton theory, 2 2 3 , 224 , 2 5 6

Micel le , 5

Mitosis. See Cell division.

Mitotic elongation, 172, 194

gelation, 183-185 , 198

index, 2 0 2 , 2 1 1

Mitotic spindle, 4 1 , 42 , 167, 168, 170,

174, 183 , 185, 193 , 194

Molecular superfolding, 9 6

Mouse, epidermis, 2 1 3 , 2 1 4

tumors, 2 0 1 - 2 0 3 , 2 1 1 , 2 1 2

Muscle , 9 6 - 1 2 8

calcium release, 2 4 4

effect of alcohol and ether on, 234 , 2 3 5

extracts, 2 1 2 , 2 1 3

exercise, 109, 2 1 3 - 2 1 5

fatigue, 104, 105, 118, 2 1 2 - 2 1 5

glycerinated, 100-102

324 SUBJECT INDEX

Ovarian extracts, 9 1 , 2 0 6 - 2 1 2

Ovary, 2 0 6 - 2 1 2

fish, 208 , 210 , 211

starfish, 2 0 7 - 2 0 9

Overton-Meyer theory, 2 2 3 , 224 , 2 5 6

Ovothrombin, 270

Ox, 8 9

Oxalate,

action on marine eggs , 141

on muscle , 8 1 , 82 , 139, 140

on nerve, 139, 141 , 142

as hastener of blood clotting, 2 6 6

effect on cortex, 39 , 4 0

in Elodea, 244

Oxidation, and anesthesia, 220

effect of clott ing on, 95 , 119-121 , 182,

247 , 2 4 8

in e g g cells, 177

enzymes , 232 , 2 3 3

in muscle , 96 , 97 , 106

increase on stimulation, 177, 2 3 1 , 2 3 2

relation to mechanism, 2, 106

Ρ

Pancreatic juice, 7 1 , 72

Papain, 116, 161 , 199

Paracentrotus egg , 2 4

Paraldehyde, 2 3 2

Paramecium, 12, 24 -27

Parasympathetic fibers, 122, 142, 150

Parathyroidectomy, 125

Peptone, 6 5

Peptone shock, 161-163 , 2 2 9

Periodate, 2 0 8

Permeability, 7

effect of calcium release on, 143, 182,

244 , 245

increase, 143, 2 3 0

of cortex, 4 0

of nerve, 143, 2 3 0

theory of excitation, 182, 230 , 2 3 1 , 2 3 6

to calcium, 108

Petechiae, 164

pH, 5 1 , 52 , 95

Phosphagen, 97

Phosphate, 97 , 139

Phosphorylation, 97 , 106

Photosynthesis , 49 , 50 , 60

Pig ovary, 211

Pigment granules, 24 , 68 , 69 , 7 1 , 74 , 79

Plasma membrane, 4 0

Plug formation, 8 0 - 8 3

Poise, 10

Poison, in fish ovaries, 208 , 210 , 211

Polarization, 145, 146

Polarization current, 47 , 137

Pollen mother cells, 174

Polymer chemistry, 4 , 5

Polysaccharide, 205 -207

Potassium, as depolarizing agent, 53 , 60

as parthenogenetic agent, 176

as stimulating agent, 222 , 241

calcium release by, 111 , 138

effect on bioelectric potential, 53 -55

on cortex, 39 , 2 4 1 , 2 4 2

on muscle , 113

electrode, 5 8

exit from nerve, 54 , 55

relation to excitation, 138, 139

release, 57 , 109

Pressure, as anesthetic, 2 2 6

Protamine, 118, 160, 162

Protease. See Proteolytic enzymes.

Protein, as antimitotic

constituent, 2 0 5 - 2 0 8

Protein chloride, 5 9

Proteolytic enzymes , activated by calci-

um, 92 , 133

clotting enzymes as, 9 2

effect of cyanide on, 198, 199

on muscle , 116

on viscosity, 189, 190, 199

in shock, 161

Prothrombin, 87 , 115, 255

Protoplasmic clotting, 84 -95

effect on oxidation, 95 , 119-121 , 182,

247 , 2 4 8

homeostatic behavior, 269-271

in excitation, 245 -247

in parthenogenesis , 78 , 79 , 177-182

relation to calcium, 92 -94 , 245 -247

Pteroylglutamic acid, 216

Puffer, 2 1 0

SUBJECT INDEX 325

Q Quarternary ammonium ions, 55 , 56

Quartz, 2 5

Quinine, 74

R

Rabbit, blood, 2 0 0

brain, 8 7

cancer, 175

heart, 152, 153

muscle , 100, 109, 110, 115, 2 4 3

shock, 161

Radiation, vacuolization caused by, 76

Rat, liver, 175

muscle , 64 , 80 , 110, 2 4 4

shock, 156-158 , 160

Rauh's nose, 104, 2 4 7

Refractory period, 2 2 6

Regeneration, 2 6 5

Rennin, 8 2

Respiration, effect on bioelectric poten-

tial, 5 3 , 6 0

increase on stimulation, 2 3 1 - 2 3 3

Response. See also Excitation.

definition, 2 1 8 - 2 2 0

repetitive, 139-142

rhythmic, 139-142

Rigor, 122

Rigidity (of cortex) , 3 8 - 4 0

Roentgen radiation, effect on dividing

cells, 2 0 3 , 2 0 4

X-ray sickness, 159

Root hairs, 74

S

Sabellaria egg , 19, 2 0

Salivary gland (of fly larvae) , 4 8

Salamander ovary, 2 1 1

Sand, 2 5

Sankyo Company, 2 1 0

Sarcode, 75

Sarcoma, 201

Sciatic nerve, 136

Sea anemone, 2 5 6

Sea cucumber muscle , 123

Sea urchin egg . See also Arbacia egg

anesthetized b y water, 224 , 2 2 6

cortex, 33 , 34

effect of calcium on, 2 4 5

of fat solvents on, 197, 2 5 1 - 2 5 3

of starfish ovary extract on, 2 0 8

of X-rays on, 2 0 3 , 2 0 4

heparin in, 204

homeostatic behavior, 269 , 270

homogenates , 9 3 , 94

parthenogenesis , 179

permeabil ity to calcium, 138

protease in, 269 , 2 7 0

s.p.r., 6 5 - 6 9 , 254 , 2 5 6

thrombin-like substance in, 84

time sequence during division, 168,

170, 171

vacuolization, 78 , 7 9

viscosity, 21 -24

Sempervivum glaucum, 2 5 0

Sepia, 131 , 132

Sepia officinalis, 132

Serratia marcescens, 2 0 5

SH group, 120, 185, 2 4 8

Shaking, 239 , 2 4 0

Shear's polysaccharide, 205 , 2 0 6

Sheep ovary, 211

Shock, 154-164

burn, 159

definition, 154, 155

loss of excitability during, 2 2 7 - 2 2 9

traumatic, 159

Sipunculus muscle , 123

Sleep, 2 2 7 - 2 2 9

Slime mold , 15, 17, 47 , 2 5 0

Sludge, 8 6

Smooth muscle , effect of acetylcholine

on, 151

of b lood serum on, 117, 118

of calc ium on, 109

of heparin on, 9 1 , 119

Snail, hibernation, 2 2 8

Snail muscle , 123

Snake muscle , 64 , 80 ,

Snake venom, 161

Sodium, calcium release by, 138

effect on muscle , 113

entrance into nerve fiber, 54 , 5 5

in muscular contraction, 107

relation to bioelectric potential, 54 , 55

326 SUBJECT INDEX

Sparrow, 7 8

Specific gravity, 18

Spermatozoa, 149

Sphaeroides maculatus, 210

Spindle. See Mitotic spindle.

Spindle fibers, 174

Spirogyra, 20 , 2 1 , 245 , 2 5 0

Spisula egg , 16, 4 1 , 186, 189, 198, 2 0 8

S.p.r. See Surface precipitation reaction.

Squid muscle , 123, 124

Squid nerve, 30 , 131-133

S-S group, 120, 185, 2 4 8

Staircase, 104

Starch paste, 25

Starfish egg , effect of ovarian extract on,

208

parthenogenesis , 180

s.p.r., 67 , 6 8

Starfish ovary, 2 0 7 - 2 0 9

Stentor, effect of calcium on, 245

of fat solvents on, 7 3 , 74 , 2 5 4

of quinine on, 7 4

surface precipitation reaction, 65 -67 ,

7 3 , 74

vacuolization, 7 8

Stimulation, 217 -220 , 2 2 2


definition, 2 1 8 - 2 2 0

relation to vacuolization, 7 8

Stimuli, 2 2 2

Stokes' law, 12-14 , 2 8

Strontium, 7 2 , 7 3

Strychnine, 109

Sugar solution, 55 , 107, 174

Sulfhydryl group, 120, 185, 2 4 8

Sun stroke, 159,

Superfolding, 96

Surf c lam, 9 0

Surface precipitation reaction, 62 -84 , 87 ,

9 1 , 9 2 , 256 , 2 5 7

effect of fat solvents on, 7 3 , 74 , 2 5 4

as homeostatic reaction, 2 6 5

in nerve protoplasm, 74 , 75 , 132

Surface tension, 3 3 , 194

Suspension, viscosity of, 23 -27

Sympathetic nerves, 142, 153

Synapse, 150, 151

Τ

Temperature. See Heat, Cold.

Tetanus, 109, 110

Tetanus toxin, 136

Tetany, 125

Tetraethylammonium, 55 , 56

Tetraodon, 2 1 0

Tetraodontidae, 2 1 0

Tetrodotoxin, 2 1 0

Thermodynamic activity, 2 2 4

Thixotropy, 26 , 37 , 2 4 0

Threshold, 139, 2 2 5

Thrombin, 84 , 87 , 117

effect in prolonging clotting t ime of

blood, 266 , 267

in muscle , 115

proteolytic activity, 92 , 116, 133

shock, 161 -163

Thrombin-l ike substances, 154

Thromboplastin, action of fat solvents on,

2 5 5

as parthenogenetic agent, 181 , 182

in cells, 87 , 8 8

in heparin rebound, 2 6 7

in muscle , 115

shock, 161 -163

Tissue culture cells, 78 , 172 , 173 , 201

Tissue extracts, 8 4 , 87 , 90 , 91

as parthenogenetic agents , 181, 182

from muscle , 2 1 2 , 2 1 3

from ovaries, 2 0 6 - 2 1 3

Toluidine blue, 8 9

Tooth, 134

Torpedo ocellata, 4 8

Tourniquet shock, 159, 162, 163, 2 1 4

Toxic factor, 157

Transmission, 148-165

neuromuscular, 150-154

Transparency, 122, 123 , 144

Treppe, 104

Trianea, 2 4 5

Trypsin, action on muscle , 116, 117

activation b y calcium, 7 1 , 7 2

as cause of shock, 161

effect of cyanide on, 199

on blood, 266 , 2 6 7

on nuclear membrane, 188

SUBJECT INDEX 327

Tumors. See also Cancer.

ascites, 202 , 2 0 3 , 2 1 1

effect of exercise on, 2 1 4 , 2 1 5

of heparin on, 2 0 1

of ovarian extracts on, 210 , 211

Turtle heart, 110

Turtle muscle , 8 0

U Ultraviolet radiation, as parthenogenet ic

agent, 176, 178, 179, 187

calcium release by, 110, 2 3 9

effect on ameba, 245 , 2 4 6

as parthenogenetic agent, 176, 178,

179, 187

Urethane, 191 , 197, 2 5 4

Uronic acid, 8 8

V Vacuolization, 74 -79 , 132 , 192, 197

in marine eggs , 78 , 79 , 176-178

Vagus nerve, 136, 152

Valonia, 6 0

Violin string, 97 , 9 8

Viscosimeters, 11 , 12

Viscosity, 10-31

anomalous, 11

apparent, 11 , 2 6

changes during cel l division, 183 , 184,

189, 190

concept , 10, 11 , 16

effect of calcium, on, 2 4 5 - 2 4 7

of fat solvents on, 2 4 9 - 2 5 3 , 259 -261

of stimulation on, 2 3 9 - 2 4 2 , 245 -247 ,

2 7 2

of X-rays on, 2 0 3 , 2 0 4

of cortex, 36 , 37

W

Water, as anesthetic, 2 2 4 , 2 2 6

W a x y degeneration, 8 0

Winlder's method, 177

Worm, toxic factor, 157

W o u n d callus, 174

X

X-ray, diffraction, 9 6

effect on dividing cells, 2 0 3 , 2 0 4

sickness, 159

Y

Yeast, 77 , 87 , 2 3 3 , 2 6 4

Yield value, 37

the dynamics of living protoplasm

Documents