REGIONAL DIFFERENCES IN CAT ESOPHAGEAL CIRCULAR SMOOTH MUSCLE: A WHITE NOISE ANALYSIS

Robert Yu

A thesis submitted in conformity with the requirements for the degree of Master of Science Graduate Department of Physiology

Institute of Biomaterials and Biomedical Engineering University of Toronto

O Copyright by Robert Yu 1999

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Regional Dinerences in Cat Esophageal Circular Smooth Muscle: A White Noise Analysis

Robert Yu Masîer of Science

Department of Physiology and Institute of Biomateriais and Biomedical Engineering

University of Toronto 1999

By using a Gaussian white noise identijïcation technique, regional differences are

demonstrated by dxerences in electrical response characteristics.

Gaussian white noise rneasurements and analysis techniques were used to obtain

input impedance functions of myocytes fiom feline esophageal body cells (EBC) isolated

from 2 and 4 cm above the lower esophageal sphincter. A whole ce11 patch-clamp

configuration was used to inject white noise current and record a voltage response. The

magnitude of the input iïnpedance of the ce11 was calculated fiom the first order kemel.

Fourier transforms of the kemel indicate a difference in input resistance. The time

constants obtained indicated that EBC4 was quicker to respond since the higher

fkequency values correspond to a fster response characteristic of the cell.

There are regional ciifferences in the passive cable properties of smooth muscle

cells fkom the smooth muscle esophagus which likely contribute to the contraction profile

and sequential nature of peristalsis.

1 would like to offer Drs, Nicholas Diamant and Berj Bardakjian my most sincere

gratitude for their guidance and wisdom. Though 1 have only worked with them for two years,

they have given me a lifetime's worüi of practical insight.

1 would also like to tfiank Dr. Salapatek and Junzhi Ji for their patience, smooth muscles

are not always cooperative. AIong with the rest of my labmates, Shuwen Xue, Dr. H. Lau, Diana

Valdez, Ahmad Muinuddin and Jervana Valdez, thank you ail for your intelligence and

friendship, it will always be cherished.

To my family, mom, dad and Sue, and my fnends, especially Elaine, without whose

encouragement and moral support, none of this would have been possible, 1 extend my deepest

appreciation and Iove.

iii

-TABLE OF CONTENTS-

Abstract Acknowledgements Table of Contents List of Tables & Figures List of Abbreviations

Chanter One 1.1 Esop hageai Anatom y and Physiology

Physiology of the Esophagus Upper Esophageal Sphincter Esophageal Body Lower Esophageal Sphincter Histology

Esophageal Motor Physiology Primary Peristalsis Secondary Peristalsis

1.2 Overview of the Control of Esophageal Motor Function Innervation of the Esophagus

Swallowing Centre i) Afferent Input ii) Coordinating Region iii) Efferent Output

Extrinsic Innervation i) Motor Innervation

Intrinsic Innervation i)Enteric Nervous System

Control of Esophageal Perïstalsis Intrinsic Myogenic Control

1.3 Regional Differences

1.4 Modeling of Physiological Systems Interpretation of Kernels

1.5 Experimental Objective Rationale and Aim of Study

Chanter Two 2. I Materials

Animal Models P harmacological Agents

iv vi vii

2.2 Methods Specific Protocols

Ceil Isolation Biophysical Measurements White-Noise Current Injection and Cell Viability Test

2.3 Data Acquisition and Analysis Computations

I st Order Kemel Fourier Transform

Statistical Methods

Chapter Three 3.1 Equipment Validation

Model Ce11 Electrode

3.2 White Noise Injection First Order Kemels Fourier Transform

Input Resistance i) C tassical Technique

Cut-Off Frequency SIope of Fourier Transform (Roll-On)

3 -3 Statistical analysis Between EBC-2 and EBC-4

Chabter Four 4.1 Discussion

Equi pment Validation Between EBC-2 and EBC-4

Input Resistance Time Constants Order of System

4.2 Future Investigations

References

Cha~te r One

Figure 1.1 The human esophagus 1 -2 Diagrammatic fepresentation of misculmis externa of the esophagus

Figure 2.1 Schematic showing procedure for obtainïng whole ce11 patch that can be studied with patch-clamp technique

2.2 Electrophysiological experiment with Gaussian white noise (GWN) input current

2.3 Total outward current voltage-dependant activation along the esophageal body

Figure 3.1 The cut-off fiequency of mode1 ce11 3.2 The time constant of the electrode 3.3 First order kemel fiom two regions 3 -4 Input resistance derived fiom input impedance plot 3 -5 Cut-off frequency and time constant of the cells 3.6 Derivation of the order of system fiom an input irnpedance plot

Table 3.1 Input resistances

Table 3.2 Surnmary of results

acetylcholine - ACh

central nervous system - CNS circuiar muscle - CM centimeter - cm current - 1 curent-voltage - IV

enteric nervous system - ENS esophageal body cell - EBC esophageal body ce11 two centimeters fiom LES - EBC-2 esophageal body ce11 four centimeters h m LES- EBC-4

fast orthogonal algorithm - FOA gastrohtestinal tract - GI tract Gaussian white noise - GWN

hertz - Hz

interstitial cells of Cajal - [CC input impedance - Z h

longitudinal muscle - LM lower esophageal sphincter - LES

megaohrn - MST millilitre - ml millimoIar - mM millisecond - ms millivolt - mV microsecond - ps molar- M

neurotransmitter - NT nitric oxide - NO

ohm -R

plus or minus - I potassium ion - K' seconds - s standard error o f the mean - SEM

upper esophageal sphincter - LES

volt - V

vii

1.1 Esophageal Anatomy and Physiology

Ph~siologv of the E s o ~ h n u s

The esophagus is a hollow muscdar tube that connects the pharynx to the

stomach. It is guarded at the proximal end by the upper esophageal sphincter W S ) and

at the distal end by the lower esophageal sphincter (LES) (Fig. 1.1). The prirnary role of

the esophagus is to propel food or fluid bolus aborally from the pharynx to the stomach.

The driving force in the aborad direction is deglutition or the coordinated contraction

induced by swallowing aided by gravity. The UES acts to prevent air entry into the

esophagus during respiration and to prevent retrograde movement of material fiom the

esophagus into the hypopharynx. The LES acts to prevent reflux of gastric secretions

into the esophagus. The interactions between the centrai nervous system (CNS), enteric

nervous system @NS) and muscular components of the esophagus that underlie the

motor reflexes of this organ are cornplex and incompletely understood.

The purpose of this thesis is to use a new technique, white noise andysis, for

measurement of passive membrane properties in order to assess if regional ciifferences

exist in the esophageal body smooth muscle.

Figure 1.1 The human esophagus. Guarded proximally by the upper esophageal sphincter ( E S ) and distaltly by the lower esophageal sphincter (LES), it serves as a hollow muscula. tube which connects the pharynx to the stomach.

Studies were performed in isolated smooth muscle cells dissociated Erom circular

muscle layers dong the esophagus. The long-term goal of our laboratory is to define

esophageal îùnction through study of its neural and muscle control mechanisms.

Therefore, a brief description of the anatomy and control mechanisms of

esophageal motor fünction is presented in this section. From a fiuictional perspective, the

esophagus can be divided into three sections, the UES, esophageai body and the LES.

Upper Esophageal Sphincter

The esophagus begins at the UES, a purely striated muscle structure, which

consists predominantly of the cricopharyngeus and caudal fibres of the inferior

pharyngeal constrictor muscle. Circular muscle (CM) fibres fiom the proximal

esophagus also contribute to the sphincter (Netter 197 1, Asoh and Goyal 1978, Welch et

al. 1979). The UES is functionally defmed as a zone of high intraluminal pressure that

separates the pharynx fiom the upper esophagus. In humans, the axial length of the UES,

as measured by intraluminal manometry, is between 2 and 4 cm (Goyai et al. 1970, Ellis

1971).

Esophageal Body

The esophagus is collapsed in its resting state and is capable of distending to

accommodate fiuid and solid material. The esophageai body begins at the inferior margin

of the cricopharyngeus muscle and extends to the LES. In the human adult, the average

length between the UES and the LES is between 23-25 cm (Csendes et al. 1993, Denardi

and Riddell199 1, Li and Rand 1989) and 18-19 cm in the cat (Reynolds et al. 1984 and

1985). Measuring fiom the incisors in the human, it is approximately 40 cm to the

gastroesophageal junction Oenardi and Riddell 199 1).

In the proximal onequarter to one-third of the esophagus, the muscle is striated in

both the inner cucular muscle (CM) layer and the outer longitudinal muscle (LM) layes

of the muscularispropria (Fig. 1.2). Striated muscle begins to be replaced by smooth

muscle about 4 cm below the UES. This marks the beginning of the transition zone

(Meyer et al. t 986). The transition zone is of variable length; striated muscle and smooth

muscle fibres continue to be intermingled up to 10-13 cm below the cricopharyngeous.

The distal one-half to one-third of the esophageal body is composed entirely of smooth

muscle in both layers (Meyer et al. 1986). Occasionally, single bundles of striated

muscle extend within the smooth muscle al1 the way to the caudal end of the esophageal

body (Arey & Tremaine 1933). The major esophageal motor disorders including that

associated with gastroesophageal reflux disease and the spastic motility disorders occur in

the smooth muscle portion.

Lower Esophageal Sphincter

The LES is located at the junction between the esophagus and the stomach

(gastroesophageal junction). It cm be identified with intraluminal manometry as a 2-4

cm zone of high pressure. The LES is composed of smooth muscle in most species,

including humans, dogs, cats and opossums. Anatomic evidence of a sphincter has k e n

difficult to obtain. Liebermann-Meffert and colleagues (1979) gave the most detailed

description of the muscular anatomy of the human sphincter. They found an asymmetric

thickening of the CM in the terminal esophagus, just above the angle of His (the junction

Vagus ncrvc Loncirudinai muscfe

Meissner's !siibmucusall

Figure 1.2 Diagrammatic representafion of muscularis externa of the esophagus. The imer circular muscle layer consists of fibres oriented dong the circderence of the tube and the outer longitudinal muscle coasists of fibres oriented dong its axis. Located between the CM and LM layers lies the Auerbach's or myentenc plexus. Meissner's or submucous plexus is found in the submucosa (From Kahrilas 1992)

of the tubular esophagus and the saccular stomach). In experimental animals, the

relationsbip behveen anatomic structure and fiuiction has been more precisely studied.

Similar thickening of circular, but not longitudinal, muscle has been observed in the

opossum and cat (Seelig and Goyal 1978, Biancani et al. 1982). In the opossum,

ultrastuctural studies have demonstrated severai differences between LES muscle cells

and those of the esophageai body. These differences may help to explain some of the

mechanisms responsible for the functional ciifferences between LES and esophageal body

contractility. Muscle cells of the LES are larger in diameter and form fewer gap junctions

(Daniel and Posey-Daniel 1984). However, neuronai ce11 bodies in the LES are smaller

than those in the esophageal body, perhaps due to the tonically contracted state of the

sphincter (Sengupta et al. 1987). Mitochondrial and smooth endoplasmic reticulum mass

is greater in the LES than in the esophageal body (Christensen and Roberts 1983).

Morphologically, the LES can also be distinguished fkom the esophageal body by the

presence of numerous intemuscular spaces containhg co~ec t ive tissue and blood

vessels (Seelig and Goyal 1978).

Hisrology

The esophageal wali, Iike other regions of the gastrointestinal (GI) tract, is

composed of three distinct layers: the mucosa, submucosa and muscularis propria (Fig.

1.2). However, what differentiates this organ nom the rest of the GI tract is the absence

of a serosal layer. The mucosal lïning is stratifi:ed squamous epithelium in al1 regions of

the esophagus except the LES, where both squamous and columnar epithelium may

coexist (Al Yassin and Toner 1977, DeNardi and Riddell 1991). The submucosa

comprises a dense network of co~ec t ive tissue within which are blood vessels,

lymphatic channels, esophageal glands and a nerve plexus called the submucous or

Meissner's plexus. The muscularis propria, the muscular wall proper, consists of muscle

which is divided into two layers: the inner circula muscle (CM) layer and the out

longitudinal muscle (LM) layer. The inner CM layer, with fibres oriented dong the

circumference of the tube, serves to constrict the esophageal lumen. The outer LM layer,

with fibres oriented dong its axis, serves to shorten the esophagus (Lerche 1950). The

combined action of these smooth muscle layers, opposed at right angles to one another, is

the basis of peristaltic contraction. Located between the CM and LM layers lies a nerve

plexus called the myenteric or Auerbach's plexus. Both the rnyenteric and submucosal

plexuses are continuos around the circumference of the esophageai wall (Sobotta and

Hammersen 1985).

Esophageal Motor Phvsiology

The major fiinction of the esophagus is to propel food and fluid into the stomach.

This is accomplished by way of sequential or peristaltic contractions of the esophageal

body in concert with an appropnately timed relaxation of the UES and LES. The

esophagus also clears any refluxed gastric contents back hto the stomach and takes part

in such reflex activities as vomiting and belching. Normal p e n d s i s is categorized into

either primary or secondary peristalsis, depending on its mode of initiation.

Primary Peristalsis

Primary peristalsis in the esophagus is a coordinated motor pattern initiated by the

act of swallowing. Continuous neuronal discharge of vagai lower motor neurons to the

UES creates a high-pressure zone through tonic contraction of the UES muscles (Asoh

and Goyal 1978, Yoshida et al. 198 1). During swallowing, this neuronal discharge

temporarily ceases allowing relaxation of the UES to occur (Van Overbeck et al. 1985).

M e r the bolus passes through the UES, the UES closes and r e m to its tonically

contracted state and a rapidly progressive CM contraction proceeds distally through the

esophagus starting in the upper esophagus and moving towards the LES. Each location

dong the esophageal axis contracts with a latency that increases gradually fkom UES to

the LES. In the upper third of the esophagus, contractions occur within 1-2 seconds afler

swallowing whereas in the lower third, this latency increases to between 5-8 seconds

(Biancani and Behar 1995).

There is a coordinated contraction of the LM and CM layers of the smooth muscle

esophagus during peristalsis, though each layer contributes to the process differently.

CM contraction produces the lumen-occluding propulsive wave front, whereas LM

contraction is associated with esophageal shortening. Sugarbaker and colleagues (1 984)

directly recorded local contractile and electrical activity of opossum CM and LM in vivo.

They found a sequential contraction of both layers in response to swallowing. With vagal

stimuiation, simultaneous LM shortening and sequential CM contractions were observed.

The LM contraction in each segment of the smooth muscle esophagus preceded and

outlasted the CM contraction, hence augmenting the amplitude of the propulsive

contraction (Sugarbaker et al. 1984). The LM contraction is associated with membrane

depolarization, whereas CM contraction is accompanied by a bimodai membrane

potential response; an initial hyperpolarization (inhibition) followed by depolarization.

The LES relaxes during swallowing and thus aiiows the aborally directed contraction

wave to pass the bolus fiom the esophagus to the stomach. AAer passage of the bolus

through the LES into the stomach, the LES closes with a prolonged contraction.

Motility studies with dry swallows reveal that contractions in the striated muscle

segment are shorter in duration (1-2 sec.) than those in the smooth muscle segment (4-7

sec). The velocity of the peristaltic wave is 3 c d s e c just above the LES (Humphries and

Castell 1977). Though the force of contractions can Vary fkom segment to segment and

even from swaliow to swallow, contractions in the distal one third of the esophagus are

also usually stronger (50- 150 mmHg) than those in the upper one-tkd (40-1 23 mmHg)

(Biancani et al. 1975). However, contractions in the proximal and distal esophagus are

stronger than those in the middle third (20-80 mmHg), perhaps because of the transition

between striated and smooth muscle esophagus in this region.

The physical characteristics of the ingested bolus also affect the speed and force

of esophageal phasic contractions. Increasing the size of the bolus tends to slow its

propagation velocity while increasing the force of contractions (Janssens et al. 1973,

Hollis 1975)- An increased viscosity dso increases the force of contractions and reduces

the speed of perktalsis (Dodds et al. 1973, Dooley et al. 1988). Bolus temperature can

also modulate esophageal peristalsis; warm boluses increase peristaltic velocity, and cold

boluses reduce it (Winship et al. 1970, Decarle et ai. 1977).

Secondury Peristalsis

Distention-induced or secondary peristalsis is the physiological mechanism

whereby reflwced gastric contents or food lefi behind d e r primary peristalsis are

propelled into the stomach. Distention-induced peristalsis is triggered by stimulation of

sensory receptors in the esophageal body.

Balloon distention (BD) is a commonly used method to induce secondary

peristalsis under experimental conditions. The esophageal response to intraluminal

distention consists of proximal excitation and distal inhibition, which serves to aid aboral

propulsion of the intraluminal bolus.

EarIy studies suggested that the esophageal response to swdowing resembled

that of bdoon distention (Craemer and Schlegel 1957, Fleshler et ai. 1959). However

more recent quantitative work by Paterson and colleagues (1 991a) has demonstrated that

contrary to earlier reports, secondary peristalsis induced by isometric distension can differ

significantly fiom primary peristalsis. This area continues to be one of considerable

debate as results fiom isobaric distension are studied.

1.2 Overview of the Control of Esophageal Motor Fuoction

Innervation of the E s o ~ h a w

The control mechanisms of esophageal peristalsis include those of the CNS, ENS,

and myogenic properties of the esophageai musculature. The esophageal innervation

provides the mechanism for the excitation and inhibition of the muscle at al1 these levels

and also provides vital sensory feedback via aflierent pathways which play important roles

in reflex modulation of the esophageal body and sphincter motor activities.

SwaZZowing Centre

CNS control of esophageal motor function resides in the swallowing centre - a

group of intercomected brainstem nuclei that receive cranial as weii as peripheral inputs.

The centre is composed of two intimately comected half centres and is located in the

medulla and pons (Jean 1984). Penstalsis cm either be triggered voluntarily, via higher

brain centres, or reflexly, by stimulation of peripheral afferents which go to the brain

stem swallowing centre (Clouse and Diamant 1988). Stimulation of the swallowing

centre activates vagal efferent nerves to the oropharyngeal and the esophageal muscle.

The vagal nerve mediates prirnary peristalsis in the striated muscle segment and

contributes to that in the smooth muscle segment of the esophagus. Conceptually, the

swallowing centre has t h e fimctional components: an afferent reception system, a

complex organizing system of neurons, and an efferent system of motor neurons

(Diamant 1989).

i) Afferent Input

Afferent information fiom the periphery enters into the solitary tract, the aEerent

reception portion of the swallowing centre. This sensory information serves to initiate

the swallowing sequence, as well as to alter previously initiated swallowing centre

activity and therefore modie ongoing motor activity. Sensory information fiom the

esophageal body and sphincters is carried in the vagus nerve, with the ce11 bodies located

in the nodose ganglion. This sensory pathway plays an important role in monitoring and

modulation of esophageal motor activity. Sensory idormation is also carried by the

sympathetic nerves to the spinal cord segments Cl to L3 (Sengupta et al. 1990, Collman

et al. 1991). This pathway is believed to primarily mediate nociception.

ii) Coordinating Region

The nucleus tract solitarii and the neighboring reticuiar substance make up the

portion of the swallowing centre that is considered responsible for the programming of

the entire swdowing sequence (Jean 1984). One level of integration (dorsal) within this

centre is invoIved in both the initiation and organization of the swaüowing sequence. A

second level of organjzation (ventrai) appears to act as a connection pathway to the

various motor neuron pools involved in the swallowuig act, including the integration of

the deglutition reflex with activity in other medullary centres, such as the respiratory

centre.

iii) Efferent Output

The motor neurons involved in the swallowing sequence lie mainly in the

trigeminal, facial and hypoglosd nuclei, the nucleus ambiguus (NA) of the vagus (for

esophageal saiated muscle), and the dorsal motor nucleus of the vagus (DMNV) (for

smooth muscle esophagus) (Fryscak et al. 1984, Carpenter 1989).

Ekîrinsic Innervarion

i) Motor Innervation

The primary extrinsic innervation of the esophagus is via the vagus nerve. Motor

fibres to the striated muscle esophagus &se fiom the vagus, originating in the upper

portion of the neck. The fibers are myelinated and make direct contact with the striated

muscle fibres through the motor end plate (Toyama et al. 1975). Acetylcholine (ACh) is

the neurotransrnitter (NT) involved at the motor end plate, exerting its effects through

stimulation of nicotinic cholinergie receptors on the striated muscle (Toyama et al. 1975).

The vagus nerves also carry preganglionic fibres to the srnooth muscle portion of

the esophagus (including the LES) where they branch out to form the esophageal plexus,

entering the esophagus at different levels (Penden et al. 1950). Preganglionic fibres

within the esophageal wall travel for several centimeters before reaching the

postganglionic neurons in the inttamural plexus, where they release ACh that activates

muscarinic and nicotinic receptors of myenteric nerve ceUs (Kravitz et al. 1978).

Sympathetic efferent pathways play a minor role in esophageal motor h c t i o n

compared to vagal (parasympathetic) efferents. The smooth muscle esophagus and LES

receive sympathetic nerve innemation that arises fiom the ce11 bodies in the

intermediolateral celi columns of spinal segments T4L2 (Coliman et al. 1992). In the

smooth muscle esophagus, sympathetic nerves affect muscle contractility mainiy through

modulation of other neurons (Seelig and Goyal 1978). The density of adrenergic

innervation varies in the esophagus and is lower in the LES than in the more proximal

esophagus (Baumgarten and Lange 1969).

InPinsic Innentaf ion

i)Enteric Nervous Sysfem

In humans, the ENS is estimated to contain 100 miuion neurons (Furness and

Costa 1987). This system controls motiiity, exocrine and endocrine secretions and the

microcirculation of the GI tract. It is also involved in regulating immune and

inflammatory processes (Goyal and Hirano 1996).

There are two major networks of nerve fibres that are intriasic to the gut: the

myenteric (Auerbach's) plexus and the submucous (Meissner's) plexus. The myenteric

plexus provides prirnarily motor innewation to the circula. and longitudinal muscle layers

and is involved in the control of peristaltic activity. Two major neurons are present, an

excitatory neuron with ACh as its neurotransmitter, and an inhibitory neuron with nitric

oxide (NO) as its main neurotransmitter. The submucous plexus contains secretory

newons that play a crucial role in exocrine and endocrine ce11 secretory control in the gut

(Costa and Brookes 1994). Sensory neurons that respond to stretch, tonicity and

temperature are also found in the enteric plexuses.

Another group of cells, the interstitial cells of Cajal (ICCs), are probably of major

functional importance. The interstitial celi was discovered in 1893 by Ramon Y Cajal,

who suggested that it was intercalated anatomically and functionally between autonomie

nerves and egector ceils. ICCs are mononudear poiymorphic cells that branch out

extensively, the branches intersechg to forrn a network throughout which the axons

extend. These branches fonn gap junctions with one another and with smooth muscle

cells and nerve varicosities (Christensen 1993). ICCs bear a certain ultrastructural

resemblance to both smooth muscle cells and fibroblasts, suggesting that they have a

mesenchpal origin which has recentIy k e n confirmed (Burns et al. 1997, Horowitz et

al. 1999). The distribution of ICCs varies throughout the GI tract. In humans and cats,

ICCs are found in both the circular and longitudinai layers of the esophageai smooth

musc le (Faussone-Pellegrhi 1 987, Faussone-Pellegrini and Cortiesini 1 987).

Contro 1 of E s o ~ h a p d Peristaisis

Control of esophageal peristalsis involves a combination of centrai (exainsic) and

penpheral (intrinsic) neuronal mechanisms using an array of putative transmitter

substances, as well as myogenic factors, varying regionaliy within the organ because of

variations in muscular composition. Both neurogenic and myogenic models have been

proposed to explain the control of esophageal peristalsis.

Intarinsic Myogenic Control

The basis for myogenic control of phasic contractility in the GI tract is manifested

by two fundamental charactenstics: i) the electrical control activity, an omnipresent

oscillation of membrane potential ("slow wave") that controls smooth muscle

excitability; and ii) the ability of the smooth muscle cells to communicate with each other

(coupling) in such a way that the entire tissue can operate as a fùnctional unit.

Esophageal contractions occur when the membrane potentid depolarizes above its

excitation threshold. Wnlike other GI smooth muscle, however, esophageal smooth

muscle is electrically quiescent (no slow wave) and does not contract spontaneously

(Crist et al. 1987). Recent studies in muscle strip and whole organ in vitro preparations

have demonstrated that a latent myogenic oscillatory mechanism for control of phasic

contractions exists in esophageal smooth muscle and that it may be activated by

nonspecific excitation of the smooth muscle membrane (Helm et al. 199 1, Helm et al.

1 WZ).

The most direct evidence that the mechanism for generation of phasic contractions

lying within the smooth muscle itself is the hding that in in viîro esophageal

preparations treated with the nerve toxin tetrodotoxin (TIX), the smooth muscle

esophagus is capable of generating propulsive movements in response to direct muscle

stimulation using electrical square waves of long pulse duration (Sarna et al. 1977, H e h

et al. 1992, Preiksaitis & Diamant in press). The myogenic mechanism of this

propagation is believed to operate partially through electrotonic spread of activation by

gap junctions present between smooth muscle cells.

Gap junctions are symmeaical patches of the membrane of two cells, occupied by

special intraceilular channels that facilitate the movement of iniracellular regulatory

molecules and ions form ce11 to celi (Perachia 1980). When an action potentiat occurs

across the membrane of a single-unit smooth muscle, it is rapidly propagated via these

gap junctions to the entire group of intercomected nuscie celis that h c t i o n eiectricaiiy

and mechanically as a fhctional syncytium.

The mechanisms involved in the integration of the myogenic and neural control

systems of peristalsis in the smooth muscle esophagus have yet to be determined. The

role of intramurai inhibitory nerves under this mode1 codd be to inhibit retrograde

peristalsis, mediate descending inhibition in advance of the penstaltic wave, and to

modulate the velocity of myogenic propagation of contractions (Clouse & Diamant

1998). Furthemore, if the ICCs serve as transducers for nerve to muscle signaling, and

as conduction paths for muscle to muscle communication, then they are likely integral

components of any myogenic control system.

13 Regional Differences

Regional ciifferences in muscle characteristics such as the resting membrane

potential have been reported in stomach, small bowel and colon. These regions dso

demonstrate regional differences in the fkequency of the electricai slow wave activity

which plays an important role in regulating cwrdinated motor activity and muscle

contraction responses when electricaliy excited (Daniel et al. 1994). As well, regional

electrophysiological differences are well described and are of fimctional importance

(Sanguinetti & Spector 1997) in many other muscle tissues. Regional diversity has been

documented in the heart (Barry & Nerbome 1996), vascular smooth muscle (Michelakis

et al. 1997), and in neurons (Storm 1993).

In the lower esophageal sphincter (LES) there are regional merences in response

to cholinergic excitation of the clasp and sling smooth muscle in both cat and human

(Preiksaitis et al. 1994, Preiksaitis and Diamant 1994). In response to cholinergic

stimulation, differences between the LES and the esophageal body in CaZ' sources

utilized has dso been shown (Biaacani et al. 1987). In both the cat (Zelcer et al. 1984,

Niel et al. 1990) and opossum (Daniel et al. 1976), RMP of the LES (-40 to -50 mV) is

consistently reported as less negative than that seen in the esophageal body (-50 to -60

mV) (Kaman et al. 1985, Crist et al. 1987, Decktor et al. 1982, Cnst ef al. 1991).

However, within and dong the esophageai body, there has been little investigation

of regional dinerences in smooth muscle properties and Uiformation remains inconsistent.

Decktor and Ryan reported a steady decrease of the RMP dong the opossum circular

muscle esophagus (-52.8 to -43.5 mV) (Decktor et al. 1982); however, others cornparhg

opossum circular muscle esophagus between 8 and 2 cm above the LES have found no

difference in the RMP (Cnst et ai. 1987, Crist et ai. 199 1 ) . Regional differences in

neural mediation have k e n described dong the esophageal body with the excitatory

(cholinergie) influence most prominent proximaily (Cnst et al. 1984, Gilbert et al. 1986,

Seno et al. 1988, Crist et al. 199 1, Dodds et al. 1979, Anad er al- 1994, Yamato et al.

1992, Paterson et al. L991) and the inhibitory (nitrergic-nitric oxide) influence most

active distally (Anad et al. 1994, Yarnato et al. 1992, Conklin et ai. 1995, Murray et al.

1995). However, there is no rnorphologicai nor biochemicd neural evidence of

differences between proximal and distal esophagus that c m explain the ciifTering neural

influences (Serio et al. 1988, Seeling et al. 1984, Rimele et al. 1979, Ny et ai. 1994 &

1 995, Murray et al. 1 994). Salapatek and colleagues (in press) have s h o w that isolated

smooth muscle cells fiom different esophageal regions display ciifferences in electricai

properties in RMP and voltage dependent K' channels. Therefore, regional differences in

muscle properties are present to provide an alternative for the gradients in excitatory and

inhibitory neural mechanisms.

The present experiments were therefore performed to provide another tool for

exploring the regional differences in muscle properties. In single cells, there exist both

passive properties and active properties. The modeling will begi. in this thesis with the

passive properties, which will be studied and presented using sub-threshold Gaussian

White Noise (GWN) and the Volterra-Wiener approach. The active properties can

subsequently be studied using high powered white noise which will evoke action

potentials, and the results c m be analyzed using the parallei NL model.

1.4 Modeling of Physiological Systems

For a linear, causal and time-invariant systems, the input-output relation is

represented by:

where x is the input, y is the output, t is tirne. The variable h(t), the impulse response

function of the system, characterizes the input-output mapping. The duration of the

impulse response function represents the linear system memory which is quantified by

the largest value of t for which the integrand of the equation above is non-zero. The input

impedance can be defmed as

where s is the complex fiequency variable of the input impedance Zjn which is the

Fourier transform (3) of h(t) when xft) is current and y@) is voltage.

The system dynamics white noise is the element of the model that represents

measurement errors, noise and possible inputs. Here we use a filtered version of an

inaccessible white noise process to identiS. the deterministic model, or kernel functional

expressions. Experimentally, th is technique relates the inputs to the outputs using

arbitrary basis functions rather than anything related to underlying physical anatomy and

physiology of the system.

Intemretation of Kemels

The system dynamics are M y described by kemel fwictions. Marmarelis and

Marmarelis (1978) described the set of GWN derived kernels as generalized combined

impulse responses of the system. This is perhaps seen as a descriptor of a system.

Marmarelis and Marmarelis fiirtber described the kemel as a provider of a quantitative

measure of the association or "cross taik" between varying stimuli.

1.5 EXPERIMENTAL OBJECTIVE

Rationale and Aim of Study

Regional differences are important in the regdation of peristaltic waves in normal

gastrointestinal fiuiction of transporthg the bolus (Salapatek et al. in press), and we

speculate that they serve a similar h c t i o n in the esophagus. The inconsistencies dong

the gut are well documented and regional differences may also have a role in myogenic

control of esophageal peristalsis. To develop tbis thesis, the patch-electrode current

clamp technique was used to measure the voltage response in isolated circular smooth

muscle cells dissociated fiom the esophagus of adult felines and to show regional

differences. The present experiment was perfonned to determine if regional differences

in the esophageal body smooth muscle could be detemillied by this biophysical

technique. White noise studies were performed in isolated smooth muscle cells

dissociated from the circular muscle layer dong the esophagus.

2.1 Materials

AnUnal Models

Current understanding of esophageal function is denved primarily fiom animal

studies. There is considerable variation in the anatomy and physiology of the esophagus

among different species. Do~s, rabbits, sheep, cows, guhea pigs, rats, lions and giraffes

have an esophageal body cornposed completely of striated muscle; whereas, primates,

hones, pigs, opossums and cats have a considerable portion of the distal esophagus

composed of smooth muscle (Diamant 1982). Hence in cats, there exists a complex

interplay of both central and intrinsic neural mechanisms, simitar to what is found in

humans. The placement of the gastroesophageal junction relative to the diaphragrn, a

significant percentage of the esophagus that is composed of striated or smooth muscle,

and the cholinergic sensitivity of the smooth muscle esophagus and LES are very similar

in hurnans and cats. For these reasons, the cat was chosen for these investigations.

P harmacoloaical Agents

The enzymes used were tiom Sigma Chernical Co. (St, Louis, MO) and al1 other

chemicals originated from Fisher Scientific Company (Fair Lawn, NI), Mallmckrodt

Specialty Chernicals Company (Paris, Kentucky), or Sigma Chernical Co. (St. Louis,

MO).

2.2 Methods

S~ecific Protocols

Cell Isolation

Adult cats of either sex were killed by intravenously injecting euthanyl(0.5

mgkg), following a protocol approved by the University of Toronto. The entire

esophagus was quickly excised and placed in oxygenated (with 95% 0, and 5% CO3

Krebs solution containing the following composition (in mM): 1 15.0 NaCl, 4.6 KCi, 1.2

NaH,PO,, 1.2 MgSO,, 22.0 NaHCO,, 2.5 CaCl,, and 1 1.0 glucose. M e r removing

connective tissues surrounding the esophagus, it was opened dong the greater cwature

of stomach. The mucosa and the circular muscle were stripped off, leaving the exposed

longitudinal muscle whose strips were dissected out and cut into squares of -2 mm2.

Three to five smooth muscle squares were then placed in 1 mi dissociation solution with a

composition of (in mM) 125.0 NaCl, 5.0 KCI, 1.0 CaCI,, 1.0 MgCl,, 10.0 HEPES, 2.5

EDTA and 10.0 glucose (pH 7.2). The dissociation solution was added 10 pl collagenase

blends F (1 30 mg/ml), 20 pl papain (500 mghl), 10 pl 1,4-dithio-L-threitol(15.4 mg/ml)

and 10 pl BSA (100 mg/ml) and then incubated at 37OC for about 45 minutes. M e r

incubation, tissues were washed wiîh enzyme-free dissociation solution 3 times and

gently agitated with a plastic transfer pipette. Spincile-shaped single SMCs were then

dispersed and used for patch clamp study within the following five hours.

Biophysical Measurements

The patch clamp technique was used to observe cellular electrical activity through

current flow and voltage response. The actual patch clamping of the cells was performed

by Dr. AM Salapatek and J Ji.

Patch pipettes were made fiom thin-walled borosilicate glass capillary tubes (OD

1.5 mm, ID 1.10 mm, Sutter Instrument Co. Novato, CA) and were heat polished prior to

use. They were pulled with a Narishige PP-83 (Tobo, Japan) two stage micropipette

puller and the raw tips were polished with a Narishige MF-83 (Tokyo, Japan) microforge.

The polishing ensures a microscopie smooth opening at the pipette tip. The reference

electrode made fiom Ag-AgC1 wire was directly connected to the bath. Pipettes were

filled with a pipette solution composed of (in mM) 140.0 KCl, 0.5 CaCl,, 1 .O MgCl,, 10.0

HEPES, 5.0 N a m (pH 7.2) pnor to attaching it to the microscope.

Pipettes were fastened ont0 an Axopatch 200B amplifier (Axon Instruments Inc.,

Foster City, Ca.) headstage electrode, which in tum was attached to an inverted

microscope (Olympus CK20, Olympus America, NY, USA). This microscope rested on

a air-cushioned table which was enclosed by a Faraday cage, minuni;ring both extemai

vibrations and electrical interference. Isolated cells in dissociation solution were placed

in a 1-ml glas-bottom dish mounted on the stage of an uiverted microscope and allowed

for 30 min. to adhere to the bottom. The ceils were then washed with extemal solution

containing (in mM) 140.0 NaCl, 5.0 KCl, 2.5 CaCl,, 1.0 MgCl,, 10.0 HEPES, and 5.5

glucose (pH 7.4). The pipette was lowered into the bath using a coarse manipdator and

placed near a SMC using a remote fine-manipulating controller.

Once in the bath, the resistance for each tip opening was calculated using Ohm's

law. Ohm's law States,

where V = potentid difference in volts, V

1 = current in amperes, A

R = resistance in ohms, S2.

The pipette resistance varied between 3 to 6 MR.

Healthy SMCs were chosen on the basis of location and appearance. The most

desired cells were individual cells that consisted of a clean membrane. Prior to patching

onto a cell, the pipette was calibrated (or zeroed) using the pipette offset knob and a "seal

test" 5 mV step was applied in the bath. This was seen as an actual step on the

oscilloscope. Using the fine-manipulating controller, the pipette was 10 wered onto a cell.

As the tip made contact and became obstructed with the ce11 membrane, a slight electrical

resistance occurred appearing as a deflection in the "seal test" current. Gentle suction

was applied on the pipette until a gigaseal(>l GS2) forms with the ce11 membrane. The

disappearance of the "seal test" step indicated that no m e r current flow existed

between the pipette and the ground, and thus a gigaseal was ensured. This is also known

as the cell-attached patch (Fig. 2. l a). Membrane potential (or chamel) characteristics

GEGAOHM

PULSE OF SUCTION OR VOLTAGE

- Whole

ce11

Fi p r e 2.1 Schematic showing procedure for obtaining whole cell patch that can be studied with patch-clamp technique (A) Configuration of ceil-attached patch. Blunt pipette tip pressed agaiast ceii membrane. With suction a gigaseal can be formed that isolates interior of pipette and surface of membrane patch fiom bath solution. (B) Configuration of a whole cell patch. By passing a small pulse of current or suction, it is possible to break the isolated patch while maintaining a gigaohm seal. This provides low-resistance access to ce11 interior. Contents of pipette rapidly exchange with cellular contents (dialyze). One now has control over ionic gradients and trammembrane potential and whole-celi currents can be quantitated.

within the patch can be observed in cell-attached patch con£iguration. Access to the

intracellular space was obtained by additional suction, thus resulting in a whole-ceil

configuration (Fig. 2.1 b). This is the configuration used in the present experiments. Due

to the nature of the technique used, the flow of ions (or current) through the ion channels

of the whole ceii could be measured with stringent accuracy and monitored with a tirne

resolution of milliseconds.

White-Noise Current Injection and Cell Viabiliîy Test

An adjustable high power level current stimulus of 0.4 second duration (400 ms =

7932 points) was injected using pClamp6 software (Fig. 2.2a). The voltage response is

captured and saved in a computer file for analysis later on (Fig. 2.2b).

Prier to and following the injection of experimental current, a standard current-

voltage (IV) protocol was applied to the ce11 to detennine the viability of both the ce11 and

the seal. The results will also be used to calculate cellular input resistance for later

cornparison. In essence, this protocol hoIds the ce11 at -50 mV for 235 ms and increases

by 20 mV every 255 ms. Throughout this process, voltage-activated chamels in properly

configured cells wili open and produce an outward current, normally at -40 mV, which

will result in a series of outward currents (Fig. 2.3).

2.3 Data Acquisition and Analysis

Whole ce11 current-clamp protocols were generated by pClamp6 software (Axon

instruments, Foster City, USA). Electmphysiological signals were relayed back to an

O 0.002 0.004 0.006 0.008 0.01

nfrœ (sec)

Figure 2.2 EZectrophysioZogicuI experimenr with Guussian white noise (GWN) input currenr. (A) The GWN input current. (B) The output voltage. (C) The nrst order kemel and close up of kemel (inset). @) The input impedance, a Fourier transform fiom time domain to frequency domain and close up of area of interest (inset).

oscilloscope where ceil characteristics were observed while data were low-pass filtered at

10 kHz by an on-board eight-pole Bessel filter before digitization with a DigiData 1200

analog-to-digital converter (Axon Instruments). Recordings were commenced 2-5 min.

after the formation of whole cell configuration in order to allow for ce11 dialysis with the

pipette solution. Al1 experiments were performed at r w m temperature of 20-22OC.

The technique chosen for kemel computation was Fast Orthogonal Algorithm

(FOA). The FOA was implemented using an algorithm developed by Korenberg (1988)

and adapted to our cornputers by William Neil Wright (1993). Utilization of the FOA is

advantageous because it requises no apriori estimates of kemel memory length. This

technique involves the creation of an orthogonal set of fùnctions fiom the actuaî input

sequence used (GWN) and these fùnctions are used to synthesize the kemel. Kernels

were calculated with 1024 lags which adequately allows the memory of the kemel to

decay to zero (Fig. 2.2~).

Fourier Transform

The kemel data was transformed fiom time domain to fkquency domain (Fig.

2.2d) in order to determine the input impedance fiom which the input resistance, the cut-

off fiequency and the order of the system c m be estimated. The input resistance is a

measure of the ce11 structure and is quantified by the magnitude of the input impedance at

zero fiequency. The cut-off tiequency is a measure of the responsiveness of a ce11 which

has low pass fiequency characteristics. The kquency that matches the 3 dB level of

impedance, a point when impedance reaches 0.707 of its maximum, is the cut-off

fiequency. The order of the system is indicative of the amount of exhibited RC circuit-

Iike characteristics which tells us the rate of attenuation of the cell. This can be shown as

sirnpiy the dope at which the impedance decreases as fiequency increases.

Statistical Methods

The data will be analyzed to determine if there is a significaat difference in input

resistance, cut-off fkequency and the order of the system. Since a small sample size was

obtained, the t-distribution should be used. Using at les t a 5% level of significance, the

nul1 hypothesis will be that the two regions, EBC-2 and EBC4, are the same.

3.1 Equipment Validation

Mode1 Ce11

First of dl, the system undenvent testing to determine its fiinctionality and its

accuracy. A model ce11 (PATCH-1 U, Axon Instruments) was provided by the

manufacturer of the amplifier dong with general information regarding its senings and

parameters. The purpose of this exercise was to see if the parameters of the model ce11

could be captured through white noise current injection and analysis. The model ce11 was

reported to have a time constant of 16.5 ms or a cut-off fiequency of 60.6 Hz. The time

constant is the inverse of the cut-off fiequency, the fiequency that matches approximately

0.707 of the maximum magnitude of impedance fiom a fiequency domain plot. The cut-

off fiequency of the model ce11 was about 65 Hz, a tirne constant of 15.4 ms (Fig. 3.1).

EIectrode

The electrode was tested to see if its response characteristics were adequate to

capture the output noise that it will be provided throughout the experiment. The effect of

the electrode shouid not dominate; in other words, the glass tip should not be too small so

Magnitude of Input lmpedance (Model Cell)

Fmquency (Hz)

Figure 3.1 The cut-offfiequency of mode2 cell. The cut-off fiequency of the mode1 celi was 65 Hz ( t h e constant 15.4 ms) which closely matches the parameters &en by the manufacturer with a t h e constant of 16.5 ms. (note: cut-off fiequency is the inverse of t h e constant)

that it causes resistance in measuring the injected current An electrode was lowered into

the bath medium and white noise current was applied in order to record the characteristics

of the electrode. Under the kquency domain, the results indicated that the electrode far

exceeded EBC nom both levels in cut-off fkquency and consequently possessed a faster

tirne constant in the ps range (Fig. 3.2). ï hus the electrode tip was small enough to patch

and yet not hinder (or swamp the noise artifact) the white noise current.

3.2 White Noise Injection

After validating the system, cells fiom both regions were patched, injected with

white noise current and its voltage response were recorded and analyzed. In total, five

cells fiom each region (ten experirnents in total) were successfully patched, studied and

reported in this section.

First Order Kemels

First order kernels were calcdated and plotted against t h e (Fig. 3.3). It is

already apparent that the two kemels show two distinct patterns generated fkom the two

regions. Generally, cells h m two centimeters above LES (EBC-2) displayed a much

slower decay curve than those isolated fiom EBC-4.

Fourier Transform

Fourier transforms of the kemels fiom time domain to kquency domain contain a

Magnitude of Input lmpedance (Elecaode vs EBC4)

Fmquency (Hz)

Figure 3.2 The time constant of the eleclrode. Under the fiequency domain, the results indicated that the electrode far exceeded EBC-4 in cut-off fkequency with a time constant of < 1 .O ms. Thus the electrode tip was srnail enough to patch and yet not hinder the white noise current.

l

O 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 4 . 1 -

Tirne (sec)

Figure 3 3 First order kernelfiom two regionS. Fùsî order kemels were calculated and plotted against t h e . Cells fkom four centimeters above LES (EBC-4) have a steeper decay cuve than those h m EBC-2.

wealth of information about the cells that can be elucidated by foilowing these methods.

Input Resistance

Input resistance of a ce11 is the impedance value at zero fiequency (Fig. 3.4). The

resistance values obtained were 3 10.8 i 42.6 MS2 and 39.1 f 36.9 MS2 for EBC-2 and

EBC-4 respectively.

i ) Classical Technique

In order to confirm these input resistance values, the numbers were compared

with input resistances derived from standard means which were accomplished by voltage

clamping the cells and recording K' outward current (Fig. 2.3). The data was taken,

plotted by linear regession and a line was fitted dong a linear area of Iow v~ltages since

nonlinear data should not be included. Small hypepolarizing pulses (-30 mV to 10 mV)

were chosen as to not take in effect of inward K+ rectifiers. The foilowing table displays

both results (Table 1).

The standard technique showed varying values of resistances with a mean value + SE of 290.3 k 147.9 MS2 and 20.7 + 1.1 Ml2 for EBC-2 and EBC-4 respectively. The

maximum percentage difference was 40 % with t-tests indicating no significant

differences between the two measurements.

Cut-Off Frequency

The cut-off fiequency is the fiequency that matches the 3-decibel level of

impedance (Fig. 3.5). The 3 dB is approxirnately 0.707 of the maximum magnitude of

Magnitude of lnput Impedance

lnput resistance

O 500 lm 1500 2000 2500 3000 3500

Frequency (Hz)

Figure 3.4 Input resistance derivedfi.om input impedance plot. input resistance of a ce11 is the impedance value at zero fiequency. The values obtained were 3 10.8 f 42.6 MR and 39.1 + 36.9 MR for EBC-2 and EBC-4 respectively.

Table 3.1 Input resistances Smooth Muscle

Type

EBC-2

E B C 4

White Noise Technique (Msr) 365 294

In calcuiation of percentage difference, standard technique was taken as tme value.

19.4 21.2 21.5

18.3 23.4 24.7

5.7 10.4 14.9

Standard Technique @lm 461 210

Percentage Dwerence

20.8 40.0

rsspsdive tim constants

3dB cut-Off

EBG2

O 0.0001 O.Oûû2 0.0003 0.OOM 0.0005 0.- 0.0007 0.0008 0.0009 0.001

Time (sec)

Figure 3.5 Cut-oflfiequency and time constant of the cells. (A) The cut-off frequency is the fiequency that matches the 34ecibel level or 0.707 of the maximum magnitude of impedance. EBC-2 and EBC-4 redteà in highly variable cut-off fkquencies of 200.6 k 55.5 Hz and 1287.0 f 1 65.2 Hz respectively. (B) The tirne constant is the time that matches the 3-decibel level of the nrst order kemel. EBC-î and EBC-4 resulted in time constants of 2.9 +, 2.2 ms and 0.9 f 0.4 ms respectively .

impedance fiom a fkequency domain plot. The t h e constant of the membrane is

inversely proportional to the cut-off fiequency. EBC-2 and EBC-4 resulted in average

cut-off fiequencies of 200.6 Hz and 1287.0 Hz respectively. These averages did not

follow the normal distribution. Therefore, more accurate tirne constants (2.9 + 2.2 ms

and 0.9 + 0.4 ms respectively) were obtained by taking the 3 dB fiom the kemei (Fig.

3.5b).

Slope of Fourier Transform (Roll Ofl

The order of the system is simply the slope at which the impedance decays as

fiequency increases. A simple technique exists for obtaining an approximate plot of the

magnitude and phase of a transfer fhction by using diagrams called Bode plots. On a

plot of decibels versus log fiequency, one can get straight-line plots or one can get the

linear regression of the most obvious downward part of a simple curve (Fig. 3.6). This

slope is a measure of the average order of a system consisting of RC networks. As the

slope gets steeper, there is an indication of a higher space constant and the presence of a

higher order in the cable properties of the cell. In al1 these cases, EBC-2 generally has a

steeper slope than EBC-4.

3.3 Statistical Analysis

Between EBC-2 and EBC-4

Using a two-tail t-test ofsignificance, al1 pairs of data (Table 2) were examined to

Order of System (EBC4)

Slope

y = -0.001~ + 3.5698 i 1 I l 1 . i

r

I

Frequency (Hz)

Figure 3.6 Derivation of the order of system f'om an input imped4nce plot. The order of the system is simply the dope at which the impedance decays as fiequency increases. The Iinear regessian of the rnost obvious dowuward part of a curve (slope) is a mesure of space constant properties of the cell. In al1 cases, EBC-2 generally has a steeper dope than EBC-4.

Table 3.2 Summary of remfis Smooth Muscle

Type

EBC-2

Average (EBC-2) ,

Average (EBC-4)

Input Resistance (MQ) 270

1 1 I - - -

Each row represents values of an individual cell,

T h e Constant (ma 1 -3

347 365 294 278

310.8 + 42.6

-0.00 1 5 -0.0009 -0.00 1 O

-0.0029 f 0.004

1

Slope of Input Impedance ( M W )

-0.0823

18.3 23.4 24.7

39.1 t 36.9

6.8 1 -0.0228 2.5 -0.0135

1.4 0.6 0.8

0.9 + 0.4

1 -9 2.0

2.9 + 2.2

-0.082 1 -0.0805

4.056 f 0.04

see if there was a significant difference betweea them. The test statistic, t, was

detennined by

where

and F = tested mean a= standard error n = sarnple sîze

In the present case, the t value for input resistance, tirne constant and slope were 10.780,

2.002 and 2.953 respectively. By using a 95 % codidence level, the hrpothesis would be

rejected if t > 1.86; therefore, a significant difference between EBC-2 and EBC-4 is

present in al1 three parameters: input resistance, cut-off fiequency and slope.

4.1 Discussion

These studies demonstrate for the fhst t h e that white noise c m be used on single

smooth muscle cells, in this case from the smooth muscle esophagus. The white noise

technique proved to be a valuable tool in detemiining two aspects of the celi not usually

characterized by other means, the cut-off fiequency (tirne constant) and the differences in

the average order of a system (space constant) between the two regions of the circular

muscle of the esophageal body.

Eaui~ment Validation

First of dl , the system undenvent tests to determine its hctionality and its

accuracy. The mode1 ce11 was docurnented to have a time constant of 16.5 ms in which

the analysis confirmed it at 15.4 ms. The 1. t rns error could have risen through

extrapolating the information from the plot where a polynornial curve was fitted, and

perhaps the curve fitting software was inadequate for our needs. Until a better curve fit

tool is acquired, a 5 % error demonstrates that the system is most reliable. Therefore the

parameters of the mode1 ce11 can reasonably be captured through white noise current

injection and analysis.

The electrode was also tested to see if the speed of electrode was adequate to

capture al1 the noise that will be provided. The results showed that the time constant of

the electrode was safely < 100 ps, shorter than the quickest celi recorded at around 0.5

ms. If the results were reverse& the electrode would not have been able to record the

cell, instead it would in essence have recorded itseff. By measuring the characteristics of

the electrode, it was determiaed that the electrode tip was suitable for experimentation.

Differences Between EBC-2 and EBC-4

This report shows evidence of regional differences in circuiar smooth muscle

properties dong the esophageal body. It was apparent by the two kemels that the cells

fiom the two regions generated two distinct patterns. Kemels of ceils fiom EBC-2 had a

much slower decay curve than those fiom EBC-4. The regionai diversity includes

differences in input resistance, ciifferences in cut-off fiequency and differences in order.

Proximally, the cells are more active, posses higher input resistaaces, demonstrate

quicker time constants (more responsive) and have smaller space constants.

Input Resistance

The input resistance is a measure of the resistive components of the cell. Input

resistances obtained were 3 10.8 f 42.6 MC2 and 39.1 + 36.9 MC2 for EBC-2 and EBC-4

respectively. Using a two-tail t-test of significance, the pair of data resulted in a

significant difference, as much as 99.9%, between them. The differences perhaps are due

in part to some ion channel differences within the ceils of both regions. Evidence of

differences dong the cat esophagus have been found and implicate diverse voltage

dependent K+ channel densities dong the esophagus (Salapatek in press). The density is

greater at EBC-4.

In order to ver* the white noise technique on isolated smooth muscle celis, input

resistances were caiculated the standard way by voltage clamping the cells. Upon

comparing the two sets, there were varying differences in magnitude among them that

range fiom 5% to 40 %. Calculating voltage-clamped input resistances were quite

arbitrary because a linear regression is fitted dong data points derived fiom a series of

hyperpolarizing pulses (-50 mV to 70 mV). The 10 to 70 voltage range, wbich represents

K+ rectiQing channel activity, was cut off leaving a few points to calculate conductance.

This method accounts for the high variability of the input resistances taken using standard

techniques. The white noise method is advantageous because from five samples, a

maximum standard error of 40 MC2 was recorded compared to a standard error 150 MR

using standard means. Upon using both white noise curent inputs and voltage outputs,

the input impedance is estimated using theoretical and mathematicd grounds, instead of

relying on numerous experimental trials and data.

Time Constants

The tirne constant is a measure of the responsiveness of a ce11 which has low pass

fiequency characteristics. EBC-2 and EBC-4 resulted in time constants of 2.9 ms and 0.9

ms respectively. Systems with lower memory normally have a higher cut-off fiequency

which also relates directly to how the ce11 responds to a given input. The striated region

is neurally mediated, which requires muscle cells to quickly respond to neural messages

and sometimes quick changes of action (Le. vomiting). Since EBC4 is doser to the

striated, these findings support the notion of a muscle gradient dong the esophagus where

reaction tirne dimiaishes as one moves distally nom striated muscle to smooth muscle.

Other regions in the gut have been found to have varying time constants. In the

canine gastric antrai circular muscles, the time constant of the circular muscle near the

myenteric plexus tended to be greater than that of the circular muscle near the submucosa,

however this clifference was not statistically different (Bauer & Sanders 1986). Their

experiment was performed on tissue where ceils are coupled.

Order of System

The average order of the system is indicative of the amount of RC circuit-like

characteristics and tells us the rate of responsiveness at which a ce11 is capable of

handling. Generally, the space constant is a fùuction of the dope of the input impedance

which is a fünction of the axial and membrane resistances of the cell. EBC-2 generally

has a steeper slope than EBC-4 meaning that EBC-2 has an overall higher space constant

and the presence of a higher order in the cable properties of the cell. Since the space

constant is the distance at which the signal travels as it attenuates, a quicker responding

ce11 shouid ideally have lower memory, shorter space constant. This lack of memory

allows for the celi to quickly respond to new stimuli. From the aspect of space constants,

regional differences exist in antral circula. muscles (Bauer & Sanders 1986). The length

constant of the circular muscle near the myenteric plexus (2.4 mm) was significantly

greater that the space constant of the circular muscle near the submucosa (1 -7 mm).

The diversity is seen as important in other muscle tissues such as those in the

heart, vascular smooth muscle and in neurons. In the esophagus, excitatory (ACh) and

inhibitory neurotransmitters (NO) have been implicated as the main mechanism for

esophageal controt by depolarking or hyperpolaripng cells, and show regional

ciifferences in effects not accounted for by the neural elernents themselves. The evidence

of regional muscle differences therefore provides a rational basis for the fiinctional

differences in neural effects, and is key in understanding how myogenic control

contributes to esophageal peristalsis. Perhaps anned with the understanding of myogenic

properties, new therapies can be developed at targeting the esophageal muscle itself in

helping people with such disorders as gastroesophageal reflux disease and spastic motor

disorders.

4.2 Future Investigations

This study should be taken on to M e r levels. The white noise technique

illustrates the dynamics of a ce11 by capturing not only the passive properties, but the

active ones. The classic way of plotting N cuves is by changing the voltage and

recording current. However, in this method, we can change the current and record the

voltage and therefore capture a nodinear c w e . This technique has been utilized in

hippocampal neurons (Bardakjian et al. 1994). Future studies in smooth muscle provide

the potential for similar assessments.

This technique may also be used to identifjr the system mode1 and to provide

passive electrïcal properties such as ce11 capacitance and coupihg resistance in a tissue of

coupled cells. This involves an optimization of the system (Fu et al. 1991), and holds the

potential to provide new insight into the coupling mechanisms of smooth muscle celis.

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