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PHYSIOLOGICAL REVIEWS

Vol. 78, No. 3, July, 1998Printed in U.S.A.

Physiological Features of Visceral Smooth Muscle Cells, WithSpecial Reference to Receptors and Ion Channels

H. KURIYAMA, K. KITAMURA, T. ITOH, AND R. INOUE

Seinan Jogakuin University, Kitakyushu, Kokura-Kita, Fukuoka; Chugai Pharmaceutical 1–135, Komakado,

Gotemba, Shizuoka; Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka;

Department of Pharmacology, Fukuoka Dental College, Fukuoka; and Department of Pharmacology,

School of Medicine, Nagoya City University, Nagoya, Japan

I. Introduction 812II. Regional and Species Differences in the Resting Membrane and Action Potentials in Visceral Smooth

Muscle Cells 813A. Resting membrane potential and passive membrane properties of VSMC 813B. Action potentials generated in VSMC 817

III. Neural Control of Membrane Activities in Visceral Smooth Muscle Cells 822A. Nerve plexuses, nerve terminals, varicosities, and cotransmitters as studied

by electrophysiological methods 823B. Features of varicosities deduced from electrophysiological investigations 825C. Multitransmitter release inferred from the generation of EJP and IJP and features

of postjunctional receptors 827IV. Characteristics of Ion Channels in Visceral Smooth Muscle Cell Membranes 839

A. K/ channels 839B. Na/ channels 846C. Ca2/ channels 848D. Cl0 channels 852E. Nonselective cation channels 853

V. Receptor-Operated Ion Channels in Visceral Smooth Muscle Cells 853A. Purinoceptor-coupled channels 853B. G protein-coupled cation channels 859C. Cytosolic Ca2/-activated nonselective cation channels 869D. Unclassified cation channels 870E. Muscarinic receptor-inactivated K/ current: M current 870

VI. Mobilization of Calcium and Maintenance of Calcium Ion Homeostasis in Visceral Smooth Muscle Cells 871A. Ca2/ concentration in the cytosol of VSMC 871B. Factors that increase [Ca2/]i in VSMC 873C. Factors that decrease [Ca2/]i in VSMC 882D. Ca2/ sensitization of smooth muscle contraction 884

VII. Summary and Conclusions 889

Kuriyama, H., K. Kitamura, T. Itoh, and R. Inoue. Physiological Features of Visceral Smooth Muscle Cells, WithSpecial Reference to Receptors and Ion Channels. Physiol. Rev. 78: 811–920, 1998.—Visceral smooth muscle cells(VSMC) play an essential role, through changes in their contraction-relaxation cycle, in the maintenance of homeosta-sis in biological systems. The features of these cells differ markedly by tissue and by species; moreover, there areoften regional differences within a given tissue. The biophysical features used to investigate ion channels in VSMChave progressed from the original extracellular recording methods (large electrode, single or double sucrose gapmethods), to the intracellular (microelectrode) recording method, and then to methods for recording from mem-brane fractions (patch-clamp, including cell-attached patch-clamp, methods). Remarkable advances are now beingmade thanks to the application of these more modern biophysical procedures and to the development of techniquesin molecular biology. Even so, we still have much to learn about the physiological features of these channels andabout their contribution to the activity of both cell and tissue. In this review, we take a detailed look at ion channelsin VSMC and at receptor-operated ion channels in particular; we look at their interaction with the contraction-relaxation cycle in individual VSMC and especially at the way in which their activity is related to Ca2/ movementsand Ca2/ homeostasis in the cell. In sections II and III, we discuss research findings mainly derived from the useof the microelectrode, although we also introduce work done using the patch-clamp procedure. These sections

8110031-9333/98 $15.00 Copyright q 1998 the American Physiological Society

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KURIYAMA, KITAMURA, ITOH, AND INOUE Volume 78812

cover work on the electrical activity of VSMC membranes (sect. II) and on neuromuscular transmission (sect. III).In sections IV and V, we discuss work done, using the patch-clamp procedure, on individual ion channels (Na/,Ca2/, K/, and Cl0; sect. IV) and on various types of receptor-operated ion channels (with or without coupled GTP-binding proteins and voltage dependent and independent; sect. V). In sect. VI, we look at work done on the role ofCa2/ in VSMC using the patch-clamp procedure, biochemical procedures, measurements of Ca2/ transients, andCa2/ sensitivity of contractile proteins of VSMC. We discuss the way in which Ca2/ mobilization occurs aftermembrane activation (Ca2/ influx and efflux through the surface membrane, Ca2/ release from and uptake into thesarcoplasmic reticulum, and dynamic changes in Ca2/ within the cytosol). In this article, we make only limitedreference to vascular smooth muscle research, since we reviewed the features of ion channels in vascular tissuesonly recently.

I. INTRODUCTION intracellular free Ca2/ ([Ca2/]i) using aequorin (see reviewby Blinks et al., Ref. 96) was first applied to VSMC by Fay

The ‘‘father’’ of the electrophysiology of visceral et al. (287) and Morgan and Morgan (775). Since then,smooth muscle cells (VSMC) was, undoubtedly, Professor similar techniques have been employed by others, usingEmil Bozler. He founded visceral smooth muscle (VSM) various dyes (see sects. IV and VI). At present, at leastphysiology by his pioneering experimental procedures us- three different procedures for measuring [Ca2/]i are ining extracellular recording, mainly from 1938 to 1948, and use, namely, Ca2/ transient measurements using varioushe recognized that VSMC could be classified as either indicators and methods using Ca2/-sensitive microelec-single- or multiunit smooth muscle cells (SMC). In particu- trodes (1188) or nuclear magnetic resonance (NMR).lar, he elucidated the myogenic propagation of excitation Combined techniques for measurements of 1) the ionicusing the rat myometrium, and he also discovered the current and Ca2/ transient, 2) the ionic current and me-basis of automaticity (the prepotential) in VSMC of the chanical response, or 3) the Ca2/ transient and mechani-intestine and ureter. In addition, differences in the shape cal response have been successfully applied to the investi-of the action potential in different VSMC (from myome- gation of VSMC activity. Furthermore, the introduction oftrium, ureter, intestine, and stomach) were first demon- the confocal microscope has enabled the determinationstrated by Bozler using extracellular recording proce- of the regional distribution and dynamic changes in thedures. He concluded that in VSMC, an all-or-none re- location of Ca2/ in the cytosol. Recent remarkable prog-sponse to a single-threshold stimulus may show either a ress in molecular biological techniques has enabled ussingle spike, a series of spikes, a plateau, or a combination to identify the primary amino acid sequence and three-of these elements. His research interests were not limited dimensional structure of the proteins that make up ionto the study of smooth muscle (SM); he also studied skele- channels and receptors, as well as giving clues to theirtal muscle, cardiac muscle, and neurons. Bozler (121) functional expression. Such procedures have also beensummarized his brilliant work on SM cells in 1948. There- successfully applied to the investigation of the membraneafter, Bozler studied skinned muscle tissues and eluci- characteristics of VSMC.dated the role of Ca2/ in skeletal muscle cells using EDTA, Visceral smooth muscle cells develop from the meso-ATP, and divalent cations (Ca2/ and Mg2/). derm, although the developmental steps involved in the

If Professor Emil Bozler was the father of SM physiol- formation of individual VSM tissues differ according toogy, the ‘‘mother’’ was definitely Professor Edith Bulbring. the type of tissue. Moreover, the membrane potential andIn 1954, she first applied microelectrode techniques to the the shape of the action potential differ by region and bymeasurement of the electrical properties of VSMC (135). species. When studying the cellular and subcellular char-Her scientific family is distributed the world over, and her acteristics of VSMC, it is necessary to know something‘‘grandchildren’’ are playing major roles in advancing SM about the basic cellular functions of VSMC (as determinedresearch. Professor C. Ladd Prosser was also one of the using the microelectrode method: resting and action po-pioneers of SM physiology, and he and his students (who tential of the membrane) before attempting to understandare mainly distributed in the United States) have made the features of the ionic mechanisms at the subcellularimportant contributions to the progress of research, level. In addition, an understanding of the neuronal modi-mainly in gastrointestinal (GI) SM physiology. fications of SMC membrane activities is essential. For this

A powerful new technique for the study of ion chan- reason, section II introduces the fundamental membranenels and receptors, the patch-clamp method, was intro- properties of VSMC measured by the microelectrode andduced by Neher and Sakmann (807) and Hamill et al. examines the ionic currents responsible for forming the(381), the first report using the method in VSMC being membrane potential and action potential. In section III,published by Benham and Bolton (71). Since then, many neuromuscular transmission in VSMC measured mainlyarticles on ion channel and receptor activities have been by the microelectrode method and some features of the

prejunctional regulation of transmitter release are dis-written. A new technique for making measurements of


July 1998 FEATURES OF VISCERAL SMOOTH MUSCLE 813

cussed. Sections IV and V review the general properties of ter, 055 to 065 mV). During gestation, cell diameter andresting membrane potential increase more or less in linethe voltage-dependent and -independent ion channels and

of the receptor-operated ion channels found in VSMC, with each other through to the last stage of gestation (21–22 days of gestation: 15–30 mm, membrane was slightlytogether with their underlying mechanisms (including the

role of GTP-binding proteins, G proteins). This area of depolarized compared with midpregnancy). The abovedata, derived by the following authors from various spe-study involves measurements made mainly by patch-

clamp methods (whole cell, cell-attached, and cell-free cies, appear to be reliable, but some experimental errormay be present in the measurements in the nonpregnantpatch-clamp recordings). Section VI deals with Ca2/ ho-

meostasis in resting and active cells, as studied mainly by uterus due to the small diameter of the myocytes. In gen-eral, the resting membrane potential in rat and mousemaking measurements of Ca2/ transients and by electro-

physiological techniques. The ultimate functions of VSMC myometriums is larger in circular muscle cells than inlongitudinal muscle cells, both before and during the earlyare control of motility and of processes allied to secretion.

These actions are induced or modulated by contractile stages of gestation. Moreover, when pregnancy is wellunder way, the membrane is hyperpolarized in both layersproteins and their regulatory proteins and are activated

by changes in [Ca2/]i and by non-Ca2/ processes. The level (‘‘progesterone blockade’’; Refs. 621, 624). However, atterm, the membrane of SMC in both layers is depolarizedof [Ca2/]i is regulated by the influx and efflux of Ca2/

across the cell membrane, and also by the uptake and to a certain extent. In the rat myometrium, at midterm,the membrane potential was 060 mV, whereas at term,release of Ca2/ from the SR. Furthermore, Ca2/ sensitiza-

tion and desensitization are discussed in relation to the this value had fallen to 045 mV (695). The highest mem-brane potential in the rat myometrium during pregnancyactions of agonists. Because the specific characteristics

of vascular SMC, together with their pharmacology, have was seen on day 16 (27, 632). In midpregnancy, the mem-brane of longitudinal SMC in the placental region showedbeen reviewed recently by the present authors (626), we

do not intend to incorporate information specific to vascu- a consistently higher membrane potential than those inthe nonplacental region (540). However, it has been re-lar SMC in this article. However, features of ionic channels

and receptor properties relevant to vascular SMC are in- ported that there is no change in the membrane potentialof SMC in the circular muscle layer during pregnancy orcluded wherever they relate to, or exemplify, the general

functions of VSMC. at term in guinea pig and sheep (882, 884).In the rat myometrium, the concentration of estradiol

increases markedly during the last 2 days of pregnancy,II. REGIONAL AND SPECIES DIFFERENCES IN whereas that of progesterone decreases. Progesterone,

THE RESTING MEMBRANE AND ACTION given intracutaneously to estrogen-primed ovariecto-POTENTIALS IN VISCERAL SMOOTH mized rats, produced a consistent hyperpolarization of theMUSCLE CELLS longitudinal SMC membrane (632). Estrogen itself given

to ovariectomized rats in the nonpregnant condition alsohyperpolarized the membrane. During pregnancy, how-A. Resting Membrane Potential and Passiveever, estrogen did not modify the membrane potential,Membrane Properties of VSMCalthough a decrease in the concentration of estrogen atnear-term occurred at the same time as a depolarization

1. Resting membrane potentials in VSMCof the membrane potential. On the other hand, in vitrotreatment with progesterone or estradiol produced differ-The resting membrane potential, as measured in vari-

ous VSMC, varies from 035 to 070 mV. Such variations ent actions on the membrane potential and contractionin circular and longitudinal muscle layers of the rat myo-in the value of the membrane potential may, partly, be

from the different experimental conditions used. Because metrium (831). Thus, on muscle membranes, progester-one enhanced electrical activity in the longitudinal musclethe resting membrane potential and action potential in

both longitudinal and circular muscle layers in the myo- layer and inhibited it in the circular muscle layer. In con-trast, estrogen produced the reverse of these actions inmetrium are greatly modified by hormonal conditions,

such as occur during the estrus cycle, during the course the two muscle layers. However, at term and postpartum,estrogen produced excitatory actions on the circular mus-of pregnancy, and also post partum, we will use the myo-

metrium as an example. cle layer.Steroid hormones, such as progesterone and estro-The cells of the myometrium are spindle shaped,

ranging in size from 2 to 10 mm in diameter and from 200 gen, may act on the cell nucleus and increase the produc-tion of mRNA. Erulkar et al. (278) concluded that gonadalto 600 mm in length. As gestation progresses, hormonal

conditions change from nonpregnant (5–10 mm in diame- steroids exert an influence on the expression of differentpopulations of ionic channels in isolated cells of the imma-ter; resting membrane potential 035 to 045 mV) through

pregnancy (15–17 days of gestation: 10–15 mm in diame- ture rat uterus. On the other hand, it is conceivable that



these steroid hormones also act directly on the surface are made of only 3–6 connexons. One of the densest dis-tributions of gap junctions has been found in the circularof cell membranes through unknown mechanisms.

Erulkar et al. (278) observed the effects of gonadal ste- muscle of the small intestine. In fact, in the circular mus-cle of the guinea pig duodenum and ileum, 0.5 and 0.2%,roids (17b-estradiol and progesterone) on K/ currents in

cells isolated from the myometrium of immature rats. In respectively, of the cell surface is occupied by gap junc-tions; these values correspond to 25 and 11 mm2/cell ortheir study, predominantly outward K/ currents with an

early transient component were recorded in response to 175,000 and 77,000 connexons/cell, respectively (329). Incontrast, in the adjacent longitudinal muscle, although thedepolarization pulses (from a holding potential of 090

mV). This current was rarely observed in adult myometrial dye Lucifer yellow will penetrate between the cells, gapjunctions are either absent or few in number (1224). Muchcells, was inactivated at 040 mV, and was blocked by

4-aminopyridine (4-AP). Depolarization also generated a the same finding has been reported in the longitudinalmuscle of the guinea pig taenia coli, although, again, asecond sustained outward current. 17b-Estradiol reduced

the probability of occurrence of the transient outward dense distribution was observed in the adjacent circularmuscle (329). Another tissue with a very high density ofcurrent, and progesterone had only a slight effect on the

currents. Treatment with 17b-estradiol (applied before gap junctions is the sphincter pupillae of rodents (actu-ally, the highest yet found). However, other SMC are virtu-isolation) led to a shorter time constant of decay for the

transient outward current, whereas progesterone caused ally, or completely, devoid of gap junctions; these includethe detrusor muscle of the rat bladder (329) and the cor-the time constant to increase.pus cavernosum of the penis (195, 196).

In electrophysiological terms (space constant and2. Passive membrane propertiesother characteristics), the role of the gap junction is notcompletely settled. In the myometrium, gap junctions areA) MORPHOLOGICAL FEATURES OF CELL-TO-CELL CONNEC-

TIONS. To understand the passive membrane properties of formed rapidly and in large numbers within a few hoursbefore parturition, and they come to occupy 0.2–0.4% ofVSMC, it is essential first to know something about the

morphological and physiological features of gap junctions the cell surface (344). Furthermore, progesterone sup-presses the formation of myometrial gap junctions,between cells. In studies of cell-to-cell connections, sev-

eral morphological structures have been described: tight whereas estrogen promotes it (226, 744). Sakai et al. (951)observed the effects of the antiprogesterone compoundsjunctions (nexuses), intermediate junctions, desmo-

somes, gap junctions, and nonjunctional membrane adhe- RU-486 and ZK-299 on cell-to-cell coupling in the guineapig myometrium during pregnancy. They found that thesions. Many VSMC possess cell-to-cell connections

through gap junctions. These gap junctions play two major myometrial SMC of guinea pigs are moderately well cou-pled before the onset of labor and that the coupling in-roles: propagation of excitation (including ion and small

molecule transport) and mechanical coupling. Moore and creases further, just before spontaneous delivery. It alsoincreases on treatment with antiprogesterones in non-Burt (768) studied the features of gap junctions and re-

ported that each junctional channel is composed of two pregnant animals. They reported an input resistance of44.6 MV on days 44–45 of gestation, falling to 22.9 MVhexamers of proteins terminating in connexins. One hex-

amer originates from each cell, and they join in the extra- on days 59–69, and then to 13.1 MV, and finally, at term,to 17.7 MV. Application of antiprogesterones reduced thecellular space to form a patent channel. Gabella (329)

reviewed the features of gap junctions in VSMC in detail input resistance to the same levels at three different stagesof pregnancy. They postulated that these events may beand described them as follows. In the gap junction, the

intercellular space is narrower than 3 nm. In contrast, if required for the synchronization and coordination of theelectrical, metabolic, and contractile events of labor. Ra-the space is larger than 60 nm, this structure is called an

adherens-type junction. The gap junction contains two mondt et al. (917) reported that estradiol administrationduring continuous infusion of naproxen (an inhibitor ofdense bands, one from each cell, linked to bundles of actin

filaments. The intramembrane particle, the connexon, is PG synthesis) increased the area occupied by gap junc-tions and improved the coordination of myometrial activ-8–10 nm in diameter, and its main component is connexin

(Cx). The presence of Cx43 has been detected in myome- ity. Thus the formation of myometrial gap junctions thatis induced by estradiol is not mediated via PG.triums and also in colonic muscle (82, 932). These parti-

cles lie always strictly parallel to each other, and there is As mentioned above, the gap junction is composedof connexins. The connexins are a gene family of at leastno encroachment of the dense material onto the cyto-

plasmic side (329, 331). The spatial density of connexons 12 members, but in vascular SMC, only two members havebeen found, namely, Cx43 and Cx40 (297, 768, 1173). Con-in gap junctions in VSM is Ç7,000/mm2, this value being

much smaller than that observed in cardiac muscles. The nexin-43, but not Cx40, has been localized to the SM layerof large vessels, whereas Cx40 and Cx43 are both clearlylargest junctions may contain some 1,400 connexons from

each of the two membranes, whereas the smallest ones present in the SM layer of some resistance vessels and in



endothelial cells (665–667, 1103). Moore and Burt (768) of Abe and Tomita (5). In these calculations, internal resis-tance (Ri) is assumed to be 125–300 Vrcm (based onreported that the connexin mRNA expressed in the rat

mesenteric artery was that related to Cx43. They reported impedance measurements in the longitudinal direction inguinea pig taenia coli, where the Ri was estimated to bea unitary conductance of 75 pS and a junctional conduc-

tance of 11.8 nS. The connexin in the pig coronary artery 100 Vrcm by Tomita (1096). In the GI tract, the l and tm

values obtained for longitudinal and circular muscle lay-was also related to Cx43, although the unitary conduc-tance was somewhat smaller (59 pS) and the junctional ers of the guinea pig stomach (antral region) were as

follows: 2.2 and 1.4 mm, and 130 and 180 ms, at membraneconductance larger (20.5 nS). In contrast, in the humancoronary artery, the mRNA expressed Cx40, and two uni- potentials of 058 and 054 mV, respectively. The calcu-

lated conduction velocities were 1.2 and 1.6 cm/s, respec-tary conductances were obtained (51 and 107 pS), thejunctional conductance being 14 nS. Moreover, cultured tively (636, 863). In longitudinal (taenia coli) and circular

muscles of the guinea pig caecum, the corresponding val-cells from the aorta of the rat (A7r5) expressed both Cx43and Cx40 and showed three different unitary conduc- ues were as follows: 1.5 and 1.7 mm and 100 and 250

ms, at membrane potentials of 055 and 052 mV, withtances (70, 108, and 141 pS) and a junctional conductanceof 11 nS. In arterioles, cell-to-cell coupling differed be- conduction velocities of 6 and 5.2 cm/s, respectively (495,

1095). In the longitudinal muscle layer of the guinea pigtween SMC and endothelial cells (769), and connexin-specific antisense oligodeoxynucleotides blocked the ex- rectum, the values of l, tm, and conduction velocity were

0.81 mm, 84 ms, and 4.4 mm/s, respectively (1279). Inpression of gap junction channels (769). Phosphorylationof connexins by protein kinase A (PKA) or by protein contrast, in the common bile duct of the guinea pig (longi-

tudinal direction), values of 0.8 mm, 100 ms, 051 mV, andkinase C (PKC), which is activated by 5-hydroxytrypta-mine (5-HT, serotonin) and by dopamine and other ago- 0.5 cm/s were reported (220). Crist et al. (225) compared

the passive and active membrane properties of the circu-nists, can apparently modify the unitary conductance andvoltage dependency of the gap junction (773, 926). lar muscle from the proximal and distal parts of the esoph-

agus of the opossum and found no differences betweenB) PASSIVE MEMBRANE CHARACTERISTICS. To aid the de-termination of the characteristic constants of each mem- the proximal and distal sites in terms of any of these

parameters.brane, various electrical models have been proposed, e.g.,the leaky condenser model and cable models (1-dimen- The passive membrane properties of the longitudinal

and circular muscle layers were compared in both thesional, 2-dimensional, and cable model for limited length).In VSM tissues, the one-dimensional model has been suc- human and dog colon by Huizinga and Chow (441). The

l value for the circular muscle layer was longer (2.14 mm;cessfully applied using the insulated partition stimulatingmethod of Abe and Tomita (5) and Tomita (1099). Thus, short axis; 0.43 mm) and tm value was shorter (160 ms)

than the corresponding values for the longitudinal muscleif longitudinal muscles of the portal vein possess a cableproperty, as suggested by the presence of many gap junc- (1.63 mm and 500–800 ms, respectively) in the human

colon, but no difference between the two muscle layerstions, the relationship between the time needed to reachthe half-amplitude of the electrotonic potential and the was observed in the dog colon. It is clear that, in the two

muscle layers, electrotonic coupling occurs, and it seemsdistance from the stimulating partition should be linearand could be expressed by t/2l, where t is the time con- to be related to the features of the gap junctions in the

human colon, but not in the dog colon in spite of the factstant of the membrane at erf-1 (measured value 420 ms)and l is the space constant of the membrane (421, 553). that gap junctions were scarce or absent in the longitudi-

nal muscle layer. It is interesting that, in the longitudinalToro (1101) measured the passive membrane proper-ties of isolated cultured SMC from the caudal artery and muscle layer of the guinea pig intestine, the l and tm

values could be calculated from electrotonic potentialsvein using the microelectrode method. In their study, thecharacteristics of cells prepared from artery and vein, re- recorded at any given distance from the stimulating elec-

trode, yet the density of gap junctions was sparse if as-spectively, were as follows: resting membrane potential,056 and 066 mV; input resistance (Rin), 590 and 450 MV; sessed using electron microscopy (329, 331).

2) Myometrium. Myometrial cells possess a syncytialmembrane time constant (tm), 19 and 19 ms; membranecapacity (Cm), 1.3 and 1.0 mF/cm2; and length (space) structure, and the passive membrane properties of this

tissue have been determined under various conditions.constant, l (measured using limited-length cable theory),900 and 1,300 mM. It is plausible that, because of the lack The values obtained for the passive membrane character-

istics have been as follows: tm, 100–300 ms in rat, guineaof a syncytial structure, the isolated cell may show a muchhigher membrane resistance and internal resistance than pig, sheep, and human myometrium in midpregnancy; l,

1–3 mm in the same species. Kuriyama and Suzuki (632)would a multicellular tissue.1) GI tract. Using multicellular VSM tissues, many reported that in the nonpregnant rat longitudinal myome-

trium, the values of the membrane potential, l, and tminvestigators have measured passive membrane proper-ties from VSMC using the partition stimulating procedure were 056 mV, 1.5 mm, and 128 ms, respectively. At the



middle stage of gestation (11–15 days), the corresponding from 200 to 300 ms, respectively (e.g., guinea pig portalvein, Refs. 496, 631; rabbit abdominal aorta, Refs. 727,values, again in the longitudinal muscle layer, were 068

mV, 2.6 mm, and 180 ms, respectively. At 18–19 days of 728; guinea pig aorta, Ref. 621; rabbit pulmonary artery,Refs. 169, 170; rabbit carotid artery, Refs. 726, 730; caninegestation, the values had changed to 064 mV, 2.6 mm,

and 198 ms, respectively. On the last day and during partu- coronary artery, Refs. 728, 731; monkey coronary artery,Ref. 731; rabbit aorta, Refs. 378, 729; for more details onrition, they were 054 mV, 2.9 mm, and 228 ms, respec-

tively, and finally, postpartum (6–10 h) they were 051 vascular SM tissues, see Ref. 626). Because elastic andcapacitive vessels do not produce action potentials undermV, 3.2 mm, and 241 ms, respectively. In midpregnancy

in the circular muscle layer, l was 1.0 mm (555). However, physiological conditions, gap junctions in these tissuesmay be more important for the transport of low-molecu-Parkington (882) reported that, in circular muscle cells

of the guinea pig myometrium, the tm value remained lar-weight substances (including ions) between cells thanfor a propagation of excitation or for tightening the struc-unchanged throughout pregnancy (days 30 and 60 of

pregnancy and in labor) but that the values were higher ture of the SM (for review, see Ref. 626).4) Vas deferens. Most electrophysiological investiga-in estrus (330 ms) than in diestrus (210 ms). In contrast,

the l value increased during the progress of gestation, tions on the vas deferens have been made because ofan interest in the innervation or in the neurotransmittersbut no change in this value was seen at term in compari-

son with the last stage of gestation. In the circular muscle involved, rather than in the function of the SMC of thevas deferens themselves. In the vas deferens of rodents,layer in sheep, despite no change in the membrane poten-

tial during gestation or at term, the l value increased such as guinea pig, rat, and mouse, the muscle is arrangedin three layers: outer and inner longitudinal layers withprogressively (Ç1 mm at 50 days of pregnancy, 2 mm at

140 days of pregnancy and 4 mm in labor, at 145 days). circular muscle in between. The morphological arrange-ment of these muscle layers is loose everywhere butIn contrast, the tm value did not change during gestation

(remained at 130 ms) but was markedly increased in labor looser in the epididymal regions than in the amplulla re-gion (144, 146, 586, 1032). The passive membrane proper-(510 ms; Ref. 882). Parkington (882) therefore postulated

that the onset of labor in the ewe is associated with rapid ties of the longitudinal muscle layer of the vasa deferentiaof guinea pig and mouse have been investigated (77, 417,and drastic changes in both the passive and active proper-

ties of the circular muscle of the uterus. Presumably, a 732). For instance, in guinea pig vas deferens, Holman etal. (427) identified two cell populations, active and inac-reduction in the Ri may contribute to these changes

through modifications of cell-to-cell connections. In the tive cells, on the basis of spike generation induced byintracellular stimulation. The former had an membraneguinea pig, although the l value increased during gesta-

tion, with no further change in labor, the values for tm and resistance of 10 kVrcm2 and a Cm of 2.5 mF/cm2, whereasin the latter, the corresponding values were 1 kVrcm2the membrane potential remained unchanged throughout

gestation and labor (881, 883). In the upper margin of the and 3 mF/cm2. When the extracellular, rather than intracel-lular, stimulating technique was used, the correspondinghuman myometrium (monolayer culture), Pressman et al.

(904) measured passive membrane properties and showed values were 10 kVrcm2 and 1 mF/cm2, respectively, anda tm of 100 ms was obtained (1097). Thus the passivethat the resting membrane potential was 049 mV, the

specific membrane resistance 6 kVrcm2, and the specific electrical characteristics of the guinea pig vas deferenswere much the same as those observed in the guinea pigmembrane capacitance 1.6 mF/cm2. In the pregnant rat

myometrium (18–19 days), Mollard et al. (766) found that taenia coli. However, they were not the same as in mice;l of the tissue was much shorter (nearly zero) in thein the longitudinal muscle layer the membrane potential

was 054.5 mV, the action potential had an overshoot mouse vas deferens, and no longitudinal propagation ofexcitation was recorded. Moreover, on membrane depo-(/7.8 mV), the specific membrane resistance was 14.8

kVrcm2, and the specific membrane capacitance was 2.3 larization evoked by electrical stimulation, the maximumfrequency of action potentials was different in the vasamF/cm2. In the pregnant human myometrium, Inoue et al.

(487) reported a l value of Ç1.0 mm and a tm of 396 ms. deferentia of the two species; it was 1–2 Hz in the guineapig but 30 Hz in the mouse (and rat) (316, 427). It is ofIn the pregnant mouse myometrium, Osa (862) mea-

sured a conduction velocity of excitation that was 20 cm/ interest that Holman et al. (427) reported that electrotonicpotentials elicited by extracellular stimulation spread ins in the longitudinal direction and 0.7 cm/s in circular

muscle cells. In the rat myometrium in late pregnancy, a longitudinal direction in the guinea pig vas deferens butthat there was no such longitudinal spread in the mouseMiller et al. (744) reported that the conduction velocity

of excitation was 9 cm/s before delivery, increasing to 11 vas deferens. Interestingly, in the guinea pig, rat, andmouse, the cells were electrically quiescent, whereas incm/s during delivery.

3) Vascular SMC. If we look at vascular SMC, we rabbit and human, the cells were spontaneously active,the cells of the former group being more sparsely inner-find that the reported l and tm values for elastic and

resistance vascular tissues vary from 1.0 to 2.0 mm and vated than those of to the latter. Friel (304) measured the



passive electrical properties of the rat vas deferens using a contraction that could be blocked by atropine. Presum-ably, the high K/ depolarized nerve terminals so that theythe whole cell voltage-clamp technique. In freshly cul-

tured cells, the membrane potential was 037 mV (a much released ACh, which then produced a contraction. Alter-natively, in this case as well as in guinea pig and porcinelower value than that measured by the microelectrode

method from the intact tissue, 058 to 065 mV, Ref. 625; coronary arteries and canine and guinea pig pulmonaryarteries, in the presence of TEA or 4-AP, the SMC067 mV, Ref. 1005). Their Rin was 2.9 GV, their C was 37

pF, and their tm was 0.1 s. After denervation, spontaneous generated only graded action potentials; Refs. 169, 464,625). Therefore, it is probable that the density of channelactivity was seen in the vasa deferentia of the guinea pig

and the rat, and it has also been reported that denervation, distribution and its ability to carry charge via voltage-operated (-dependent) Ca2/ channels (VOCC) is low inby sectioning the hypogastric nerves, led to an accelerated

propagation of excitation, presumably because of an inthese tissues.Figure 1 shows various types of action potentialscrease in the number of tight junctions (994). Not only

female reproductive organs, but also male reproductive recorded from longitudinal and circular myometrialcells of rats under different physiological conditionsstructures, such as the vas deferens, are modulated by

hormones. For example, automaticity and conductivity (pregnancy, at delivery, and post partum) and after treat-ments with estradiol and progesterone. In the nonpreg-were increased after castration in male guinea pig, and

such increases were suppressed by treatment with testos- nant rat, mouse, and guinea pig myometriums, circularmuscle produced slow potential changes with or withoutterone (993).initial spike. In the nonpregnant conditions, longitudinalmuscle produced bursts of spikes with variable quies-

B. Action Potentials Generated in VSMCcent intervals. In the rat myometrium, at the middlestage of gestation (10th-15th day of pregnancy), circular

1. Spike potentialsmuscle generated only a slow plateau potential with orwithout spike potentials, as gestation progressed, theSome VSMC generate spontaneous spike potentials

(e.g., longitudinal SMC of the pyloric region of the stom- amplitude of the plateau became smaller and eitherbursts of spikes of irregular amplitude or slow oscilla-ach, ileum, and taenia coli, pyloric region of the ureter,

myometrium, and portal vein in all experimental animals tions occurred on the plateau potential; at term, burstsof spikes of uniformal amplitude masked the generationtested). However, some VSMC are electrically quiescent

(longitudinal SMC in the stomach fundus, SMC of conduit of the plateau (882). In contrast, in the rat longitudinalmuscle layer, in the middle stage of gestation, burstsand capacitive vessels, and the trachea). Quiescent SMC

generate electrical activity on field stimulation of periph- of spikes with irregular amplitude occurred on a smallplateau potential. However, as gestation progressed toeral nerves; these include SMC in the mesenteric arteries

and vasa deferentia (for review, see Refs. 106, 426, 625). term, the amplitude of the plateau potential becamemuch smaller, and bursts of spikes of regular amplitudeIn the trachea of many species, such as guinea pig, dog,

and cow, peripheral nerve stimulation generates a slow were superimposed on it at regular intervals. Much thesame pattern of electrical activity as that observed inpotential change without spike generation. However, after

treatment with tetraethylammonium (TEA), 4-AP, or pro- the last stage of gestation can be induced during themiddle stage of gestation by treatment with estradiolcaine, oscillations or spike potentials can be recorded,

after direct or indirect (nerve) stimulation, from canine (3–4 days, 0.1 mg/day) without any marked change inthe membrane potential (Fig. 1). On the other hand,and bovine tracheas (467, 497, 571). It is known that, in

spontaneously active VSMC, the amplitude of the spike when progesterone (2–3 days, 5 mg/day) was given onthe 22nd day of gestation in the rat, SM membranes werepotentials recorded is irregular (except in a few examples,

such as the estrogen-dominated, or at-term, rat myome- hyperpolarized in both longitudinal and circular musclecells. Moreover, cells of both layers showed a plateautrium; Ref. 625), and an overshoot potential is not consis-

tently observed. However, after treatment with TEA or 4- potential of reduced amplitude, with spike dischargesof irregular amplitudes superimposed on it. Further-AP, spikes of regular amplitude with an overshoot can

be recorded from intestinal and vascular SMC (625). In more, in the middle stage of gestation, the rate of risein the spike recorded from rat and mice myometriumsquiescent VSMC, such as those in the canine trachea, the

Ca2/ antagonist-sensitive L-type Ca2/ current could be re- was greater than that observed in the last stage of ges-tion and, after treatment with progesterone, the rate ofcorded after treatment with TEA or 4-AP. This implies

that, under normal conditions, a large outward K/ current rise was increased more than after treatment with estro-gen (with progesterone from Ç10 to Ç20 V/s; Ref. 621).(Ca2/ sensitive and delayed rectifying) may mask the in-

ward Ca2/ current and prevent the generation of spike In the mouse myometrium, much the same responsescould be observed as in the rat myometrium both duringpotentials (625, 785). However, in bovine and human iris

dilator SMC, high concentrations of K/ solution produced gestation and also after separate treatment with estro-



FIG. 1. Spontaneous or induced electrical activitiesrecorded from longitudinal (A) and circular (B) musclecells during gestation, at delivery, postpartum (18 h afterdelivery), and with ovarian hormone treatments. Estradiol(E; 0.1 mg/day) was injected from the 19th day of gestation(3 days). Progesterone (P; 5 mg/day) was injected fromthe 20th day of gestation. [From Kishikawa (577).]

gen or progesterone (834, 861, 864). When studying the before, or at the onset of, myometrial contraction. Intrigu-ingly, mRNA was markedly decreased at parturition. Howeffect of estrogens on the Ca2/ current in cultured SMC

of the late pregnant rat myometrium, Yamamoto (1200) changes in isoform expression occur during labor is notyet known, but multiple unknown factors probably con-found that b-estradiol inhibited the Ca2/ current, in a

voltage-dependent manner, and shifted the steady-state tribute to the initiation of parturition. Figure 2 shows theeffects of 17b-estradiol on ionic currents in excised preg-inactivation curve toward more negative potential lev-

els. A synthetic estrogen, diethylstilbestrol, also had ac- nant rat myometrium.Parturition is a complex process involving the inter-tions on the Ca2/ current similar to those of b-estradiol,

at a lower concentration, may directly act on the surface play of several endocrine factors. Kishikawa (577) re-ported that in the rat, progesterone content rapidly de-membrane, and inhibited VOCC more potently than de-

layed rectifying K/ current. creased and estrogen content increased 1 day before de-livery. Therefore, the ratio of progesterone to estrogenIn the pregnant rat myometrium, Miyoshi et al. (764)

reported that the inward currents consisted of Ca2/ and decreased rapidly on that day. The events occurring inthe last stage of gestation and during delivery differedNa/ currents, whereas the evoked outward current con-

sisted of a fast TEA-insensitive current and a large TEA- from those seen in the middle stage of gestation. At theend of gestation, a plateau potential was not apparent insensitive delayed rectifying outward current. Mershon et

al. (733) examined the hypothesis that parturition is asso- the longitudinal muscle layer, and the amplitude of theplateau potential was reduced in the circular muscle, de-ciated with significant changes in the expression of the

a1-subunit of the VOCC at the mRNA or protein level. spite the increase in the estrogen content of the sera. Inaddition, the amplitude of the plateau potentials seen inThey concluded that the number of L-type Ca2/ channels,

although large in the rat myometrium, does not change both muscle cell types did not increase with estrogen



2. Pacemaker potentials and slow potential changes

A pacemaker potential (i.e., a gradual depolarizationwhich, when it reaches a threshold level, triggers a spikepotential) occurs in some VSMC. The first description ofthe typical pacemaker potential was published by Bozler(121), who used an extracellular electrode on the dogureter. In pacemaker cells (sinoatrial node) in the heart,the current responsible for generating the pacemaker po-tential has been called a ‘‘funny’’ current (If) or hyperpo-larization-activated current (Ih; Refs. 828, 1204).

A) SLOW WAVE. In many VSMC, the membrane potentialexhibits slow spontaneous changes in activity in the ab-sence of any influence from nerve activity or endogenoussubstances [mostly examined in the presence of tetrodo-toxin (TTX)]. Slow potential changes are defined here asthose changes that occur very slowly up to 020 to 030mV and exhibit repolarization also with a very slow timecourse. The total time course of such slow potentials ex-ceeds 1 s, and they occur in the GI tract, urinary bladder,myometrium, and oviduct. In some cells, these slow po-tential changes evoke a spike or spikes when the depolar-ization exceeds threshold, but in other cells, spike genera-tion does not occur on slow potential changes (e.g., ovi-duct and stomach fundus). In other words, generationof a spike potential is not necessarily a function of thegeneration of slow potential changes.

The spontaneous slow potential change (slow wave)that occurs in the GI tract is also called a pacemakerpotential, because this potential occurs without the gener-ation of a prepotential. The features of this slow wave

FIG. 2. Effects of 17b-estradiol (10 mM) on Ca2/ and K/ currentsrecorded from the GI tract have been reviewed exten-in excised pregnant rat myometrium (longitudinal muscle at 19 days ofsively (954, 1066, 1100). Confusingly, the slow waves gen-gestation). A: control. B: 17b-estradiol. Depolarization pulses to 030,

010, and 30 mV from a holding potential of 050 mV were applied (500 erated in the GI tract are resistant to Ca2/ channel block-ms). (From K. Okabe, unpublished observations.)

ers, but their amplitude and rate of rise are dependent onthe Ca2/ concentration. To try to resolve this discrepancy,a role for the T-type VOCC in the generation of the slowtreatment but did increase with progesterone treatmentwave might seem worthy of investigation. Unfortunately,in the last stage of gestation.however, the presence of the T-type Ca2/ channel in theIn the pregnant rat myometrium, the placental re-GI tract has not yet been confirmed (L type alone foundgion shows different electrical properties from the non-in newt stomach, Ref. 156; dog stomach, Ref. 985; rabbitplacental region. Thus the membrane potential mea-ileum, Ref. 490; dog proximal colon, Ref. 1160). In thissured from the placental region showed a consistentlycontext, an analysis has been made of the features ofhigher value than that from the nonplacental region upthe anomalous L-type Ca2/ channel and of the voltageto the last stage of gestation. Moreover, the conductiondependency of the L-type Ca2/ channel. Huizinga et al.velocity of excitation was consistently low in the placen-(442, 443) reported in canine colon that the generationtal region (about one-tenth of that in the nonplacentalof the slow potential change involves a non-L-type Ca2/region; days 7–22). Furthermore, propagation of excita-conductance. In freshly dispersed human uterine SMC,tion seems to be blocked from the cells of the nonplacen-Young and Herndon-Smith (1217) suggested that the T-tal region to the placental region, but not vice versa. Thetype current may be primarily involved with action poten-shape of the spike in the nonplacental region changedtial transmission and the L-type current primarily withmarkedly during the progress of gestation, whereas theincreasing [Ca2/]i by bulk Ca2/ transport. However, as yet,electrical activity of muscle cells in the placental regionthere is insufficient experimental data for us to determinewas not consistent. However, the electrical activity ofthe validity of this idea. In cat colon circular muscle, Ven-muscle cells recorded at full term was much the same

in both regions (550). kova and Krier (1124) studied the feature together with



the effects of norepinephrine on slow wave in the pres- to oscillations in [Ca2/]i that appeared to be secondary tothe periodic influx of Ca2/. Lee and Sanders (652) furtherence of TTX with atropine and concluded that the circular

SMC near the submucosal border had a resting membrane observed that when currents recorded from the ICC werecompared with those recorded from SMC from the samepotential of 076 mV and exhibited electrical slow waves

(frequency, 4–6 Hz; upstroke potential,040.7 mV; plateau region of circular muscle in the canine colon, depolariza-tion of both types of cells initiated a transient inwardpotential, 044 mV, and duration, 4.9 s), and the circular

SMC near the myenteric border had a resting membrane current followed by a slowly inactivating outward current.When the inward current and Ca2/-dependent outwardpotential of 051 mV and did not exhibit electrical slow

waves. current were both blocked, the ICC then generated a more4-AP-resistant outward current (rapidly activated and in-In fact, the mechanisms underlying the generation of

slow waves in the GI tract have been investigated by many activated current; A-like current) than did circular SMC.Furthermore, the outward currents recorded from the ICCresearchers over the past 30 years. Prosser’s group (213)

postulated that the periodic appearance of slow waves in were deactivated at a more negative potential (half-inacti-vation at053 mV) than were those recorded from circularintestinal SMC may correspond to oscillations in the activ-

ity of the electrogenic Na/-K/ pump. In contrast Tomita’s SMC (020 mV). Thus the features of the outward currentsin the two types of cell are not exactly the same. Further-group (835, 836) postulated that the slow waves in stom-

ach circular muscle consist of two components: pace- more, when the inward currents were compared in termsof their current-voltage relationship (0100 mV to /20maker activity, triggered by a non-voltage-dependent pro-

cess, and a later, ionic conductance. Experiments using mV), the ICC showed the presence of both L- and T-typeVOCC (generation of a hump at potentials within the rangea multicellular double sucrose gap method with the parti-

tion stimulating procedure of Abe and Tomita (5) revealed 070 mV to about 040 mV), whereas SMC showed the L-type current alone. The function of ICC is generally agreedthat changes in the membrane potential modify the ampli-

tude of the slow wave but not its frequency of generation to be difficult to investigate in intact GI muscles becauseof the structural complexity of the tissues. Attempts to(in circular muscle of guinea pig stomach, Ref. 629). Also

in guinea pig stomach, Tsugeno et al. (1108) observed the remove ICC surgically or chemically may fail to removethe cells in sufficient quantities or may damage adjacenteffects of phosphodiesterase (PDE) inhibitors, such as

caffeine, theophylline, IBMX (a nonselective PDE inhibi- SMC or neurons, or introduce nonspecific pharmacologi-cal effects (952). Sanders and Ozaki (956) thus postulatedtor, Ref. 996), and rolipram, as well as the effects of cAMP

synthesis accelerators, such as isoproterenol, forskolin, that this voltage-dependent T-type Ca2/ channel and anegative potential-activated outward current may com-dibutyryl cAMP (DBcAMP), and 8-bromo (8-BrcAMP).

They reported that an increase in cAMP reduces slow bine to generate the slow potential changes seen in theGI tract. Recently, Sanders (953) reviewed recent viewswave frequency without changing either the membrane

potential or the slow wave configuration. They concluded concerning roles of ICC as a pacemaker cell: 1) electro-physiological experiments on dissected muscle stripsthat an increase in intracellular cAMP inhibits the pace-

maker activity of the slow waves. In their view, 1) an show that slow waves originate from specific sites. Thesepacemaker areas are populated by networks of ICC thatincrease in K/ conductance is unlikely to be a major factor

in this inhibition, and 2) slow waves appear to represent make gap junction with SMC. Removal of pacemaker re-gions interferes with slow wave generation and propaga-compound electrical activity in a groupof muscle cells,

which is likely to be disintegrated by xanthine derivatives. tion. 2) Chemicals that label ICC histochemically can dam-age ICC and abolish rhythmicity. 3) Isolated ICC are spon-B) INTERSTITIAL CELLS OF CAJAL. Some elegant work has

been carried out by Sanders’ group (644, 645, 652, 906, taneously active, and several voltage-dependent ionchannel are expressed. 4) Interstitial cells of Cajal are952, 953) and also by other investigators to clarify the

origin of pacemaker activity in the GI tract in relation to innervated by enteric neurons, and they respond to neuro-transmitters. Interstitial cells of Cajal may produce nitricthe interstitial cells of Cajal (ICC). Their conclusion was

that it actually lies in the activity of ICC. For example, oxide (NO) and amplify inhibitory neurotransmission. 5)Some classes of ICC fail to develop in animals with muta-Langton and co-workers (644, 645) reported that ICC iso-

lated from the pacemaker region of the canine colon tions in c-kit or stem cell factor, the ligand for c-kit recep-tors. Without ICC, electrical slow waves are absent.showed spontaneous electrical activity of an amplitude

and with characteristics similar to those of the electrical Xue et al. (1189) studied the expression of NO syn-thase (NOS) in ICC of the canine proximal colon. Theyslow waves recorded from muscle strips. They suggestted

that the ion channels responsible for the electrical events concluded that the submucosal component of the circularmuscle layer, which lies along the surface of the septumare Ca2/ channels and Ca2/-dependent K/ channels. Publi-

cover et al. (906), using freshly dispersed and cultured in the myenteric region between the longitudinal and cir-cular muscle layers, expresses a constitutive form of NOS.ICC prepared from the canine colon, reported that the

spontaneous electrical activities they recorded were due They therefore suggested that the synthesis of NO by ICC



may influence electrical rhythmicity and may serve to am- on the ACK2-treated murine intestinal tract was to in-crease both the contractile responses and receptor sensi-plify and even propagate enteric inhibitory neurotransmis-

sion. tivity of its longitudinal SMC and that ACh produced alarger depolarization in the SMC of the ileum in the ACK2-C) C-KIT IN RELATION TO THE PACEMAKER ACTIVITY. Re-

cently, the involvement of ICC in pacemaker activity was treated mice than in controls. Circular SM responses tothese substances, as measured by changes in intraluminalelegantly demonstrated (682, 960, 1094, 1101, 1155). They

made use of c-kit, which is a proto-oncogene encoding a pressure, were not altered by ACK2 treatment. They con-cluded that ICC play an important role not only in thetyrosine kinase receptor, c-kit, a member of a platelet-

derived growth factor (PDGF)/mast cell colony stimulat- development of the pacemaker system of the small intes-tine but also in the functional development of the contrac-ing factor (CSF) receptor family, namely, the PDGF/CSF-

1 receptor family (1212). Maeda et al. (682) reported that tile properties of intestinal SMC. Liu et al. (667) studiedthe relationship between the SR Ca2/ and periodicity ofsome of the cells in the SM layers of the developing intes-

tine express c-kit, and blockade of its function for a few the biochemical clock to determine the pacemaker fre-quency using canine colon and reported that since cyclo-days postnatally by an antagonistic anti-c-kit monoclonal

antibody (ACK2) results in a severe anomaly of gut move- piazonic acid (CPA) inhibited the SR Ca2/ pump and re-duced the pacemaker frequency, the Ca2/ refilling cyclement, which in BALB/c mice produces a lethal paralysis.

On this basis, they postulated that c-kit plays a crucial of the inositol trisphosphate (InsP3)-sensitive Ca2/ storesassociated with the myoplasma membrane may determinerole in the development of a component of the pacemaker

system that is required for the generation of automatic the frequency of the pacemaker activity generated by thesubmuscular ICC-SM network.gut motility. Torihashi et al. (1100) confirmed the above

view using cells that express c-kit-like immunoreactivity On pacemaker cells of proximal colon, Kobayashi’sgroup (597, 795) described that, in the canine proximalin the mouse small intestine and colon. Furthermore, in

the colon they found cells that were labeled with ACK2 colon, tissue near the submucosal surface of the circularmuscle layer (innermost pacemaker muscle cells; P layer)in the region of the myenteric plexus as well as deep

muscular plexus of the small intestine and in the subser- produces spontaneous mechanical contractions, synchro-nized with electrical slow waves. The P-layer SMC haveosa (in the myenteric plexus region), within the circular

and longitudinal muscle layers, and along the submucosal slow distinguishable features from the bulk circular SMC:1) flattened and shorter shapes of the cell and nucleus,surface of the circular SMC. The distribution of cells that

expressed c-kit was the same as that of interstitial cells. 2) numerous caveola on the cell surface and abundantmitochondria, and 3) frequent gap junction formation.A decrease in ICC was accompanied by a loss of electrical

rhythmicity in the small intestine and reduced neural re- Neither slow waves nor spontaneous mechanical rhyth-micity was recorded from the submucosal or connectivesponses in the small bowel and colon. Ward et al. (1155)

and Huizinga et al. (444) suggested that the c-kit function tissue or from the bulk circular muscle. They concludedthat the inner sublayer characterized by special SMC withmay be important in the development of the network of

ICC, because W/WV and W// mutant mice (179, 346) had a delicate nerve plexus is essential for producng spontane-ous activities of circular muscle coat in the canine proxi-few ICC in the myenteric plexus region, and this was

associated with a loss of slow electrical activity. Further- mal colon. Furthermore, they (795) reported that thesespecified SMC in the slow wave (pacemaker) was inhibi-more, when the excitability of c-kit-expressing cells in

primary culture was examined using the nystatin-perfora- tory innervated by nitrinergic nerves, and NG-nitro-L-argi-nine methyl ester (L-NAME) enhanced the spontaneousted patch-clamp procedure, the majority of c-kit-express-

ing cells showed rhythmic current waves with an ampli- mechanical rhythms, whereas L-arginine suppressed theL-NAME-induced enhancement. In fact, the ICC and P-tude and frequency of 263 nA and 2.3 cycles/min, respec-

tively, when the membrane was depolarized to 040 mV. layer cells are the same cells.To conclude, the pacemaker activity that generatesThe reversal potential level of the rhythmic current was

close to the Cl0 equilibrium potential. These electrical the slow wave (slow potential change) may indeed proveto be originating from the ICC. Although the ICC wasrhythms were blocked by SITS, and such responses were

not observed in tissue samples containing SMC alone. originally thought to be neural origin (914), they are nowknown to be specialized SMC and derived from the neuralTherefore, they concluded that the intestinal c-kit ex-

pressing cells, but not SMC, exhibit a rhythmic Cl0 current crest and to develop independently from the enteric ner-vous system (650). However, it is possible that the originoscillation, which suggests that they participate in the

pacemaker activity responsible for peristaltic gut move- may differ in different tissues in the GI tract (on the basisof their biophysical features). More detailed informationments. Slow wave was D600 (gallopamil) resistant, and

thus differentiation between action potential and slow can be expected in the near future on pacemaker activityin the GI tract in relation to activities of individual ICC.wave can be differentiated (Fig. 3). Moreover, Sato et al.

(962) found that the effects of ACh, bradykinin, and PGF2a At present, unsolved problems remain; for example, how



FIG. 3. Action potentials recorded from small intes-tine of normal (c-kit positive) and W/Wv mutant (c-kit neg-ative) mice. ///, Preparation from wild-type mice havingnormal interstitial cell of Cajal network; W/Wv, preparationfrom W mutant mice. In normal small intestine, slow wavegeneration occurred constantly and was resistant to D600,a Ca2/ channel blocker, whereas in c-kit-negative intestine,no constant and stable waves were seen. [From Huizingaet al. (444).]

is the activity of individual ICC cells coordinated (the role procedure, the generation of a plateau potential was inhib-ited when Na/ was removed from the extracellular me-of the ICC network), how do the actions of ICC correlate

with those of the myenteric plexus (not only by nitric dium (593, 630), and when extracellular Ca2/ was in-creased, the amplitude of the spike was enhanced,oxide), how does the heterogeneous distribution of ICC

in ileum and cecum relate to functional differences in the whereas the duration of the plateau potential was reduced(620). However, such responses induced by changes ininitiation of pacemaker activity, and so on. We also need

more detailed data to clarify the features of pacemaker Na/ or Ca2/ have yet to be confirmed using the patch-clamp procedure. Incidentally, the spike potential gener-activity in spontaneously active SMC.ated in the ureter is due to activation of VOCC.

In guinea pig ureter, the contribution made by the Ca2/-3. Plateau potentials

dependent K/ current to action potential formation appearsto be relatively small, and a delayed rectifying K/ currentWith the use of the microelectrode method, a plateauis lacking; as a consequence, spike potentials appear repeti-phase of high amplitude that is generated after a spike ortively at depolarized potential levels and produce a plateauspikes can be recorded in guinea pig and rabbit uretersphase by virtue of the slow inactivation of the Ca2/ current(592, 593, 630), whereas a plateau phase of low amplitude(466). These authors also postulated, with regard to thecan be recorded in the uterus (estrogen-treated longitudi-plateau potential and from the action of norepinephrine onnal and circular muscle layers; Refs. 693, 834). Zhang etthe ureter, that Ca2/ channel activity is regulated by dualal. (1226) classified the cells of the guinea pig renal pelvismechanisms: a Ca2/-dependent inhibition and a G protein-into three types on the basis of their electrical activity: 1)mediated potentiation. Under physiological conditions,pacemaker cells (10% of the population), with a simpleCa2/-inward and Ca2/-dependent K/ currents were bothaction potential comprising relatively slow rising and re-reduced by norepinephrine. However, the reduction in thepolarizing phases triggered on top of a slowly developingCa2/-dependent K/ outward current was much larger thanprepotential; 2) driven cells (75%), with a complex actionthat in the Ca2/ current, thus resulting in an increase in netpotential comprising a rapid initial spike, followed by ainward current during the action potential plateau and aperiod of membrane oscillation and a plateau of 0.2–2 sprolongation of the action potential’s duration (788). In theduration; and 3) intermediate cells (15%) that fired actionureter, Aickin et al. (17) reported the presence of Na//Ca2/potentials with an initial rapid phase and then a long pla-exchange diffusion, and Kazarian et al. (558) suggested thatteau phase. The generation of a spike potential is a prereq-Na//Ca2/ exchange diffusion may contribute to the forma-uisite for plateau potential generation in the ureter, buttion of a plateau potential. It is plausible that plateau forma-not in the colon, where a plateau-potential-like sustainedtion in VSMC may not be due to activation of a specific iondepolarization occurs with or without an initial spike. Fur-channel but may be due to the concerted activation andthermore, in the case of the myometrium, early pregnantinactivation of several ion channels.circular muscle cells generated only slow potential

changes, but later in the progress of gestation, there wasa typical sustained depolarization after spike generation. III. NEURAL CONTROL OF MEMBRANEAt term, a burst of spikes of regular amplitude was super- ACTIVITIES IN VISCERAL SMOOTHimposed on a low-amplitude plateau potential. In the myo- MUSCLE CELLSmetrium, the duration of the plateau phase was reducedin high extracellular Ca2/ concentration. In guinea pig Neurotransmitters (endogenous agonists) activate in-

dividual receptors distributed on VSMC, and this inducesand cat ureters, in experiments using the microelectrode



two main actions. Transmitters released from nerves 1) contained calretinin plus substance P. There were sevenclasses of neurons that innervated myenteric ganglia;modify the ionic permeability directly, with or without

activation of G proteins, and induce either depolarization these contained NOS, VIP, NOS plus VIP, NPY, calretininplus calbindin, substance P, or 5-HT. In the large intestine,or hyperpolarization of the membrane (ionotropic action)

or 2) activate receptors leading to the synthesis of second there were five major classes of neurons that containedNOS, NOS plus VIP, GABA, substance P, or calretininemessengers through the action of a G protein and enzymes

such as phospholipase C (PLC) or adenylate and guanyl- plus substance P, and seven major classes of neurons thatinnervated myenteric ganglia and contained NOS, VIP, cal-ate cyclases (metabotropic action). These synthesized

second messengers phosphorylate various proteins in in- retinin plus calbindin, calretinin, substance P, GABA, or5-HT. Distributions of galanin-containing neurons weredividual cells and also indirectly regulate ion-permeable

channels. also detected in the canine GI tract, i.e., galanin-like immu-noreactive (GAL-Li) nerves and cell bodies were distrib-uted in esophagus, stomach, and intestine. Thus it was

A. Nerve Plexuses, Nerve Terminals, Varicosities,estimated that GAL-Li nerves are distributed more widely

and Cotransmitters as Studied byin the canin enteric nervous system than previously recog-

Electrophysiological Methodsnized.

In the canine intestine, according to Furness et al.1. GI tract

(317), VIP fibers run anally in the myenteric plexus ofboth small and large intestine, whereas substance P fibersA) MYENTERIC PLEXUS. In the myenteric plexus of the

proximal colon of the guinea pig, Messenger et al. (735) run orally in the large intestine and both orally and anallyin the small intestine. The innervation of the muscularisclassified plexus neurons electrophysiologically as either

afterhyperpolarization-forming (AH) neurons (morpho- mucosa and mucosa by substance P- and VIP-containingfibers was not affected by myectomy or extrinsic denerva-logically these cells typically had a large, oval soma and

several long tapering processes that sent branches into tion, and these structures are, therefore, likely to be inner-vated from nerve cells in the submucous ganglia. Furnessmany adjacent ganglia) or S neurons (these neurons were

uniaxonal and many of them had axons ending in an et al. (315) reported that the myenteric plexus containsboth excitatory and inhibitory nerves, of which the formerexpansion bulb in the myenteric plexus; they typically had

broad, lamellar processes or short, spiny processes). The contain ACh and tachykinins such as substance P andneurokinin A (NKA), whereas the latter contains VIP, pep-AH neurons were characterized electrophysiologically by

the presence of a slow afterhyperpolarization after their tide histidine methionine, and peptide histidine isoleucine(PHI). The myenteric reflex response to distension of theaction potential. Internodal strand stimulation in the intes-

tine evoked the slow excitatory synaptic potential, but guinea pig small intestine has been extensively investi-gated, by means of electrophysiological methods, bynot a fast excitatory synaptic potential. The S neurons

lacked a slow afterhyperpolarization, and internodal Smith and co-workers (1000, 1001). Distension of the tis-sue generated excitatory junction potentials (EJP) in thestrand stimulation usually evoked fast excitatory synaptic

potentials (although some neurons did show slow excit- oral region but inhibitory junction potentials (IJP) in theanal region. Crist et al. (221) studied circular SMC of theatory synaptic potentials). A subpopulation of AH neurons

displayed a rhythmic oscillation in their membrane poten- guinea pig distal ileum and found that intramural nervestimulation produced a fast EJP and a late EJP at oraltial (which could trigger an action potential). The S neu-

rons could be subdivided into tonic and phasic firing sites as well as an initial IJP and EJP and a prolongedhyperpolarization at anal sites; there was a also a late IJP,types. About 80% of the AH neurons examined by Messen-

ger et al. (735) were immunoreactive for calbindin, as which was observed only at sites anal to the stimuluspoint, and a late EJP, which was observed only at siteswere 10% of S neurons. A further 17% of S neurons, but

no AH neurons, were calretinin immunoreactive. Roughly oral to the stimulus point. The neural inhibition of circularand longitudinal colonic SMC (in relation to the submuco-equal proportions of S neurons had orally or anally di-

rected projections. However, almost all the S neurons that sal ICC) have also been investigated, using electrophysio-logical procedures by Huizinga et al. (439). Endogenouswere immunoreactive for calbindin or calretinin projected

orally. alkaline phosphatase activity is located in enteric neurons(Dogiel type 1, but not type II) of the guinea pig smallSang and Young (959) studied the presence and colo-

calization of a range of putative transmitter in myenteric intestine, and this activity is associated with NOS-con-taining neurons. The latter includes inhibitory motoneu-plexus of the small and large intestine of mouse. They

classified the two major classes of circular muscle moto- rons to the circular muscles and anally directed interneu-rons to other myenteric and submucous neurons. Pater-neurons in the small intestine: one class was characterized

by the presence of NOS, vasoactive intestinal polypeptide son and Indrakrishnan (891) investigated in opossumesophageal SMC whether or not the distension-induced(VIP), plus neuropeptide Y (NPY), and the second class



esophageal peristaltic reflex involves a polysynaptic path- there are a greater number of morphological and chemicalcategories. After studying neurons and their projectionsway. They concluded that this reflex is not polysynaptic,

but rather involves long descending neurons that depend in the proximal colon of the guinea pig, Messenger (734)reported that the myenteric and submucous plexuseson NO as a final mediator.

B) SUBMUCOSAL PLEXUS. In the submucous plexus of were not uniform around the enteric circumference. Atthe mesenteric aspect of the colon, there was almost nothe guinea pig small intestine, Bornstein et al. (114) have

classified neurons according to their synaptic inputs. They longitudinal muscle, and the circular muscle was unusu-ally thick and cordlike. In this region, there was no tertiarystudied 130 neurons and found that 82 cells were VIP

reactive, of which 23 cells were NPY sensitive. Electrical plexus of fibers, and the ganglia of the myenteric andsubmucous plexuses were elongated in the direction ofstimulation of internodal strands evoked EJP (lasting 20–

30 ms; fast response) in all 130 neurons. Most VIP-reactive the circular muscle. Neurons containing VIP, gastrin-re-leasing peptide, galanin, calbindin, or NADPH-diaphoraseneurons and some VIP-negative neurons, but no NPY-sen-

sitive neurons, exhibited inhibitory synaptic potentials all lay in anally projecting pathways within the myentericplexus, whereas enkephalin and somatostatin appeared(ISP; amplitude in the range 2–30 mV, and lasting 150–

1,500 ms). Most VIP-reactive neurons exhibited a slow in orally projecting nerve pathways. Few NPY neuronswere present within the myenteric plexus of the proximalexcitatory synaptic potential (ESP) that could be evoked

by a single stimulus, lasted 5–20 s, and was associated colon. The longitudinal muscle was innervated by fiberscontaining VIP, substance P, enkephalin, or NADPH di-with an increase in input resistance. In contrast, in some

NPY-reactive neurons, a single stimulus evoked an ESP aphorase. The circular muscle was innervated by axonseach containing one of the substances investigated, withlasting 500–1,500 ms with an associated fall in the input

resistance. Therefore, Bornstein et al. (114) concluded the whole list, except NPY, being represented. Galanin,NPY, somatostatin, and VIP fibers, all dense in the mu-that neurochemically distinct populations of submucous

neurons can be distinguished physiologically on the basis cosa, largely arose from nerve cell bodies in the submu-cous plexus. Messenger et al. (735) concluded that theof the particular combination of synaptic inputs they re-

ceive. chemically specified neural population in the proximalcolon is more similar to that of the distal colon than thatEvans et al. (281) also studied the guinea pig submu-

cous plexus and reported that five distinct groups of cells of the ileum, but that neurochemical and anatomic differ-ences exist between the populations of the proximal andcould be distinguished. These were as follows. 1) S cells

(neurons) with an inhibitory input (73 specimens Å 61%) distal colon. Furthermore, it has been reported that, inthe myenteric and submucous ganglia, the GABA fiberswere immunoreactive to VIP and showed a Dogiel type

III morphology; their varicosities and tufts of varicosities contained NOS activity (823).were observed surrounding other cell bodies as well asblood vessels. 2) S cells without an inhibitory input (19%), 2. Esophagusof which most were immunoreactive to NPY, also showedDogiel type III morphology but possessed a shorter axonal In the canine and human esophageal sphincter, Tsu-

mori et al. (1108a) found that VIP and NPY are synthesizedprojection. 3) AH cells (8%), which most likely containedsubstance P, lacked a synaptic input and exhibited Dogiel in the same neuron, stored in the same axon terminal,

and released together to act on sphincter SMC. Singaramtype II morphology; they branched more extensively thanthe S cells and also formed varicose tufts within other et al. (993) reported that, in the human esophagus, most

myenteric neurons (55%) were nitrergic. Most (96%) re-ganglia. 4) S-AH cells (5%), which combined the electro-physiological properties of the type of S cells with an ceived terminals containing VIP, calcitonin gene-related

peptide (CGRP) (80%), and galanin (56%). Furthermore,inhibitory input (i.e., type 1 above) with those of AH cells,did not show consistent morphological characteristics. 5) of neuronal somata, 14% contained VIP, whereas 10% con-

tained galanin. They postulated that NO is an inhibitoryGlial networks were typical and unusual networks. On thisbasis, they drew three conclusions: that VIP-containing S (I) nonadrenergic noncholinergic (NANC) mediator and

has a possible interactive role with the peptidergic system.cells may act as interneurons, mediating a slow ESP; thatNPY-containing S cells, which are known to be choliner- In the feline extrahepatic biliary tree, the distributions of

substance P, VIP, and cholecystokinin (CCK) have beengic, may play a role as cholinergic interneurons mediatingthe nicotinic fast ESP; and that AH neurons, too, may investigated by Dahlstrand (227) using immunohisto-

chemical procedures. Nerve terminals containing sub-provide cholinergic innervation to other submucosal neu-rons in addition to their dual projection into the mucosa stance P were distributed to the SM layer and to acetyl-

cholinesterase (AChE)-positive ganglion cells in the in-and the myenteric plexus.From these observations, it is clear that both plex- trinsic plexuses. Substance P was also located in cell

bodies of the intrinsic plexuses as well as in vagal axons,uses contain at least five different types of cells, on thebasis of their electrophysiological properties, but that and VIP had a similar distribution. Using opossum esopha-



gus, Christensen et al. (197) investigated distributions of of one varicosity per cell is not necessary because of thesyncytial structure of SMC. These varicosities in arteriesNADPH-diaphorase and concluded that, in the circular

muscle layer, NADPH-diaphorase-positive fibers were and veins are Ç2 mm long, 1 mm in diameter, and arespaced at 5- to 10-mm intervals. The terminal branches ofmost abundant at the cephalic end of esophageal body

with a significant decline toward and through the esopha- each individual fiber bear several hundred varicosities,and the number of varicosities varies with the size ofgogastric sphincter. In the longitudinal muscle layer and

the longitudinally oriented muscularis mucosae, these the vessel. Varicosities contain two types of dense-coredvesicles: small dense-cored vesicles (600–1,000 A) maynerve fibers were most abundant at the esophagogastric

sphincter, with a significant decline toward and through contain norepinephrine (NE) and ATP, and large dense-cored vesicles (1,200–1,500 A) may contain NE, ATP, andthe striated-smooth muscle junction. Constitutional nitric

oxide synthase (cNOS) immunoreactivity colocalized with neuropeptides, such as CGRP (241), NPY, or VIP (32, 260).Luff and McLachlan (674) studied the sympathetic neuro-NADPH-diaphrase activity, and CGRP-Li were distributed

like the NADPH-diaphorase-positive fibers. They further de- muscular innervation of arterioles (diameter 50 mm) inthe guinea pig ileum and found that 13% of the varicositiesscribed that fibers stained for immunoreactivity to the VIP,

galanin, and substance P showed no clear differences in made contact with SMC and that most of these (92 or82% in 2 preparations) formed junctions. Iontophoreticallydistribution along the esophagus in any of the muscle layer.applied NE and nerve stimulation produce much the same

3. Airwaydepolarization, in terms of shape. Stjarne (1036), who

In trachea of sheep, Cocoran (192) investigated distri- studied the release of neurotransmitters in the rat tailbutions of nerve fibers containing CGRP, VIP, NPY, sub- artery, estimated that the average varicosity containedstance P, and the catecholamine (CA)/enzyme maker do- 25 large dense-cored vesicles and 500 small dense-coredpamine b-hydroxylase (DBH) and concluded that moder- vesicles whose contents are released by exocytosis. Inate to large numbers of CGRP-Li nerve fibers were present the former case, release was from ‘‘random sites,’’ and inin all parts of the trachea. Substance P nerve fibers had the latter from a ‘‘preferred release site’’ on the varicositya similar distribution to CGRP, but they were absent from membrane (1037). Stjarne et al. (1037) noted that theepithelial cells and only small numbers of fibers were nerve impulse is propagated along to the terminal at 0.5present in other areas. Moderate numbers of VIP-Li and m/s, invades all the varicosites, and activates the voltage-NPY-Li were present in SMC. Large numbers of DBH-Li dependent N-type Ca2/ channel. Within the terminal, itnerve fibers were present in the SMC, and they had a travels only at Ç0.5 mm/s, and release of transmitter maysimilar distribution to NPY. The presence of both NPY require Ca2/ with a permissive factor, ‘‘x.’’ The ATP re-and DBH in most DBH-Li nerve fibers was established in leased in this way acts locally and probably only on post-the SMC and dense interconnecting network. In plexus of junctional SMC, whereas the released NE acts both onthe ferret trachea, Dey et al. (245) studied neuroanatomic postjunctional SMC and on the prejunctional nerve termi-features and concluded that 1) most cholinergic nerve nal (uptake of NE and activations of a2-adrenoceptorsdo not contain VIP, NOS, or substance P; 2) cholinergic occurs at nerve terminals). Recently, Stjarne and Stjarneneurons are predominantly located in the longitudinal (1038) reviewed the geometry, kinetics, and plasticity oftrunk ganglion; 3) VIP, NOS, and substance P are predomi- release and clearance of ATP and NE, as well as theirnantly located in the superficial muscular plexus ganglia; roles in neurogenic contraction in the vas deferens of theand 4) nerve terminals containing exclusively substance guinea pig and mouse and in the rat tail artery. They againP, suggesting possible sensory origin, are closely associ- emphasized their hypothesis (‘‘the string model’’) aboutated with some neurons in the plexus. In human airway, the quantal release of ATP and NE: 1) most sites ignoreFischer and Hoffman (296) investigated NOS-containing the nerve impulse and only a few (õ1%) release a singlefibers and concluded that the innervation density of air- quantum of ATP and NE, 2) the probability of monoquan-way SMC by NOS-containing nerve fibers decreased sig- tal release is extremely nonuniform, but 3) there is a highnificantly from trachea to large-diameter bronchi to small- probability from sites on ‘‘active strings,’’ and 4) an im-diameter bronchi, whereas NOS-containing nerve fibers pulse train causes repeated quantal release from thesewere completely absent from bronchioli. Colocalization sites. Concerning exocytosis, they proposed that the coin-of NOS with VIP but not with substance P was frequent cident presence of at least two factors, Ca2/ and specificin these nerve fibers. cytosolic proteins, may be required to move a ‘‘fusion

clamp,’’ form a ‘‘fusion complex,’’ and trigger exocytosisB. Features of Varicosities Deduced Fromof a sympathetic transmitter quantum and that the avail-Electrophysiological Investigationsability of these proteins may regulate the probability of

1. Features of varicosities release.The morphology and electrophysiology of the vari-The nerves innervating all VSMC carry many varicosi-

ties on their peripheral portions; however, a distribution cosities distributed in mouse vas deferens have been ele-



gantly investigated by Bennett’s group (77, 546, 646–648). cose segments vary in length and diameter, with the nar-rowest ones accompanying the more clear-cut varicosi-Using the fluorescent dye 3,3-diethyloxardicarbocyanine

iodide [DiOC2(5)] or Faglu fluorescence (catecholamine ties. Some intervaricose segments are as small as 50 nmin diameter. Intracellular recordings were made from in-staining), they visualized varicosities and catecholamine

distribution while simultaneously recording excitatory tramural neurons in the urinary bladder of guinea pig(383). From these procedures, the firing patterns werejunctional currents (EJC). For this they used a small mi-

croelectrode (4–6 mm; placed over 1–3 varicosities) and classified into two groups: prolonged firing of action po-tentials (tonic type; 86%) and one to three action poten-a large-diameter microelectrode (20–50 mm; placed over

3–7 varicosities), each placed on the same nerve fiber. tials (phasic type) by depolarization of nerves. Single ac-tion potentials were followed by fast hyperpolarization,The size of the varicosities and the intervaricosity dis-

tance obtained using DiOC2(5) were 1.09 and 5.53 mm, and the repetitive firing of action potentials was followedby delayed, slow hyperpolarization, which were dimin-respectively. With the use of Faglu fluorescence, corre-

sponding values were 1.05 and 5.12 mm, respectively. They ished by 4-AP and Ca2/-free and high-Mg2/ solution. Elec-trical stimulation of nerve fiber tract evoked fast EJP.further found that the mean and variance of the evoked

EJC was similar to that of the spontaneous EJC, sug- Presumably, two types of excitatory neurons may contrib-ute in this tissue.gesting that a given varicosity secreted at most one quan-

tum of transmitter(s) on arrival of a nerve impulse. Fur- Because the features of synaptic vesicles incorpo-rated in nerve terminals have already been extensivelythermore, they found that the mean quantal content of

the EJC declined by over threefold along the length of a reviewed (52, 157, 561, 920, 965), here we do not intendto describe the features of synaptic vesicle biogenesis,single sympathetic nerve terminal between adjacent sets

of varicosities. Moreover, they described close-contact docking, fusion, and exocytosis in relation to biochemicalprocesses.varicosities (Ç50 nm from the muscle) and loose-contact

varicosities (at a greater distance). They interpreted thesefindings in relation to the generation and shape of EJP; 2. Presence of cotransmitters in nerves distributedthus 1) an EJP may result from transmitter release from on VSMCclose-contact (formed intermittent fast component) orloose-contact (formed nonintermittent slow component) Until about 30 years ago, it was thought that most

VSMC are innervated by ACh-containing (cholinergic)varicosities, and 2) each of the varicosities could secretea quantum of transmitter with a particular probability, and/or NE-containing (adrenergic) nerves. After pioneer-

ing investigations carried out by Burnstock (142, 148, 149),after a delay that is characteristic for that varicosity (77).Klemm (585) reported on the ultrastructure of the it became apparent that cotransmitters were also released

from these nerves. The physiological roles of cotransmit-projections to the longitudinal SMC of the guinea pig il-eum from nerves of the teriary plexus. It was found that ters in adrenergic, cholinergic, I-NANC, and excitatory

(E)-NANC nerves have been extensively investigated bythere were two different types of neuromuscular junction;two-thirds of the junctions had many vesicles aggregated many workers. For example, in vascular tissues and vasa

deferentia, NE and ATP are released together. However,toward the area of junctional contact. Some 20% of thesejunctions had prejunctional membrane specializations. for the generation of an EJP after peripheral adrenergic

nerve stimulation, ATP, rather than NE, seems to be theThe remaining junctions were smaller than others usuallyfound and covered a small area of membrane and con- main transmitter in these nerves and, thus ‘‘NANC pu-

rinergic nerve’’ would seem a more suitable name fortained only a few small vesicles; prejunctional membranespecializations were not found on these junction. Thus it them. On the other hand, for the mechanical response,

NE seems to play the major role, and not ATP. Thus, onwas estimated from these observations and other physio-logical experiments that nearly released transmitters acti- that basis, they should be termed ‘‘adrenergic nerves.’’ In

contrast, in the GI tract, ATP seems to be the I-NANCvate a different subset of receptors to externally appliedtransmitter substances. transmitter in the guinea pig taenia coli (137) and jejunum

(50, 51, 385), and this substance may or may not be in-Gabella (330) studied on the structural relations be-tween nerve fibers and SMC in the urinary bladder of the volved as a cotransmitter with NE. In the GI tract, a single

field stimulus evoked an apamin-sensitive inhibitory junc-rat, i.e., upon penetrating into the musculature, the nervebundles branch repeatedly, and almost all turn into single tion potential (IJP), whereas NE overflowing from the

myenteric plexus on repetitive field stimulation may gen-fibers; their axons become varicose, and the separationbetween axonal membrane and SMC membrane is re- erate a slow hyperpolarization of the SMC membranes

through b-adrenoceptor stimulation (50, 51, 155). In SMCduced to tens of nanometers. Varocosities contain mostlyof the agranular type of vesicles. Terminal varicosities are of the guinea pig ileum, Bauer and Kuriyama (50, 51) con-

cluded, on the basis of electrophysiological and pharma-often devoid of Schwann cell sheath and lie closes to amuscle cell (gap is often reduced to Ç10 nm). Intervari- cological procedures, that E-NANC fibers are more



densely distributed in the terminal than in the proximal corded from the GI tract (for review, see Refs. 142, 143,145, 416, 615). For example, from guinea pig, rat, andregion, whereas in the case of I-NANC fibers, the situation

is the reverse. Circular muscle cells are, however, homo- rabbit mesenteric arteries, and from rabbit ear and rat tailarteries, EJP and spontaneously generated miniature EJPgeneously innervated by I-NANC nerves.

In SMC of the guinea pig urinary bladder, Hashitani (sometimes depolarization exceeded 15 mV) were re-corded. A mutual relationship between spontaneouslyand Suzuki (389) reported that nerve stimulation releases

ACh and VIP, and the former produces contraction with- generated EJP and evoked EJP has been inferred fromstudies of the properties of varicosities. Compared without changes in the membrane potential, whereas the latter

generates the EJP that triggers an action potential and that seen in the rabbit mesenteric artery, the EJP evokedby perivascular nerve stimulaton in the rabbit mesentericthus elicits contraction.

In the airway SMC of many species, a dual adrenergic vein exhibited a slower rising phase; however, in theguinea pig mesenteric vein, repetitive stimulation evokedand cholinergic innervation in many species has been re-

ported, but the distribution of adrenergic nerves is not only a slow fused depolarization (1057). The slow natureof the responses in the latter two cases may be explainedconsistent. For example, adrenergic nerves are found in

all parts of the canine airway system (886) but not in by a lack of, or a low-density distribution of, P2x receptorsor by the presence of larger numbers of loose-contacthuman bronchi (893). Recently, the densest form of in-

nervation, cholinergic excitatory fibers, has been found varicosities than of close-contact varicosities at peripheralnerve terminals (77). In the canine basilar artery, perivas-to contain not only ACh but also I-NANC substances such

as VIP and NO. The presence of I-NANC nerves had been cular nerve stimulation evoked EJP with a prolongedsmooth hyperpolarization. Repetitive stimulation fusedpostulated in airway SMC from the pharmacological ef-

fects on either electrical or mechanical responses of ad- the hyperpolarizations, but within several seconds, thefused hyperpolarization depolarized went back to the rest-renergic and cholinergic receptor blockers (guinea pig,

Refs. 203, 930, 931; cat, Ref. 498; pig, Ref. 760; baboon, ing level because of a depression of the response. Bothtypes of potential changes were blocked by TTX, but theRef. 740). Recently, the heterogeneous inhibitory actions

of NO and VIP in cat central and peripheral airways were hyperpolarization was not blocked by apamin or by b-adrenoceptor blockers (308). In the spontaneously activedescribed by Takahashi et al. (1069). Ward et al. (1154)

observed the distribution of human I-NANC bronchodila- guinea pig portal vein, perivascular nerve stimulationfailed to evoke any depolarization of the membrane. Intor and NO-immunoreactive nerves and concluded that I-

NANC neural relaxation appears inhomogeneously in air- the rabbit ear artery (535), histograms plotting the genera-tion of spontaneous EJP (recorded by the microelectrodeway system because of a decrease in the density of ni-

trergic innervation in the peripheral region. These obser- method) show a skew distribution pattern in most cases,but in a few penetrations, a bell-shaped distribution pat-vations may indicate that neither the amount nor the sites

of transmitter release in the airway SM system exhibits a tern was observed (628). In mesenteric and tail arteries,and in the vas deferens, EJP show facilitation when thehomogeneous distribution.stimulus frequency exceeds 0.1 Hz, and when the depolar-ization reaches threshold, a spike potential is superim-

C. Multitransmitter Release Inferred From theposed on it. The tm of the falling phase of the EJP re-

Generation of EJP and IJP and Features ofcorded from the guinea pig mesenteric artery was Ç200

Postjunctional Receptorsms (at 357C), whereas that of the resting membrane wasÇ130 ms (and l was 0.8 mm in a longitudinal direction).

1. EJP and IJP recorded from VSMCTherefore, the decay of the EJP may not solely reflect apassive decay of the EJC. However, the decay of a sponta-Since 1959, pioneering electrophysiological investiga-

tions involving the recording of EJP and IJP from vasa neous (miniature) EJP may mostly depend on the passiveproperties of the membane (tm).deferentia, GI tract, and vascular tissues have been car-

ried out by Australian groups such as those of Burnstock In the urinary bladder of most experimental animals,and humans, Brading and Inoue (122) concluded thatand Holman (150) and now by Hirst’s group and Bennett’s

group. there is a dual purinergic (ATP) and cholinergic (ACh)excitatory innervation. The former depolarized SMC mem-Field or transmural peripheral nerve stimulation

evokes EJP (EJC) or IJP (IJC) in some VSMC, but not in branes and increased the spike frequency (EJP-forming),whereas in most species, ACh did not modify the mem-all. The clearest excitatory potential changes (depolariza-

tion), especially EJP, were recorded from resistance vas- brane potential, yet still produced contraction. However,in some species, a small depolarization was seen after thecular tissues, vas deferens, urinary bladder, or iris sphinc-

ter, as were the clearest inhibitory potential changes. The purinergic EJP (218). This response may reflect ATP-NEcotransmission in vascular SMC.EJP was first recorded by Burnstock and Holman (150)

from guinea pig vas deferens, and the IJP was first re- In the GI tract, Komori and Suzuki (602), who studied



the circular muscle of the guinea pig stomach, reported have been identified, whereas in the rat thoracic aorta, thea1A- (antagonist, SNAP-5089), a1B-, and a1D-adrenoceptorsthat in the presence of guanethidine, transmural nerve

stimulation evoked an atropine-sensitive EJP (reversal po- (antagonist, BMY-7378) have been found. In human vasdeferens, it was estimated that a1A-adrenoceptors contrib-tential of 018 mV) in the fundus region and an apamin-

sensitive IJP in the antrum region (reversal potential of ute for mechanical responses induced by adrenoceptoragonists (320). It is not yet clear, however, what role is089 mV). After additional application of atropine to the

bath, an IJP was evoked by transmural stimulation in both played by a1A- and a1D-adrenoceptors (132, 187, 788, 849).Concerning actions of NE on VOCC in vascular SMC,regions. After application of AChE inhibitors, such as phy-

sostigmine or neostigmine, cholinergic stimulation Benham and Tsien (76) reported that NE increase the L-type Ca2/ channel of the rat mesenteric artery, and thisevoked an enhanced EJP and a subsequently produced

depolarization of the membrane in the fundus region. In action was enhanced by GTP. It was also reported usingthe same tissue by Nelson et al. (810) that NE shiftedcontrast, in the antrum region, the amplitude of the slow

potential changes was further enhanced. Experiments the Ca2/ activating potential level (threshold) to morenegative potential level and enhanced the L-type Ca2/ cur-with exogenously applied ACh showed that the threshold

concentration for the depolarization of the membrane was rent. Loirand et al. (671) observed the effects of NE onthe rat portal vein that this agent enhanced the fast com-1,000 times higher in the antrum region (1 mM) than in

the fundus region (1 nM). Thus whether an EJP or IJP ponent of VOCC, and this action was further enhancedby guanosine 5*-O-(3-thiotriphosphate) (GTPgS). In SMCis generated in a given region could depend on its ACh

sensitivity. In SMC of the guinea pig stomach fundus, prepared from cultured rat aorta, Pacaud et al. (874)observed that NE enhanced the dihydropyridine (DHP)-transmural nerve stimulation would normally evoke a cho-

linergic EJP, but in the presence of atropine, it would insensitive (presumably T-type) Ca2/ channel, and NE in-hibited the DHP-sensitive Ca2/ channel via an increaseproduce a NANC-IJP. Suramin enhanced EJP amplitude

and inhibited the IJP with no significant effect on the in the cytosolic Ca2/. However, Droogmans et al. (252)reported that NE inhibited L-type Ca2/ current. Furthermembrane potential. Ohno and co-workers (839, 840) pos-

tulated a possible involvement of ATP in the generation investigations are required to clarify this controversialobservation.of the NANC-IJP.

A) CATECHOLAMINE RECEPTORS AND RELATED SUB- When discussing vascular SMC, Stjarne et al. (1038)concluded that released ATP generates an EJP, whereasSTANCES. The a1-receptor is subdivided into a1A , a1B , a1C ,

and a1D (although a1C Å a1D), and the presence has been NE generates a slow and small depolarization via the a2-adrenoceptor. In contrast, the a1-adrenoceptor does notclaimed of a1L and a1N . The a2-receptor is subdivided into

a2A , a2B , and a2C , and the b-adrenoceptor is further sub- appreciably change the membrane potential (627), as alsoconcluded by Nally and Muir (802), working on the rabbitclassified into b1-, b2-, and b3-adrenoceptors (273).

1) a-Adrenoceptors. When studying the actions of NE saphenous artery. Moreover, Cheung and Fujioka (193)found that in the rat saphenous vein, perivascular nervevia a1A- and a2A-adrenoceptors in rat portal vein myocytes,

Macrez-Lepretre et al. (680) tried to identify the type of stimulation produced an EJP and sustained depolariza-tion, and the latter, slow depolarization was due to activa-PLC involved. They found that an inhibitor of PL-PLC, U-

73122, inhibited the release of Ca2/ from the store sites tion of the a2-adrenoceptor, not the a1-adrenoceptor. Inthe rat tail artery, exogenously applied NE markedly depo-induced by activation of the a1A-adrenoceptor related

InsP3 production, whereas an inactive analog, U-73343, larized the membrane, and this depolarization wasblocked by the a2-adrenoceptor antagonist yohimbine, buthad no effect on the NE-induced release of Ca2/ from its

stores. In contrast, both U-73122 and U-73343 inhibited the not by the a1-adrenoceptor blocker prazosin.In the dilator muscle of the rat iris, Hill et al. (409)L-type Ca2/ channel. An inhibitor of phosphatidylcholine

(PC)-PLC, called D-609, had no direct inhibitory effect on reported that there are three different muscle cell types.In two of these, which were assumed to be myoepithelialthe L-type Ca2/ channel, but it inhibited the a2A-induced

stimulation of Ca2/ channels, which had already been cells, sympathetic nerve stimulation evoked EJP (with along latency of several seconds and duration of severalshown to be independent of phosphatidylinositol hydroly-

sis. They therefore postulated that the a2A-adrenoceptor seconds). These EJP were blocked by prazosin and wereinsensitive to yohimbine; furthermore, they were blockedactivates a PC-PLC in vascular myocytes. However, it

must be said that D-609 had other sites of action as it by chloroethylcholine. Thus the receptor involved wascharacterized as the a1B-adrenoceptor. Furthermore, EJPblocked both NE- and caffeine-induced Ca2/ release from

the stores. evoked by sympathetic nerve stimulation were blocked byreducing the external concentration of Cl0. Consequently,The presence of various a1-adrenoceptor subtypes

has been described in vascular SMC. However, in the rab- they concluded that EJP are generated in this tissue byactivation of Ca2/-dependent Cl0 channels through activa-bit thoracic aorta, only a1B-adrenoceptors (antagonist, AH

11110A) and a1L-adrenoceptors (antagonist, JTH-601) tion of the a1B-receptor and that Ca2/ released from the



SR and subsequently synthesized InsP3 are involved in the and concluded that cAMP and the a-subunit of the relatedG protein act independently, and also that channel open-generation of EJP and of contractions.

A mutual relationship between the hypogastric and ings they induced differed in terms of their kinetics.Therefore, they suggested that b-adrenoceptor stimula-pelvic nerves has been demonstrated, in as much as sym-

pathetic nerves may end on ganglion cells in the pelvic tion of maxi-K/ channel activity and relaxation of tone inthis tissue both occur, in part, independently of cAMPplexus and act inhibitory nerves (238). In response to

hypogastric nerve stimulation, the urinary bladder shows formation. Recently, Kotlikoff and Kamm (609) reviewedrecent data of b-adrenergic relaxation of airway SMC; b-slight relaxation through the actions of mainly the b2-

adrenoceptor, and in many species, the urethra shows adrenoceptor stimulation results in the opening of Ca2/-dependent maxi-K/ channels. Coupling between receptorbiphasic response (contraction and subsequent relax-

ation) evoked via a2- and b2-receptors, respectively. How- and channel occurs by phosphorylation-dependent and-independent mechanisms. Furthermore, b-adrenoceptorever, in the rabbit urethra, the a2-adrenoceptor was more

dominant (29). In the urinary bladder of dog, rabbit, and agonists can decrease the Ca2/ sensitivity of contractileproteins. This desensitization does not result from theguinea pig, and after treatment with a1- and a2-adrenocep-

tor blockers, pelvic nerve stimulation produced a large phosphorylation of myosin light-chain kinase (MLCK) butmay be associated with the activation of a MLCK phospha-EJP, presumably because of the release of ATP.

2) b-Adrenoceptors. The presence of the b-adreno- tase.B) MUSCARINIC RECEPTORS AND RELATED SUBSTANCES.ceptor in VSMC was described many years ago; activation

of this receptor was found to hyperpolarize SMC mem- Muscarinic receptors sensitive to ACh are subdivided intofive subtypes (M1-M5) (43). For example, the guinea pigbranes, probably via the synthesis of cAMP in the guinea

pig vas deferens (138, 622). Moreover, Kuriyama and Ma- airway contains M2 and M3 receptors, whereas, in humans,the receptor subtype is M3 and the presumed subtype iskita (627) reported that isoproterenol (Isop) increased the

amplitude of EJP through an action at a prejunctional M3 in the bovine airway (445, 664, 688). However, in bothhuman and porcine airway SMC, use of a cDNA probereceptor. Thus the b-adrenoceptor may have both a pre-

and postjunctional distribution. In urogenital organs, Isop has revealed the presence of the mRNAs for both M2 andM3 receptors (688, 689). Activation of the M receptorsand terbutamine each blocked the response evoked by

nerve field stimulation, and these inhibitory responses expressed by m1, m3, and m5 genes stimulated phosphati-dylinositol turnover, whereas activation of those ex-were reserved by the application of sotalol. In the GI tract,

b-adrenoceptors have been identified in the guinea pig pressed by m2 and m4 gene decreased cAMP synthesis. Incardiac muscle, a K/ current was modified by a receptorileum (365), and this receptor is thought to be the b3-

adrenoceptor (1118). In airway SMC, there is a sparse expressed by the m2 gene and acting via by protein kinaseA (89). Thus m1 and m3 genes were associated with activa-innervation by sympathetic (adrenergic) nerves, and a-

adrenoceptors are more sparsely distributed than b-adre- tion of Gq/11 (synthesis of InsP3), and m2 and m4 genescorresponded with activations of Gi/o (inhibition of synthe-noceptors (44, 45). Consequently, the contraction induced

by a-agonists was weak, being only 10–20% of the maxi- sis of cAMP, direct activations of K/ channels, and otheractions) (111, 131, 250, 446). In addition, the presencemum response elicited by ACh or histamine (590). Both

a1- and a2-adrenoceptors appear to be distributed in air- has been deduced of the M1 receptor in parasympatheticganglia and of the M2 receptor on parasympathetic nerveway SMC, but with different densities (422). The canine

trachealis, however, seems to be unusual in that the a2- endings (688).Selective antagonists for muscarinic receptors havereceptor is apparently more densely distributed than the

a1-adrenoceptor (44). The distributions of b-adrenocep- been introduced. These include the following: for the M1

receptor, pirenzepine and telenzepine; for the M2 receptor,tors in airway SMC have been elucidated in several spe-cies, and the receptor most responsible for airway dilation methoctramine and himbacine; for M3, hexahydrosiladi-

fenidol and p-hurohexahydrosiladifenidol; and for M4,is the b2-adrenoceptor. However, the terminal airways andalveoli contain a mixture of b2- and b1-receptors (355). tropicanide. A selective antagonist has not yet been intro-

duced for the M5 receptor.Carstairs et al. (167) reported that the density of b2-adre-noceptors in airway SMC increases progressively from the Acetylcholine, in many VSMC, is known to act as

an excitatory transmitter. However, in the lingual artery,trachea to the small bronchioles. It was reported in airwaySMC that PKC markedly enhanced the relaxation induced Bevan and Brayden (81) reported that peripheral nerve

stimulation induced relaxation and hyperpolarization ofby b-adrenoceptor activation. On the other hand, vagal(cholinergic) nerves densely innervate airway SMC (39– the membrane and that these inhibitory responses were

blocked by atropine. This phenomenon indicates that cho-44, 930). In tracheal SMC, Ca2/-dependent maxi K/ activ-ity was induced by cAMP synthesized in response to epi- linergic inhibitory nerves may be distributed in some vas-

cular tissues.nephrine or forskolin (616–618). Kume et al. (617) furtherinvestigated the action of cAMP on the maxi-K/ channel 1) Urogenital organs. Cholinergic innervations have



been identified in the vasa deferentia of various species. bromide (4-DAMP) but facilitated by methoctramine. Incontrast, IJP were blocked by methoctamine but unaf-Fukushi and Wakui (311–313) and Wakui and Inomata

(1143, 1144), who studied the guinea pig vas deferens, fected by 4-DAMP. These results indicate the involvementof two different receptors. The former action may be me-reported that during muscarinic blockade, high concentra-

tions of nicotinic agonists produced a depolarization. diated by activation of the M3 receptor and the latter bythe M2 receptor. In guinea pig ileum and trachea, interac-However, the interpretation of this result is not simple

because it is known that many small ganglionic cells in tion between M2 (inhibition of adenylate cyclase) and b-adrenoceptor (stimulation of adenylate cyclase) has beenperipheral hypogastric nerves are distributed in this tis-

sue. To establish the presence of the nicotinic receptor estimated. In guinea pig esophageal SMC, lack of interac-tion between M2 receptors and b-adrenoceptors but anin SMC in vasa deferentia would require more detailed

experiments. It is clear that ACh in vasa deferentia acti- interaction between M3 receptor and b-adrenoceptor hasbeen suggested (1164).vates the postjunctional M2 receptor and produces excit-

atory effects, but this action on SMC is weak by compari- 4) Iris. Dual innervation by adrenergic and choliner-gic nerves occurs in SMC and is examplified by the caseson with those of NE and ATP released from the adrener-

gic innervation. On the other hand, ACh also activates the of the iris sphincter and dilator muscles. The mammalianiris dilator seems to be innervated by excitatory adrener-M1 receptor distributed on adrenergic prejunctional nerve

terminals and negatively controls the release of NE (301). gic nerves, but investigators have revealed that this tissuealso receives a cholinergic inhibitory innervation in catsBrading and Mostwin (123) recorded the electrical

and mechanical responses of guinea pig urinary bladder (266), rats (813), and cattle (1063). In human iris dilator,Yoshitomi et al. (1215), confirming that this tissue is inner-SMC to nerve stimulation and observed that the mechani-

cal response to excitatory nerves is biphasic. The early vated by adrenergic excitatory and cholinergic inhibitorynerves, further suggested that the cholinergic inhibitoryresponse to the transmitter, which takes the form of EJP

and an evoked spike (E-NANC), is attenuated by the de- innervation may support cholinergic meiosis, as reportedin cattle and rats (803, 1063). Apparently, in the monkey,sensitization of purinoceptors. The late response is medi-

ated through muscarinic receptors, involves little mem- iris dilator muscle receives 75% adrenergic and 25% cho-linergic nerve terminals (829). Furthermore, in the bovinebrane depolarization, and is unaffected by desensitization

of purinoceptors. iris dilator muscle, excess extracellular K/ produced abiphasic response that was blocked by simultaneous treat-2) Airway. In the airways, by means of an autoradio-

graphic procedure (using [3H]QNB), the presence has ment with phentolamine and atropine (when phentol-amine was applied alone, it produced a slight relaxation).been demonstrated of M2 and M3 receptors in the guinea

pig, and of the M3 receptor in humans (687). However, Thus the contraction induced by extracellular K/ is dueto joint activations of adrenergic and cholinergic nervesusing a molecular biological pocedure, Maeda et al. (681)

and Mak and co-workers (688, 689) have reported that the and is not due to a direct action on SMC through anactivation of VOCC (1162, 1163). Furthermore, YoshitomimRNA for both M2 and M3 receptors are present in porcine

and human airway SMC. Furthermore, with the use of and Ito (1216) reported a double reciprocal innervationin dog iris sphincter and dilator muscles. Thus, in bothpharmacological procedures, involving antagonists of M2

(AF-DX 116, methoctramine) and M3 (hexahydrosiladifen- sphincter and dilator muscles, nerve stimulation evokedan initial phasic contraction followed by relaxation. Atro-oidol) receptors, it was concluded that the M3 receptor

possesses a more potent action than the M2 receptor on pine selectively suppressed the phasic contraction of thesphincter and the relaxation of the dilator muscle,human, guinea pig, and bovine airway SMC (936, 937,

1087). It is thought in the rabbit airway SMC that M3 recep- whereas guanethidine selectively blocked the relaxationof the sphincter and the contraction of the dilator. In thetors have been associated with SM contraction and M2

receptors have been implicated in Gi-coupled inhibition rat iris sphincter, after treatment with phentolamine, aninitial contraction followed by a relaxation was evoked byof adenylate cyclase. Schramm et al. (970) reported that

there exist inherent age-dependent differences in both the transmural nerve stimulation or ACh (1197). Furthermore,Watanabe’s group (703) concluded that only relaxation ofairway relaxant responsiveness to b-adrenoceptor stimu-

lation and muscarinic functional antagonism of b-adrener- the dilator muscle is related to activation of the pertussistoxin (PTX)-sensitive G protein (Gia) and that contractiongic relaxation, and the latter is attributed to mechanisms

other than ontogenetic alteration in M2 receptor function in the dilator muscle may be induced via M3 or M3-likereceptor activation, as also obtained by Yamahara et al.or Gi protein expression in maturing rabbit tracheal SMC.

3) GI tract. The role of muscarinic receptors in the (1197). The contraction induced by ACh may be mediatedby PTX-insensitive G protein.parasympathetic control of colonic activity in the rabbit

was investigated in vitro by Blanquet et al. (92), who found In iris sphincter and dilator muscles, at least fourdifferent M receptors seem to be distributed (M1-M4). Thethat EJP evoked by parasympathetic (vagal) nerves were

blocked by 4-diphenyl-acetoxy-N-methylpiperidine metho- M2 receptor plays a role, on prejunctional cholinergic



nerves, in the autoregulation of transmitter release (103, Wagner and Sjostrand (1139) reported that excitatory re-sponses in human and dog vasa deferentia (in which the104), whereas the M3 receptor predominantly mediates

contraction through activation of PLC, leading to the syn- innervations are rather sparse in comparison with thatof rodents) are blocked by a-adrenergic blocking agents.thesis of InsP3 and diacylglycerol (DAG). In the bovine

iris sphincter, the mRNA encoding the M3 receptor is the Moreover, after denervation, the response induced by hy-pogastric nerve stimulation in the guinea pig vas deferenspredominant form found (429). Direct evidence in support

of the idea that the M5 receptor subtype mRNA and the was blocked by phentolamine. Cheung (191) studied theguinea pig vas deferens to try to elucidate the role ofcorresponding M receptor subtype has not been found in

the iris sphincter, but Bognar et al. (103) reported that a the nerve action potential in synaptic transmission; theyconcluded that transmitter release is intimately related toreceptor other than the M1-M4 receptors contributes to the

mediation of the contraction in the rabbit iris sphincter. presynaptic nerve activity.During excitatory transmission in sympatheticAbdel-Latif (2) recently reviewed cross talk between

cAMP and the polyphosphoinositide on signaling cascade nerves, the cotransmitters ATP and NE may act coopera-tively under physiological conditions. In the isolated vasin iris sphincter. Namely, cholinergically synthesized InsP3

and adrenergically synthesized cAMP showed a mutual deferens of rat, rabbit, and guinea pig, Sneddon (1005)and Sneddon and Machaly (1007) reported regional varia-interaction through metabolic path; cAMP inhibits PLC

activation and stimulates InsP3 3-kinase activity, both of tion with respect to purinergic and adrenergic responses:1) NE was more potent in producing contraction in epidid-which can result in 1) reduction in InsP3 concentration

and 2) reduction in InsP3-dependent Ca2/ mobilization, ymal segments than in prostatic segments, whereas 2)ATP and a,b-methylene ATP were more potent in produc-which may lead to muscle relaxation. In addition to InsP3-

induced Ca2/ mobilization, changes in [Ca2/]i are the re- ing contraction of prostatic segments than epididymal seg-ments. Furthermore, they noted that in guinea pig vassult of the interplay of many processes that may also serve

as potential sites for cAMP inhibition. deferens, the resting membrane potential was greater inSMC in the prostatic than in the epididymal region. Excit-C) PURINOCEPTORS AND THEIR RELATED SUBSTANCES. In

VSMC, receptors sensitive to purine derivatives, purino- atory junction potentials in both regions were of similaramplitude and were almost abolished by the P2x purino-ceptors, have been classified as either PI (P1) or PII (P2)

receptors. The former, P1 , has been subdivided into A1, ceptor antagonist suramin, but phentolamine had no ef-fect. Furthermore, distributions of the SR (i.e., Ca2/ ho-A2A, and A2B, and the latter, P2 , has been subdivided into

P2t , P2x , P2z (these 3 types are ionotropic receptors), P2u , meostasis) differed by regions in vas deferens (1128).McLaren et al. (720) measured the membrane potentialand P2y (these 2 types are G protein-coupled types). Brake

et al. (125) claimed that the sequences of their cloned P2x and the contraction elicited in guinea pig vas deferens inthe presence of the P2x purinergic antagonist pyridoxal-receptor predicted a subunit structure (P2x1-P2x3) resem-

bling that of voltage-insensitive cation channels. phosphate-6-azophenyl-2*,4*-disulfonic acid (PPADS).This agent produced a small potentiation of the phasic1) Urogenital organs. The innervation of the vas def-

erens in many species (guinea pig, rat, mouse, rabbit, dog, contraction induced by field stimulation (at 0.1 mM; sug-gesting a predominantly purinergic excitatory compo-and human) has been investigated in detail using histo-

chemical, electron microscopic, and pharmacological pro- nent), but high concentrations (3–10 mM) produced inhi-bition. Furthermore, PPADS inhibited contractionscedures, leading to prediction about the distribution of

adrenergic and cholinergic terminals and receptors. There evoked by a,b-methylene ATP. On the other hand, thisagent had no effect on the tonic component of contractionis a relatively dense innervation by adrenergic nerves in

vasa deferentia, with the a1-adrenoceptor being located (predominantly noradrenergic component). At 10 mM,PPADS reduced the amplitude of EJP and depolarized thepostjunctionally on SMC and the a2-adrenoceptor being

predominantly prejunctional on nerve terminals (212). membrane. However, the PPADS-induced depolarizationwas not modified by suramin. This implies that ATP andThe former produces excitatory responses, and the latter

produces inhibitory responses through inhibition of NE NE released from nerve terminals may play different rolesin excitatory transmission. With regard to the action ofrelease. However, EJP and mechanical responses evoked

by hypogastric nerves are not completely blocked by a- PPADS, Piper and Hollingsworth (899) reported that thisagent antagonized the action of ATP in the isolated guineaadrenoceptor blockers, and the presence of a cotransmit-

ter, ATP, was deduced. Burnstock (146, 147) hypothesized pig vas deferens but had no effect on responses to ATPin the guinea pig taenia coli, indicating that this antagonistthat ATP is stored with NE in the small-cored vesicles

seen in adrenergic nerves, and also together with ACh in induced selective for P2x over P2y purinoceptors.It has been reported in rabbit prostate SMC (972)the small dense-agranulated vesicles seen in cholinergic

nerves. This means that no purinergic fibers distributed that this muscle tissue contains two muscle bundles: oneforms the capsule and the other runs longitudinally in theto the vasa deferentia and that the role played by this

substance is a cotransmitter (1037, 1038). However, outermost layer of the prostate. The former was con-



tracted by exogenously applied NE and the latter by ACh. drugs, they concluded that ATP releases Ca2/ from theintracellular stores without involving InsP3, but via a Ca2/-In fact, the NE-sensitive muscle receives an adrenergic

excitatory and a NANC inhibitory innervation, whereas release mechanism activated by Ca2/ influx through anATP-gated channel (see sect. V).the ACh-sensitive muscle receives a cholinergic excitatory

and a NANC inhibitory innervation. In the rat vas deferens, In SMC of the rabbit facial vein, perivascular nervestimulation produced a biphasic response: a depolariza-Kurz et al. (634) reported that electrical- and agonist-

evoked ATP overflow correlated well with the contractile tion followed by a hyperpolarization. This hyperpolariza-tion was blocked by a b-adrenoceptor blocker, implyingresponses.

In the urinary bladder, the idea that ATP is an excit- that NE activates a b-adrenoceptor distributed postjunc-tionally. In the rabbit ear artery, ATP activated the P2xatory cotransmitter (142) has been supported electrophys-

iologically (158, 209, 305). It is possibly released with ACh receptor and evoked a transient inward current (69, 72,75), whereas in the portal vein, ATP activated P2x and P2yfrom the parasympathetic (pelvic) nerves (1084). Further-

more, pelvic nerves, which innervate the lower urinary receptors (1184) and evoked a biphasic response. Withthe use of the whole cell voltage-clamp procedure, it hastract, contain many neuropeptides, such as NPY, VIP, so-

matostatin, substance P, and CGRP. However, none may been found that activation of the P2x receptor by ATPproduces a transient phasic inward current mediated viabe a primary neurotransmitter in the detrusor; rather, they

may act as potent modulators of sympathetic and para- cation channels (mainly Na/ and Ca2/), whereas activa-tion of the P2y receptor evokes a sustained tonic inwardsympathetic transmission (432).

2) Vascular tissues. There is considerable variation current that is mediated via nonselective cation channel.3) GI tract. Crist et al. (221–223) studied guinea pigbetween tissues in terms of the receptors they carry. For

example, the rabbit saphenous artery has been found to ileum and opossum esophageal circular muscles and re-ported that the electrical events elicited by nerve stimula-have approximately equal contributions from P2x purino-

ceptors and a1-adrenoceptors, whereas the ileocolic ar- tion could be classified as one of four types: early EJP orIJP and late EJP or IJP. The late IJP is recorded only attery has mainly P2x purinoceptors with a smaller contribu-

tion from a1-adrenoceptors, the plantaris vein has mainly sites anal to the stimulus, and the late EJP is recordedonly at sites oral to the stimulus. A slow NANC-IJP wasa2-adrenoceptors with a small contribution from P2x puri-

noceptors and a1-adrenoceptors, and the saphenous vein thought to be generated by a decrease in the Cl0 conduc-tance (in the esophagus). Furthermore, in guinea pig ileumhas only a1-adrenoceptors (626, 679). In the human saphe-

nous vein, Rump and Kugelgen (945) reported that a1- and circular muscle, Crist et al. (224) reported that both ATPand VIP are inhibitory transmitters and that they may bea2-adrenoceptors and ATP all contribute to the generation

of vasoconstriction. In contrast, Evans and Suprenant the transmitters responsible for the fast IJP and the slowIJP, respectively. Bridgewater et al. (128) reported in the(283), studying guinea pig submucosal arterioles, thought

that postjunctional responses to sympathetic nerve stimu- guinea pig taenia coli that reactive blue 2, a purinoceptorantagonist, attenuated the fast IJP, suggesting that thelation were mediated solely through the action on the P2x

receptor of ATP. In fact, Starke et al. (1028), reviewing NANC inhibitory transmitter responsible for the fast IJPis an ATP. Using circular SMC of the guinea pig ileum,the contribution of ATP to neurogenic vasoconstriction,

concluded that it varies from 0 (in the rabbit pulmonary He and Goyal (391) found that field stimulation induceda fast IJP that was blocked by TTX, apamin, and a,b-artery) to 100% (in the rabbit small jejunal artery) (915).

The picture is further complicated by the assertion by methylene ATP and, after application of apamin and sub-stance P, a slow IJP that was blocked by TTX and NG-Maynard et al. (712) that chronic electrical stimulation of

the greater auricular nerve supplying the rabbit central nitro-L-arginine (L-NNA). Consequently, they concludedthat NO is not involved in the fast IJP (which was medi-ear artery may lead to a selective alteration in the post-

junctional P2x purinoceptor, whereas the effects mediated ated by ATP). However, NO is involved in the slow IJP,which is actually mediated by VIP and NO acting in series.by postjunctional a1-adrenoceptors remain unchanged.

These accumulated results from work on vascular SMC The hyperpolarizing effects of VIP and the slow IJP areboth normally masked by an overlapping depolarizationindicate that the relative contribution made by the recep-

tors for ATP (P2x receptor) and NE (a1- and a2-adrenocep- due to the concomitant release of substance P inducedby the peptide VIP. In rat anococcygeus muscles, Byrnetors), with respect to the production of excitation and

contraction, varies by tissue and also by species (as re- and Large (151) reported that field stimulation evokedthree types of TTX-sensitive electrical responses, a fastported by Starke et al., Ref. 1028). Recent work in the rat

portal vein by Pacaud et al. (876) has shown that ATP, in EJP (latency õ100 ms and time to peak of 300 ms), aslow EJP (latency several hundred milliseconds and timethe presence of gallopamil and at a holding potential of

060 mV, induces an inward current and increase [Ca2/]i to peak 1–2 s), and an IJP. Iontophoretically applied ATPproduced a fast EJP-type response (latency õ30 ms and(as measured using indo 1). On the basis of results ob-

tained in various ionic environments and with various time to peak 150–300 ms), and ionotophoretically applied



NE produced a slow EJP-type response (latency 470 ms induced relaxation is due to the synthesis of NO. In thecanine jejunum, Stark and co-workers (1025, 1026) alsoand time to peak 860 ms). These responses are much the

same as those observed in vascular tissues (626). concluded that NO mediates NANC neural inhibition andmay act as a I-NANC transmitter. However, Stark et al.(1025) found, in the human and canine jejunum (circular2. Heterogeneous distributions of receptors, especiallymuscle), that nerve stimulation evoked a rapidly devel-on nitrergic receptors, estimated from IJP andoped IJP followed by a late sustained hyperpolarization.hyperpolarizationExogenously applied NO mimicked only the late responsein the case of the human jejunum, whereupon nerve stimu-In recent years, NO has come to be a popular candi-

date for the role of inhibitory substance in VSMC such as lation evoked an IJP that consisted of a fast hyperpolariza-tion, alone. When the effect of NOS inhibitors (L-argininethose of the GI tract, including the anococcygeus muscle,

and those in vascular beds. It is also thought to act in the derivatives) on these hyperpolarizations was tested, theywere found to reduce the amplitude of the initial IJP insame way in airway and in other VSMC.

A) GI TRACT. After studying the NANC-IJP in the GI the canine jejunum but to reduce only the late sustainedhyperpolarization in the human jejunum. Thus these au-tract, Sanders’s and Boeckxstaens’s groups (98–102) ex-

tensively reviewed its features in relation to NO and con- thors concluded that NO mediates neural inhibition in thecircular muscle of both the human and canine jejunums,cluded that many of the criteria necessary for NO to be

considered a neurotransmitter had been satisfied (956). but through different mechanisms. Serio et al. (975)thought that, in circular muscle of the rat proximal colon,In the canine proximal colon, Thornbury et al. (1091) dem-

onstrated that NO and the NO carrier S-nitrosocysteine while NO plays an important role in NANC-IJP generation,another mechanism, peptidergic in nature, is also in-can mimic the hyperpolarization induced by nerve stimu-

lation. Daziel et al. (236), using the same preparation, volved. Presences of nitrergic inhibitory nerves in dogsphincter of Oddi have been elucidated (1077).suggested that the NO synthetic processes is involved in

I-NANC neurotransmission and that the NANC-IJP is gen- In the GI tract, NO may contribute as a relaxing sub-stance, but distributions of nitrergic nerves and NO varyerated by released NO. Ward and Sanders (1158, 1159)

found that S-nitrosocysteine breaks down fast enough to markedly by species, strains, and regions, and also aging.For example, NO hyperpolarized the membrane in the dogcause an IJP-like hyperpolarization and that oxyhemoglo-

bin (oxy-Hb) blocks the hyperpolarization response to proximal colon (236, 980, 1025, 1092) but not in the ratproximal colon (1055) and guinea pig gastric fundus (685).NO, S-nitrosocysteine, and the NANC-IJP. They further

noted that NO enhanced the open probability of Ca2/- Nitric oxide increased the synthesis of cGMP in manyregion of GI tract and hyperpolarized and relaxed tissues,activated K/ channels, which produce hyperpolarization

in response to NANC neurotransmission in colonic mus- but in some cases, increased cGMP did not correlate withrelaxation (1075). Using JCL Wistar (Wistar), SLC Wistar-cle. In this tissue, their group also reported that the effects

of NANC nerve stimulation and NO may be mediated by ST (Wistar-ST), and Sprague-Dawley (SD) lines of rats (8wk old), Hata and Takeuchi (390) compared differences incGMP. In the canine proximal colon (between the pyloric

sphincter and the sphincter of Oddi, and using prepara- participation of NO in relaxant responses of the intestineamong intestinal regions and strains, and also the effectstions of circular muscles from near the myenteric and

submucosal surfaces), Bayguinov et al. (55) reported that of aging (4, 8, and 50 wk old) on NO-mediated relaxantresponses of intestine of Wistar rat. They concluded thatcells near the submucosal surface showed predominantly

EJP, whereas those near the myenteric border showed 1) in longitudinal SMC of jejunum, NO-mediated and L-arginine reversed relaxing component were 30% of thepredominantly IJP or biphasic responses (small EJP fol-

lowed by IJP). The EJP were blocked by atropine, and total relaxing response in Wistar-ST, 0% in Wistar, and60% in SD rats; those in longitudinal SMC in rectum werethe IJP were apamin sensitive. Furthermore, in the canine

circular pyloric sphincter, Bayguinov and Sanders (53) 0% in Wistar-ST, 0% in Wistar, and 65% in SD rats, and inlongitudinal SMC in proximal colon were 90% in Wistar-reported that the apamin-sensitive IJP they recorded were

attenuated by L-arginine derivatives (analogs) and that ST, 90% in Wistar, and 70% in SD rats. Thus differencesin contribution of NO on the relaxing responses observedsuch inhibitions were reversed by L-arginine. Oxyhemo-

globin partly blocked and the combined application of in these strains indicate that NO seems to play a dominantrole in the proximal colon, but it becomes less in moreoxy-Hb and NG-monomethyl-L-arginine (L-NMMA) com-

pletely blocked IJP. Exogenously applied NO hyperpolar- oral region or more anal region of GI tract. 2) When theeffects of aging were compared in longitudinal SMC inized the membrane, and this action was blocked by

apamin. jejunum of Wistar rats, NO-mediated relaxing componentswere changed from 40% of the total relaxing response inOsthaus and Galligan (87), who studied nerve-medi-

ated relaxation in the guinea pig ileum, concluded from 4-wk-old rats to 0% in 8-wk-old rats and to 0% in 50-wk-old rats. Those changes in longitudinal SMC of rectumthe actions of antagonists of NOS that inhibitory NANC-



were 0, 0, and 0%, respectively, and those changes in longi- hydrochloride, were both more strongly inhibited by qui-nine than by apamin, but that TEA, charybdotoxin, 4-AP,tudinal SMC of proximal colon were 80, 90, and 30%, re-

spectively. Thus age-dependent change in NO distribution and glibenclamide had no effect on these hyperpolariza-tions. Thus the IJP evoked in the esophagus depends onare also apparent (390). Much the same age-dependent

reduction in relaxing response has been reported in the cGMP elevation and is secondary to activation of quinine-and apamin-sensitive K/ channels. Hydroquinoline inhib-rat stomach fundus (1002) and ileum (1003).

In anococcygeus muscle, the involvement of NO is ited only the hyperpolarization induced by S-nitrosocyst-eine. Apamin partly inhibited IJP and the hyperpolariza-well documented (351, 354, 363). However, Gibson et al.

(350) questioned whether nitrergic transmission simply tion induced by NO, but not the nitroprusside-inducedhyperpolarization. In addition, 8-bromo-cGMP also pro-involves release of NO. They detailed several possibilities:

1) NO is released attached to a carrier molecule, perhaps duced hyperpolarization, and this hyperpolarization wasnot inhibited by apamin, TEA, or glyburide. Furthermore,in the form of a nitrosothiol; 2) NO is released in a modi-

fied redox form; 3) NO is released as a free radical, but Jury et al. (530) reported that NO release activates K/

outward currents in the opossum esophagus circular mus-is protected within the neuroeffector junction by othersubstances that preferentially interact with scavenger cle, which may depend on Ca2/ release from the SR stores.

C) GENITAL ORGANS. In male genital organs, J. Gilles-molecules; and 4) NO is released as a free radical which,because of a rapid and unhindered rate of diffusion over pie’s group (243, 246, 732, 733) detected and studied a

relaxing substance released from genital and anococcy-a short distance, is less susceptible than exogenous NOto scavenger molecules. As yet, they concluded, there is geus muscles. This substance was named ‘‘inhibitory fac-

tor’’ (IF). Inhibitory factor is soluble in water, is not aninsufficient experimental evidence to decide which, if any,of these explanations is correct. Bridgewater et al. (128), adenine nucleotide or a peptide, is inhibited by hemoglo-

bin, and increases the synthesis of cGMP but not that ofin the guinea pig taenia coli, recorded two distinct IJP(fast and slow) under treatment with hexamethonium, cAMP. It is now clear that these properties of IF are the

same as those of endothelium-derived growth factor. Theatropine, v-conotoxin GVIA, or apamin. That the abilityof v-conotoxin GVIA to selectively abolish the fast IJP, presence of cNOS has been demonstrated in male repro-

ductive tissues, using immunohistochemical methods orleaving the slow IJP intact, suggests that separate nervesare involved in mediating these responses. NADPH-diaphorase staining [the activity of cNOS is de-

pendent on Ca2/, calmodulin (CaM), and NADPH]. ThusB) ESOPHAGUS. Murray and co-workers (789, 790) re-ported that, in opossum esophageal SMC, NO activates cNOS has been found in nerves of the bovine and rat

retractor penis (228, 977), in nerves innervating penilethe maxi-K/ (Ca2/-dependent) channel via cGMP-depen-dent G protein pathways. Moreover, using the same tissue, arteries and cavernosal SMC, and also endotheliums of

arteries and cavernosal sinusoids (7, 141). ParticipationCayabyab and Daniel (178) observed that the IJP evokedby NO was dependent on cGMP elevation and the activa- of NO in excitatory neurotransmission in rat vas deferens

was also evident (1131). In a bovine retractor penis prepa-tion of quinine- and apamin-sensitive K/ channels. Robert-son et al. (936) have studied in vascular SMC that the ration, L-NMMA has been reported to have a partially ago-

nistic action (392, 393; although it inhibited the actions ofPKG-mediated activation of the Ca2/-dependent K/ chan-nel may be involved in relation with the action of NO and L-NNA and L-NAME, Refs. 699, 700). In the bovine retrac-

tor penis and in the penile artery, L-NNA blocked theother nitrovasodilators. The presence of NADPH-diapho-rase in the intramural plexuses of the esophagus of the cat relaxation induced by activation of NANC nerves, as it

did in the human and rabbit corpus cavenosum (reviewsand opossum has been detected by Fang and Christensen(285). Du et al. (254) and Conklin and co-workers (210, in Refs. 31, 586). It seems likely, but not certain, that there

is a relationship between penile erection and the action211) also concluded that NO, or a NO-like compound,probably mediates nerve-induced hyperpolarization and of I-NANC nerves. In the deep penile artery of the horse,

Simonsen et al. (985) found that relaxation induced byalso that this hyperpolarization is induced via cGMP. Inopossum esophagus-body SMC and in canine intestinal NANC nerve stimulation involves NO or a NO-like sub-

stance released from nitrergic nerves. Papka et al. (877)circular SMC, Christinck et al. (199) postulated that thegeneration of NANC-IJP is due to release of NO as final looked at the source of NOS-containing nerves supplying

the rat uterus and which other peptides might coexistmediator. Christinck et al. (198) further reported that theresponse in the former tissue was apamin insensitive, with NO in these nerves. They concluded that NOS-1/

NADPH-diaphorase reactivity coexisted with VIP and sub-whereas in the latter it was partially sensitive to apamin.The L-arginine analog L-NAME abolished the IJP in both stance P in parasympathetic nerves, but not in tyrosine

hydroxylase-1 neurons of pelvic parasympathetic ganglia.tissues. In opossum esophagus circular muscle, Cayabyaband Daniel (178) reported that the IJP evoked by electrical Nitric oxide synthase-containing nerves in the uterus are

autonomic and sensory and could play significant roles.field stimulation and the hyperpolarization induced by NOdonors, such as nitroprusside and 3-morpholinosydnimine It would be interesting to know whether the NO in-



volved in nitrergic neurotransmission is continously re- remaining part being sensitive to substance P) aftercon-traction (‘‘off-contraction’’; Refs. 47, 48). Much the sameleased under resting conditions, or is released only during

nerve excitation in a constitutive manner when the cyto- nitrergic off-contraction in response to electrical stimla-tion of I-NANC nerves has been reported in the opposumsolic Ca2/ is increased. In the rabbit anococcygeus mus-

cle, Kasakov et al. (547) found that NO is continously esophageal body and in the cat distal colon (1124). Fur-thermore, not only a rebound contraction, but a directlyreleased from nitrergic nerve terminals so as to maintain

a tonic relaxation. Moreover, they found that NO is also NO-induced contraction apparently occurs in the longitu-dinal muscle of the rat ileum and in the longitudinal mus-released during short-term (1–60 s) and long-term (10–

120 min) electrical field stimulation. This NO further re- cle of the opossum esophagus (47, 48). In fact, Barthoand Lefebvre (49), working on the rat ileum, found thatlaxed the preparation and modulated sympathetic trans-

mission. It was further reported by Shuttleworth et al. contraction occurred with a lower concentration of NOthan did relaxation. This NO-induced contraction in the(982) that sustained nitrergic neurotransmission in enteric

neurons is due to the synthesis of NO through functional opossum is related to the synthesis of cGMP (as is theNO-induced relaxation), but this is apparently not the caserecycling of citrulline, a coproduct of NO, to L-arginine

(the precursor of NO). in the rat. The NO-induced contraction is not related tothe ryanodine-sensitive Ca2/ channel but is sensitive toD) IRIS. In the isolated rabbit iris sphincter muscle,

Chuman et al. (200) observed the role of NO in the post- blockers of the L-type Ca2/ channel. According to thesame authors, primary contractions due to NO can alsojunctional regulation and concluded that cholinergic con-

traction is NO sensitive, whereas tachykinergic contrac- be observed in the rat whole ileum and in rat cecal longitu-dinal muscle, whereas a rebound contraction induced bytion is NO insensitive. Therefore, they estimated that, in

rabbit, the cholinergic and tachychinergic responses have NO can be seen in the rat descending, transverse, andsigmoid colon, as well as in cat ileal longitudinal muscle.different features for the fine adjustment of the iris sphinc-

ter muscle tone. Furthermore, in the muscle at the neck of the urinarybladder in the sheep, Thornbury and Peake (1090) andE) URINARY TRACT. Ehren et al. (267) investigated the

localization of cNOS activity in the human lower urinary Thornbury et al. (1089) found that NO plays a role inpoststimulation contraction (rebound contraction) andtract and its correlation with neuroeffector responses;

they concluded that in areas where a marked relaxation that this phenomenon depended more on release of Ca2/

from stores than on Ca2/ influx through L-type Ca2/ chan-was elicited by nerve stimulation, there was a high NOactivity. Thus NO seems to be the mediator for neurogenic nels.dilation of the bladder neck and urethra during the mictu-rition reflex. Almost the same conclusion in the same 3. Relationship between NO-inducedtissue have been reached by others (30). hyperpolarization and apamin

F) AIRWAY. Recently Zhou et al. (1234) reported, inChinese hamster tracheal SMC, using the cell-attached In the GI tract, most of the IJP generated in the GI

tract are sensitive to the bee venom apamin; those evokedpatch, that 8-(4-chlorophenylthio)-cGMP (an activator ofPKG) enhanced the open-state probability of the Ca2/- in the opposum esophagus (199) were resistant to apamin.

It was suggested that the generation of IJP in the GI tractdependent maxi-K/ channels. These actions induced byPKG were inhibited by the PKG inhibitor KT-5823 and is due to activation of K/ channels (1098), and the target

K/ channel for apamin was reported to be a small-conduc-protein phosphatase inhibitors (microcystin and okadicacid). The catalytic subunit of protein phosphatase 2A tance K/ channel (140, 517, 765, 1042). However, more

recently, Thornbury et al. (1090) demonstrated that appli-mimicked the effects of the PKG on the open probabilityin excised inside-out patches. Therefore, Zhou et al. (1234) cation of NO to canine colonic SMC enhanced the open

probability of Ca2/-activated maxi-K/ channels. The re-concluded that activations of the maxi-K/ channels byPKG, but not by PKA, required the activity of protein lease of NO has been demonstrated from inhibitory en-

teric neurons (127, 1156, 1157), and a TTX-sensitive re-phosphatase 2A. In cat airway SMC, Takahashi et al.(1069) reported that free radical NO and NO-containing lease of NO can be induced by field stimulation in the GI

tract: gastric fundus (240, 737), intestine (102), internalcompounds are involved in the L-NAME-sensitive NANCrelaxation, but that only free radical NO has a prejunc- anal sphincter (181). In the canine proximal intestine and

the pyloric sphincter (53–55), apamin blocked evokedtional action.Although NO is normally considered to have inhibi- IJP. This finding was supported by experiments carried

out on the ‘‘knockout’’ mouse by Huang et al. (435), whotory actions, it has been reported that this substance mayalso act as an excitatory substance. Thus Bartho and bred mice that lacked the b-NOS gene (knockout). They

reported that b-NOS expression and NADPH-diaphoraseLefebvre (49) reported that, in the longitudinal layer ofthe guinea pig ileum, NO produced a moderate relaxation staining were absent in the mutant mice but that the pres-

ence of a very low level of residual catalytic activity couldfollowed by a TTX-sensitive (partly atropine-sensitive, the



be detected. Thus other enzymes may also generate NO whose synthesis is triggered by influxes of Ca2/ throughactivated cNOS, activates the release of VIP at nerve ter-to a minor extent. However, these mutant mice did not

show any histopathological abnormalities in the central minals and that the released VIP activates two differentreceptors (VIP-specific receptor and VIP-preferring recep-nervous system, and the most evident effect of disrupting

the neural NOS gene was the development of a grossly tors). The former recognizes VIP and blocked pituitaryadenylyl (adenylate) cyclase activating peptide (PACAP),enlarged stomach, with hypertrophy of the pyloric sphinc-

ter and circular muscle layer. They considered that this but not PHI, whereas, the latter recognizes VIP, PHI, andPACAP. The former receptor is thought to synthesizephenotype resembles the human disorder known as infan-

tile pyloristenosis. cGMP through activation of NO via activation of a NOSthat is tightly bound to CaM in the plasma membrane.In general, NO released from nerve terminals induces

a slow relaxation in precontracted VSM tissues. The main The latter receptor is thought to synthesize cAMP throughactivation of a G protein. To test the truth of this hypothe-inhibitory actions of NO on VSMC are thought to be due

to the inhibitory action of cGMP on contractile proteins sis in the GI tract, we need the answer to many unsolvedproblems. For instance, Desai et al. (244) reported thatand to activation of K/ channels distributed on postjunc-

tional cells, and also to inhibition of excitatory transmitter NO, but not VIP, is a vagal inhibitory neurotransmitter ofthe guinea pig stomach.release from nerve terminals. In some tissues, such as the

GI tract, this agent is thought to generate an IJP or a slow In airway SMC, VIP and NO are both thought to beinhibitory transmitters, but recently a general consensushyperpolarization. However, the evidence is not straight-

forward, in that the actions of NO antagonists and apamin has emerged that NO probably plays the more dominantrole as a relaxant (66, 246, 642). The release of NO occurson these two inhibitory electrical processes are not con-

sistent. For example, in canine proximal colon and jejunal independently of the presence of epithelial cells in airwaySMC (1165). In equine airways, Yu et al. (1219) found thatmuscles, L-arginine antagonists (L-NNA or L-NMMA)

partly, but not completely, blocked IJP, with this inhibi- adrenergic innervation is limited to the cranial part of thetrachea, whereas NO-containing I-NANC nerves supplytion being reversed by L-arginine (1026, 1092, 1156, 1157).

In SMC, it is thought that L-arginine antagonists block both the trachea and the central bronchi. Furthermore,Ward et al. (1154) and Belvisi et al. (65), who studied thecGMP-activated Ca2/-dependent maxi-K/ channels (90,

1092), as also observed in studies of the actions of cAMP distribution of human I-NANC bronchodilator and NO-immunoreactive nerves, and also the effects of variouson the maxi-K/ channel (612). Furthermore, it was re-

ported by Gerland and McPherson (347) that NO does NO-related agents in the main, proximal, and distal air-ways of isolated human airways, concluded that I-NANCnot induce hyperpolarization of the membrane in the rat

mesenteric artery. However, Kitamura et al. (580) found nerves innervate most densely the distal region and lessso the proximal region. It is thought that the synthesis ofin the rat gastric fundus that NO-cysteine and sodium

nitroprusside hyperpolarized the membrane and that NO in airway SM tissue occurs in sensory nerves, endothe-lial cells, vascular and airway SMC, inflammatory cells,these responses were sensitive to apamin (but not in cat

airway, Ref. 526). Kitamura et al. (580) further postulated and the airway epithelium (805). Kannan and Johnson(542) investigated whether or not NO released from I-that NO may activate both apamin-sensitive and -insensi-

tive K/ channels. Unfortunately, however, neither the NANC nerves is involved in producing SMC relaxationthrough the action of synthesized cGMP in airway SMC.apamin-sensitive K/ channel nor the apamin-insensitive,

L-arginine antagonist-sensitive K/ channel has yet been They concluded that NO-induced relaxation is due to acti-vation of both charybdotoxin- and iberiotoxin-sensitiveidentified (Fig. 4).Ca2/-dependent K/ channels. In the human trachea, Bel-visi et al. (65) and Ellis and Undem (271) demonstrated4. Mutual relationship between NO and VIPthat NO completely relaxed the contraction evoked byelectical field stimulation. On the other hand, Ito’s groupA mutual relationship between NO and VIP in the GI

tract has been hypothesized. According to this hypothesis, postulated that VIP and NO are distributed differentlyfrom each other in cat airway SMC and exert coordinatedduring GI tract electrical stimulation with EJP sup-

pressed, a fast large-amplitude IJP may be generated by inhibitory actions on cholinergic excitatory transmission.In fact, in the guinea pig, administration of a-chymotryp-ATP and a sustained low-amplitude hyperpolarization by

VIP and NO (391, 1025, 1027, 1071). Thus VIP and NO sin or incubation with VIP antiserum reduced the mechan-ical response evoked by electrical stimulation (by 20–would be functionally linked cotransmitters as well as

postjunctional regulators of SM activity. The NO released 70%; Refs. 270, 650), the remaining relaxation being abol-ished by NOS inhibitors (660, 1110). In the dog trachea,from nerve terminals by Ca2/ after nerve activation would

regulate VIP release and, in turn, VIP would regenerate but less so in the cat, the amplitude of fused EJP wasevoked by repetitive stimulation (20 Hz) increased in aNO by activating a NOS present in the target SMC, ac-

cording to Murthy et al. (792). They postulated that NO, stimulus frequency-dependent manner (379, 497). After



FIG. 4. Effects of tetrodotoxin(TTX), NG-nitro-L-arginine (L-NNA), andL-arginine on inhibitory junction poten-tials by a constant-intensity electric fieldstimulation in rat gastric fundus. [FromKitamura et al. (580).]

treatment with VIP antiserum or with VIP antagonists, esophagus, Ref. 86). In the cat gastric fundus, NO is a maintransmitter, but during sustained relaxation, VIP may actthe EJP recorded from the cat trachea showed marked

summation in response to repetitive field stimulation as a NANC neurotransmitter. In opossum internal analsphincter, the increase in NO production in response to(1182). Moreover, in the same tissue, S-nitrosocysteine

and VIP did not modify either the membrane potential or VIP occurs mainly from the myenteric neurons, with somecontribution from the SMC (182). Furthermore, Daniel’sinput resistance of SMC, but did relax the tissue (498,

526). From these accumulated results, Ito’s group (497, group (182, 231, 529), working on the canine and opossumlower esophageal sphincters, reported that VIP did not498) postulated that VIP and ACh probably coexist in the

same nerves and act as cotransmitters with each other contribute to the generation of IJP, whereas NO did (theseconclusion was also supported in the rat proximal colon,(39, 40, 247, 640), just as VIP and NO are thought to coexist

in the same nerve terminals (216, 246). In airway SMC, Ref. 1054). Furthermore, evidence for coexistence of ATPand NO in NANC inhibitory neurons in the rat ileum, co-excitatory cholinergic transmission may be regulated by

two inhibitory substances (VIP and NO) released from lon, and anococcygeus muscles was presented (63, 1004).It has also been postulated on VIP receptors that thethe same excitatory vagal nerve fibers (526). In isolated

stomach of the guinea pig, Desai et al. (244) investigated a IJP is a VIP-induced response in esophageal circular SMC(224), and VIP has been nominated as an inhibitory I-possible link between NO and VIP in mediating relaxation

induced by vagal stimulation. They concluded that NO NANC transmitter in the GI tract (230, 231, 362, 369).However, this idea was not supported by experiments onhas a neural origin and support NO, but not VIP, as a

major neurotransmitter of vagally induced gastric relax- dog ileum, opossum esophagus, or guinea pig taenia coli(144, 199, 231, 529, 690). In airway SMC, although thereation. Ideas about the role of NO in bronchial hyper-

responsiveness have been reviewed by Nijkamp and is evidence in favor of NO as an I-NANC transmitter (64,65, 542), VIP immunoreactivity and VIP receptors haveFolkert (815).

On the basis of electrophysiological experiments, it also been identified. Moreover, after studying the circularmuscle of the canine proximal colon, Keef et al. (559)has been postulated by Hakoda and Ito (379) that in the

dog penile artery and vein, VIP acts as an I-NANC sub- concluded that the enteric plexus releases two I-NANCtransmitters, NO and VIP, as cotransmitters released instance; the EJP was inhibited by VIP, but not by a,b-

methylene ATP. Hakoda et al. (378), also using electro- parallel from the enteric inhibitory nerves. Their evidencewas that 1) the electrical and mechanical effects of VIPphysiological procedures, investigated the possible role

of VIP as a cotransmitter with ACh in the cat trachea after did not depend on NO synthesis, 2) the VIP-inducedchanges in [Ca2/]i did not depend on NO synthesis, andit had been immunized against a conjugate of VIP-BSA.

In in vitro experiments, EJP were markedly enhanced, so 3) VIP did not cause the release of NO. Cotransmissionvia VIP and NO has also been suggested in the rat gastricVIP was postulated to be an I-NANC substance (these

observations were also supported in the rabbit lower fundus (240), whereas distributions of ATP and NO, as I-



NANC substances, in guinea pig colon have been docu- ulation were markedly suppressed by capsaicin, andtherefore, Saito et al. (950) suggested a role for CGRP asmented (1221).

With the use of immunohistochemical techniques, an I-NANC substance in inhibitory nerves. Possibly, thelong-lasting hyperpolarization observed in dog basilar ar-distributions of PHI and peptide histidine-methionine

(which possess structural similarities with VIP) have been tery (307) may be of the same nature. In human myome-trial cells, Azadzoi and Szenz de Tejada (36) found thatidentified in airway SMC (247, 380, 709, 933). Furthermore,

VIP receptors are found predominantly in large rather CGRP induced a direct G protein-mediated activation ofK/ channels, leading to hyperpolarization and SM relax-than small airways (166). In airway SMC of the cat, VIP

antagonists dose dependently enhanced the amplitude of ation. Santicioli and Maggi (960) reported that, in theguinea pig ureter, electrical field stimulation evoked aEJP without changing either the membrane potential or

input resistance, although in those of the dog, such antag- graded hyperpolarization that was blocked by TTX andprevented by capsaicin desensitization (capsaicin itselfonists had no effect (1182). Thus this effect in the cat

seems to be an example of VIP acting on prejunctional produced a slight hyperpolarization). Exogenously ap-plied CGRP also produced hyperpolarization, but this wasnerve terminals.insensitive to TTX. Furthermore, an antagonist of ATP-sensitive K/ (KATP) channel openers, glyburide, blocked,5. Role of CGRPbut low K/ solution enhanced, both IJP and the CGRP-induced hyperpolarization. Thus CGRP appears to be re-As well as VIP, other peptides also act on VSMC as

cotransmitters released from cholinergic and adrenergic leased from the peripheral endings of capsaicin-sensitiveprimary afferent neurons, causing hyperpolarization duenerves (675, 1037). For instance, the immunohistochemi-

cal localization of CGRP and other cotransmitters was to activation of KATP channels. Distributions of inhibitoryCGRP neurons in the guinea pig ileum (46) and rabbit anddescribed in a subpopulation of postganglionic neurons

in the porcine inferior mesenteric ganglion by Majewski rat colon (710) have been elucidated.Calcitonin gene-related peptide has been detected inand Heym (687). They subdivided these neurons ac-

cording to size and cotransmitter content. Thus CGRP- sensory and motoneurons in the GI tract (683, 935). Calci-tonin gene-related peptide enhanced contraction in theimmunoreactive (CGRP-IR) neurons were demonstrated

to belong to the population of nonadrenergic neurons (ty- guinea pig small intestine through an acceleration of AChrelease (428, 684) and also in guinea pig proximal androsine hydroxylase and DBH negative). Virtually all of the

CGRP-IR neurons exhibited colocalization of CGRP with distal colon and taenia coli (716), but it caused a concen-tration-dependent inhibition of [3H]ACh release from thesomatostatin, and some of them with NPY. Majewski and

Heym (687) further reported that NPY-, somatostatin-, and rat stomach antrum. Furthermore, CGRP enhanced thecontraction evoked by NANC nerve stimulation in theNPY/somatostatin-IR subpopulations of adrenergic and

NANC neurons were present. In the rat vas deferens, exog- guinea pig small intestine (901). In the guinea pig proximalcolon, Kojima and Shimo (601) likewise reported thatenously applied CGRP (human) did not modify the EJP

but did inhibit contraction through an inhibitory actions CGRP enhanced the contraction evoked by NANC nervestimulation (in the presence of a- and b-adrenergic andon postjunctional SMC (361). However, the same group

(948) reported that, in the large cerebral artery of the cat, muscarinic inhibitors), in this case through a prejunc-tional mechanism via a non-CGRP-1 receptor (242, 901)1) immunoreactive CGRP-like material was contained not

only in nerve axons but also in boutonlike structures and located on intramural tachykininergic neurons. In the ca-nine lingual artery, Kobayashi et al. (591) reported that,2) that transmural nerve stimulation caused relaxation

and NANC-IJP, responses that were blocked by capsaicin, on electrical stimulation, NANC nerves released CGRP,but not NO, inducing relaxation by activation of theas observed for responses induced by applied CGRP. Thus

they concluded that CGRP is involved in the mediation of CGRP-1 receptor. Using the sucrose gap method, Maggiet al. (684) reported that CGRP produced a TTX-resistantvasodilator responses in this tissue (501). Somatostatin

has been reported to act as a negative regulator of transmembrane hyperpolarization that was partly blocked byeither TEA or CPA while being unaffected by glyburide.mitter release from nerve terminals in the canine ileal

circular muscle (524) and in the esophagus. Moreover, They concluded that CGRP produces a direct relaxationof circular SMC of the guinea pig proximal colon throughRattan et al. (921) postulated an inhibitory role for CGRP

in neuromuscular transmission, and in the guinea pig ure- activation of Ca2/-dependent K/ channels and Ca2/ re-lease and uptake from the SR.ter, Santicioli and Maggi (960) reported that CGRP-in-

duced inhibition of IJP evoked by nerve stimulation is due In GI tract, distributions of PACAP, as a relaxing sub-stance, have been elucidated in the guinea pig taenia colito activation of glibenclamide (glyburide)-, ATP-sensitive

K/ channels. (525) and stomach (549), rat colon (368), human and catesophageal sphincter (833), and rat distal colon (576). InIn large cerebral arteries of cats, CGRP hyperpolar-

ized the membrane and IJP evoked by electrical field stim- the trachea, the PACAP-induced relaxation was inhibited



TABLE 1. K/ channels in visceral smooth muscle cells

UnitaryConductance, Reference

Classification Cells Type Structure pS mRNA in SMC IC50 No.

Delayed Canine colon KV1.2 499 aa; 6 TM 14 GI tract 75 mM (4-AP); ú10 mM (TEA) 385rectifier K Canine colon KV1.5 599 aa; 6 TM 10 GI tract, vascular 211 mM (4-AP); ú10 mM (TEA) 868

cells, heartCanine colon Native 19 70 mM (4-AP); ú10 mM (TEA) 162Porcine airway Native 13 120Canine trachea Native 70 mM (4-AP); 3 mM (TEA) 785

Maxi K Bovine trachea mslo 62 kDa (a) 240 Trachea, aortae Charybdotoxin sensitive 34331 kDa(b)(191 aa; 2 TM)

Canine colon Native 70–120 0.3 mM (TEA) 160Guinea pig Native 274 693Urinary bladder

ATP-sensitive K uKATP 424 aa; 2 TM 70 GI tract Glibenclamide insensitive 468Porcine urethra Native 43 Glibenclamide sensitive 1080Guinea pig Native 10 Glibenclamide sensitive 108Urinary bladder

GI, gastrointestinal; 4-AP, 4-aminopyridine; TEA, tetraethylammonium; aa, amino acids; TM, transmembrane segment; SMC, smooth musclecells.

by charybdotoxin (415); presumably this agent activates current-like properties, whereas Shaw and Shab K/ chan-nels exhibit delayed rectifier-like properties (197). The a-Ca2/-dependent maxi-K/ channels.subunits of Kv1.1, Kv1.2, Kv1.5, and Kv1.6 in Shaker-typechannels bestow delayed rectifying properties, but these

IV. CHARACTERISTICS OF ION CHANNELS same channels show A current-like properties with a b1-IN VISCERAL SMOOTH MUSCLE CELL or b3-subunit (274, 275, 974). The a-subunits in Kv1.3 andMEMBRANES Kv1.4 of the Shaker type bestow A current-like properties,

and coexpression of the b1-subunit enhanced the timecourse of inactivation (770, 1220). Kv3.1, of the Shaw type,

A. K/ Channelscan be classified as the delayed rectifier type, whereasKv3.3 has inactivating properties. Moreover, Kv2.1 and

1. Subtypes of K/ channelsKv2.2 of the Shab type, possess delayed rectifying proper-ties, but those of the Shal type had A current-like proper-The recent development of molecular biological tech-ties. When the effects were observed of the muscarinicniques has enabled the development of a genomic and(m3) receptor on Kv1.2 and Kv1.5 channels cloned origi-structural classification for K/ channels (see Table 1).nally from canine colonic SMC, they were found to causeClassical voltage-dependent K/ channels (including Ca2/-a poorly reversible decrease in the opening probability ofactivated, delayed rectifying, and A-like K/ channels) formthese channels (1135). In contrast, it has been reportedan ionophore with a tetramer of subunits having six trans-that possession of the b1-subunit has no effect on non-membrane (TM) domains. Although these K/ channelsShaker-type K/ channels (974, 1220).could be functionally expressed as a homomultimer, the

The inward rectifying K/ channel possesses a two trans-presence of variation in K/ channel properties has led tomembrane structure. Other members of this group includethe idea that the K/ channels are actually expressed as athe KATP channel, Na/-sensitive K/ channel, and pH-sensitiveheteromultimer, the components being encoded by differ-K/ channel, as well as the inward rectifying K/ channel.ent types of K/ channel genes (217, 946). This type of K/

Although the pH-sensitive K/ channel (RACTK1) has not beenchannel has an S4 region in its structure that gives itidentified in intestinal SMC, this channel has been identifiedvoltage dependency, and the channels form a super fam-in cardiac and arterial SMC, as well as in collecting duct cellsily. Genetically, the voltage-dependent K/ channels makein the kidney (1060). These types of K/ channel have noup four families (Shaker, Shab, Shaw and Shal), whichvoltage dependency, because of their lack of the S1-S4 regionshave been subdivided into many Kv subfamilies (183, 184,found in the classical voltage-dependent K/ channels. Al-372). In terms of function, these K/ channels can be di-though these K/ channels lack the voltage sensor, their in-vided into two large groups (delayed rectifier and tran-ward rectifying properties emerge when Mg2/ is present insient K/ channel types). The relation between familythe cytosol (inward rectifying K/ current, Ref. 830; KATP cur-membership and function is not a simple one. For exam-

ple, Shaker and Shal (Kv4)-type K/ channels exhibit A rent, Ref. 630; pH-sensitive K/ current, Refs. 1060, 1061).



In whole cell experiments, membrane depolarization delayed rectifying K/ channels. This TEA-sensitive out-ward current (sustained component) was charybdotoxinproduces transient and sustained outward currents, after

the Ca2/ inward current. The transient component of the and iberiotoxin sensitive, indicating a contribution of theCa2/-activated (maxi-) K/ channels to the delayed rectify-outward current is formed by the activities of two differ-

ent K/ channels. One of them is a Ca2/-activated K/ chan- ing outward current in these SMC.Concerning channel phosphorylation, Carl et al. (162)nel (mainly maxi-K/ channel), and the other is an outward

rectifier K/ channel. In pharmacological experiments, reported a Ca2/-activated K/ channel of 260 pS that wasactivated by phosphorylation via PKA. Calyculin A alsoTEA, which at low concentrations blocks the maxi-K/

channel relatively selectively, inhibited part of the tran- augmented this channel’s activity, but to a lesser extentthan PKA, suggesting modulation of the channel openingsient current, but not all of it. Although intracellular appli-

cation of InsP3 had no effect on the amplitude of the by cAMP or adenylate cyclase. Because calyculin A short-ened the slow wave’s duration (1160), these authors pos-transient outward current, ryanodine, an open blocker of

the Ca2/-induced Ca2/ release channel in the SR, abol- tulated that phosphorylation and dephosphorylation ofthis Ca2/-activated K/ channel might contribute, at least inished the transient outward current, thus indicating a ma-

jor contribution by Ca2/ release from the SR to the activa- part, to slow-wave formation. In addition, they speculatedthat the phosphatase that acts on the Ca2/-activated K/tion of this channel (64, 850, 950, 1229). The remaining

component of the transient current is blocked by 4-AP, channel in colonic SMC might not be of type I or type II,judging from the potencies of calyculin A and okadaicknown to be a blocker of the A current. In canine and

guinea pig gastric muscle cells, the transient K/ current acid. In this channel, G protein-coupled channel modula-tion has also been reported (205, 206). In fact, phorbolwas reported to be a Ca2/-activated K/ current, but its

sensitivity to TEA was lower than that of the classical 12,13-dibutyrate (PDBu), a phorbol ester and a PKC acti-vator, inhibited the transient outward current, and 1-(5-maxi-K/ channel (824, 985). In the circular muscle layer of

the guinea pig ileum, Duridanova and Boev (257) classified isoquinolinylsulfonyl)-2-methylpiperazine (H-7; a PKC in-hibitor) abolished this action of PDBu (581). Single-chan-voltage-dependent K/ currents into three components on

the basis of drug actions: 1) at a holding potential of 050 nel current recording then revealed that PKA, but notPDBu, enhanced channel activity at the unitary currentmV, apamin inhibited 29% of the whole cell K/ current

(IK) at a 0 mV membrane potential. This current disap- level, suggesting the presence of an indirect mechanismfor channel activation, rather than direct channel phos-peared after blockade of the inward Ca2/ current. 2) Gly-

buride inhibited 31% of the total current at 0 mV. 3) Other phorylation by PKC.In vascular SMC (rabbit portal vein), Isop and for-components formed 36–46% of the IK in cells clamped

at a holding potential of 080 mV; this was inhibited by skolin each enhanced the amplitude of the 4-AP-sensitivedelayed rectifying K/ channel, possibly through an activa-charybdotoxin (maxi-K/). Adda et al. (12) studied the ex-

pression and function of voltage-dependent K/ channel tion of PKA (18). Furthermore, there is evidence thatKv1.2 and Kv1.5 channels, both of which are 4-AP-sensi-genes in human airway SMC. RNA from airway SM tissues

revealed the presence of Kv1.2 and Kv1.5 transcripts, as tive delayed rectifying K/ channels, have PKA consensussites in their cytosolic region (NH2 and COOH terminals)well as Kv1.1 mRNA. The available voltage-dependent K/

current in human airway myocytes was insensitive to (564). Moreover, the rat cardiac Kv1.2 channel was foundto be augmented by Isop and PKA via phosphorylation of acharybdotoxin but blocked by 4-AP. Dendrotoxin, cha-

rybdotoxin, and glybenclamide had no effect on the rest- single site in the NH2-terminal region (436). These findingsindicate that, in VSMC, delayed rectifying K/ channels areing tone of muscle cells. Conversely, 4-AP increased rest-

ing tension with an EC50 equivalent to that observed for also modulated by metabolic processes. Walsh and Kass(1146) noted that PKA caused a shift to the left in thecurrent inhibition. They concluded that human airway my-

ocytes express mRNA for several members of the Kv1 channel’s activation curve and an enhancement of currentamplitude by PKC, both in cardiac delayed rectifyingfamily; the channel that underlies the predominant volt-

age-dependent K/ current and regulation of basal tone channels. They concluded that PKA and PKC both aug-mented the delayed rectifying channel, but did so indepen-appears to be Kv1.5.

The sustained component of the outward current is dently.The KATP channel can be activated via various recep-generally called the delayed rectifying outward current.

However, as mentioned previously, the properties of the tors, such as those for CGRP, adenosine, somatostatin,and galanin. In rabbit mesenteric artery and guinea pigsustained outward current differ between tissues. In fact,

Snetkov et al. (1008) reported that, even in the same tissue gallbladder, CGRP opens the KATP channel through an acti-vation of PKA (910, 1226). In fact, the KATP channel has(human bronchial SMC), individual cells possessed phar-

macologically different K/ channels; some cells had 4-AP- potent PKA and PKC phosphorylation sites in the COOH-terminal region. Indeed, muscarinic receptor stimulationsensitive (Ca2/-independent) delayed rectifying K/ chan-

nels, whereas others had TEA-sensitive (Ca2/-dependent) inhibited KATP activity through PKC stimulation.



Kim et al. (567) reported that individual Kv1.4 chan- been published on the unitary current of this delayed recti-fying channel. The reported unitary conductance (esti-nels, which are Shaker-type K/ channels, gather together

to form a cluster when this channel is coexpressed with mated by noise analysis) is Ç5–10 pS (rabbit portal vein,Refs. 58–60; rabbit coronary artery, Ref. 1136), these val-PDS-95 protein. They concluded that the PDZ-1 region of

the PDS-95 protein and the TAV or TDV of the COOH ues being slightly smaller than those obtained for naturaland cloned delayed rectifying K/ channels from the canineterminal are essential sequences for interaction. The PDS-

95 protein is a guanylate kinase and has a SH3 domain colon (160–164, 385, 868). A similar value for unitary con-ductance has been reported for the anomalous rectifiernear the kinase domain. Therefore, it is plausible that

interaction between Kv1.4 channel-PDS-95 protein com- K/ channel in tunicate egg cells (5.2 pS, Ref. 314). Theunitary conductance of cloned delayed rectifying K/ chan-plexes may produce additional functions by phosphoryla-

tion of the protein. A clustering of channels caused by nels was 14 pS (CSMK1, Kv1.2) or 10 pS (Kv1.5) in sym-metrical 140 mM K/ solutions (385). These values arecytoplasmic protein has also been reported for the ACh

receptor, as has coexpression of 43K/rapsyn and the gly- much the same as those obtained for native delayed recti-fier K/ channels recorded from SMC of the porcine airwaycine receptor with gephyrin in neurons (574, 895).(13 pS) and canine colon (19 pS) (120, 161). Northernblotting of CSMK1 and Kv1.5 channel mRNAs has shown2. Delayed rectifying K/ channelsthat the Kv1.5 channel is common throughout the entireGI tract as well as in several vascular tissues and theThis is a common K/ channel current observed in a

variety of SMC, including vascular cells. It is activated by heart, whereas the Kv1.2 channel is restricted to the GItract (385, 868). These data indicate that the delayed recti-membrane depolarization to 030 or 020 mV in a Ca2/-

free medium. 4-Aminopyridine and TEA are used as block- fier K/ channels observed in various cells do not makeup a uniform population. In colonic SMC, the IC50 valueers of this current; however, both are required in concen-

trations of a few millimolar for channel block, and these for the action of 4-AP on the delayed rectifying K/ channelwas 70 mM, but it was 300 mM in the trachea and in pulmo-drugs more selectively inhibit the transient (A-like) and

Ca2/-activated K/ currents, respectively. In rabbit ileal nary artery cells (162, 785, 854). Delayed rectifying K/

channels highly sensitive to 4-AP have also been reportedmuscle, the delayed rectifying K/ current was inhibitedby 1 mM TEA but not by 1 mM 4-AP, whereas the same in rabbit portal vein and in porcine and canine airway

SMC (59, 120). Cloned Kv1.2 and Kv1.5 channels had IC50current in the rabbit pulmonary artery was inhibited by 1mM 4-AP but not by 10 mM TEA (849). Another blocker values for the action of 4-AP of 75 and 211 mM, respec-

tively (385, 868). The dissociation constant (Kd) value ob-of the delayed rectifying K/ current is a-dendrotoxin, asnake venom peptide of 60 amino acids from Dendroaspis tained with 4-AP (270 mM) for cloned Kv1.5 (hPCN1) chan-

nels was similar to that found for K/ channels in colonicangusticeps (DTX; Refs. 173, 1024, 1167), and this toxinhas also been reported to block a transient K/ current in SMC. However, the Kd value (590 mM) for cloned Kv1.2

(NGK1) channels was quite different from that found inthe hippocampus, as does 4-AP (249). Studies of aminoacid mutations in the DTX-sensitive delayed rectifier K/ colonic Kv1.2 channels. This suggests that we should al-

ways take into consideration any small differences in the(RBK1) channels has shown that Ala352-Glu353 and Tyr379

in the H5 region between the S5 and S6 transmembrane protein sequence as well as heteromultimeric channel for-mation when studying drug sensitivity.domains are important residues for the toxin sensitivity

of this channel (449). The activation of this current is It has been reported that there are delayed rectifyingK/ channels resistant to 4-AP in the gastric antrum, taeniaboth time and depolarization dependent, but it is not, in

general, inactivated during a short depolarizing pulse. coli, colon, ureter, and urinary bladder of the guinea pig,rabbit ileum, rat anococcygeus muscle, canine coronary,However, a very long depolarizing pulse (longer than sev-

eral seconds) does induce a slow current inactivation. pulmonary and renal arteries, and feline cerebral artery(for review, see Ref. 626). In fact, for inhibition of thisIn addition, 4-AP blocked a cloned K/ channel (Kv1.5),

classified as a delayed rectifying K/ channel, which was delayed rectifying K/ current, high concentrations of TEAwere required (IC50 Å 3 mM in rabbit ileal longitudinalexpressed in Xenopus oocytes and in mammlian cells

(116, 289, 1036). A cloned channel (CSMK1) from canine muscle, Ref. 849; 3 mM in canine tracheal muscle, Ref.785;ú10 mM in canine colonic circular muscle and clonedcolonic circular muscle was also inhibited by 4-AP, at

submillimolar concentrations (385). This SM delayed rec- Kv1.2 and Kv1.5 channels, Ref. 868). After studying RCK2,a Kv1.6-type delayed rectifying K/ channel found in rattifying channel has a very high homology with others of

the Kv1.2 subfamily [90–91%; rat atrial K/ channel (RAK), brain, Kirsh et al. (573) reported that the channel had twodistinct TEA-binding sites (on inner and outer margins ofrat brain K/ channels (RBK2, RCK5)]. In fact, block by 4-

AP of this K/ channel has been demonstrated in both the pore). Furthermore, of the other delayed rectifying K/

channels, Kv2.1 had a high sensitivity to TEA at the inter-open and closed states. However, perhaps because of itsvery small unitary conductance, only a few papers have nal site, whereas Kv3.1 was sensitive at the outer site



(572). Although the blocking of delayed rectifying K/ (643). Its pharmacological and biophysical characteristicsare similar to those of the A current in neurons and tochannels by 4-AP and TEA is voltage dependent, the differ-

ent sensitivities to 4-AP and TEA suggest that different those of the transient outward current found in cardiaccells (9, 528). The unitary current conductance of thistypes of delayed rectifying K/ channels might be ex-

pressed in different SM tissues. Recently, Horowitz et al. transient K/ current is 14 pS under quasi-physiologicalionic conditions (465). Although the transient outward(430) showed that coexpression of Kv1.2 and Kv1.5 chan-

nels in oocytes and other cells resulted in the formation current recorded in SMC such as those of the ureter andportal vein had a similar 4-AP sensitivity to that of theof heterotetramers of both channel subunits. Heterogenei-

ties, in terms of the properties of delayed rectifying K/ delayed rectifying K/ current, its inactivation kinetics dif-fered from the delayed rectifier in these preparations. Thischannels, between tissues and between cloned and native

K/ channels might be explained by the formation of heter- suggests that the two channels might differ in structure(the Shaker-type K/ channel has a ‘‘ball and chain’’ struc-omers involving different subunits.

4-Aminopyridine (2 mM) or TEA (5 mM) prolongs ture at the NH2 terminal, whereas the delayed rectifiertype K/ channel does not, Ref. 1220). Schwarz et al. (972)plateau duration (guinea pig gall bladder, Ref. 1227) or

produces an action potential on membrane depolarization have already reported that the a-subunits of both A-type(Kv1.3 and Kv1.4) and delayed rectifying (Kv1.1, Kv1.2(guinea pig pulmonary artery, Ref. 79; canine trachea, Ref.

467). Thus it is clear that the delayed rectifying K/ current and Kv1.5) K/ channels are produced by alternative splic-ings from the same gene. The evidence indicates thatserves as a repolarizer of cells in a way that controls

action potential duration. Indeed, inhibition of this current these K/ channels have the same ‘‘core’’ sequence (6 TMand H5 regions), but different NH2 and COOH terminals.(pharmacologically or by removal of cytosolic K/) pro-

longs action potential duration and leads to the formation Indeed, deletion of the amino acid sequence at the NH2

terminal in the A-type channel prevented the current’sof a plateau potential (849, 1146). Furthermore, as men-tioned above, delayed rectifying K/ channels in cardiac inactivation, and insertion of a synthesized peptide having

the same sequence as the NH2 terminal reproduced thecells were found to be regulated by stimulation of variousreceptors via PKA or PKC activation, or directly via G inactivating property of this channel (1223). Sewing et al.

(976) also showed that coexpression of the a,b-subunitproteins. In vascular SMC, PKA has been reported to acti-vate the delayed rectifying K/ channel after b-adrenocep- with an a1-subunit of the delayed rectifier type Kv1.5 chan-

nel transferred the delayed rectifier property to anothertor stimulation (18). These reports indicate that the de-layed rectifying K/ channels in SMC, including visceral K/ channel having an A-like channel property. These re-

ports strongly indicate that the various inactivation ratescells, can be modulated by agonist stimulation and mem-brane depolarization via multiple pathways. of transient outward currents might be due to the forma-

tion of a heteromultimeric channel by different a- and b-subunits (951, 970).3. Transient outward K/ currents

Transient outward K/ currents in SMC can be classi- 4. Inward rectifying K/ channelfied as either Ca2/-dependent or -independent K/ currents.Their relative sensitivity to drugs has also revealed the An inward rectifying K/ current has not yet been

recorded in VSMC (other than in blood vessels), and thispresence of two types of transient outward current.Charybdotoxin and TEA are rather specific blockers for K/ current has been studied mainly in cerebral, mesen-

teric, and coronary arteries (for review, see Ref. 626). Inthe Ca2/-activated K/ current, and as low concentrationsof TEA, but neither 4-AP nor apamin, effectively block middle cerebral and pial arteries, an increase in extracel-

lular K/ causes vasodilation by two different mechanisms:the Ca2/-dependent transient K/ current, this is possiblya current produced by activation of the maxi-K/ channel. an ouabain-sensitive mechanism and a Ba2/- and Cs/-sen-

sitive one (636, 715). Furthermore, Edwards and HirstIndeed, in rabbit portal vein, periodic activations couldbe recorded of the maxi-K/ channel under certain condi- (261) reported that the vasodilation induced by an eleva-

tion in extracellular K/ was related to the membrane hy-tions in the inside-out membrane patch (1185). Caffeineand ryanodine, but not InsP3, also effectively inhibited perpolarization observed under the same conditions. This

in turn resulted from activation of the inward rectifierthe Ca2/-dependent component of the transient outwardcurrent, together with a slight inhibition of the VOCC, K/ channel distributed in the distal region of the middle

cerebral artery; indeed, a depolarization, rather than asuggesting an involvement of the Ca2/-induced Ca2/ re-lease (CICR) mechanism in the Ca2/-dependent transient hyperpolarization, occurred in the proximal region of the

same artery, where the inward rectifier K/ channel wascurrent (850, 950).In the guinea pig ureter, a Ca2/-independent K/ tran- not recorded (261, 262). The resting membrane potential

of the distal cerebral artery is reported to be around 043sient current can be recorded (465, 643), and this currentcan be blocked by 1 mM 4-AP, but not by 5 mM TEA mV (262), indicating that most of the inward rectifier K/



channels are closed. However, because Ba2/ (0.5 mM), the unitary conductance for the KATP found in rabbit andguinea pig ventricular cells (Ç75–80 pS in symmetricala blocker of the inward rectifier K/ channel, caused a

depolarization of the membrane (262), the inward rectifier 140 mM K/ solutions; Refs. 284, 382, 844, 1093) was closeto those of the cloned KATP channels. Moreover, the uni-K/ channel did make some contribution to the resting

membrane potential. Possibly, closure of this channel may tary conductances of the KATP channels found in pancre-atic b- and insulin-secreting cells were, respectively, 88be more important than its opening. Closure might be

important for depolarization induced by vasoconstrictors pS (mouse b-cell) and 50–55 pS (RINm5F, Ref. 927; HITT15, Ref. 816). On the other hand, smaller unitary conduc-or for keeping a relatively high membrane potential (depo-

larized condition), whereas opening of this channel would tances have been reported for guinea pig urinary bladder(10 pS), porcine coronary artery (35 pS), porcine urethraset the resting membrane potential at a relatively low

level. It is interesting and well established that the prop- (43 pS), and rabbit portal vein (24 and 50 pS) (for review,see Ref. 626). Conversely, larger values were obtained inerty of inward rectification is not a property of the channel

itself but depends on a unidirectional channel block rabbit mesenteric artery, canine aorta, and rat ventrome-dial hypothalamic neurons (135 pS, Ref. 1023; 130 pS,caused by intracellular Mg2/ and polyamines (706, 1207).

Recently, gene cloning has led to the identification of simi- Ref. 610; 150 pS, Ref. 34) in symmetrical K/ (140 mM) orasymmetrical high K/ conditions (60 mM/140 mM K/).lar K/ channels with inward rectifying properties, such

as the ATP-regulated K/ channel (ROMK1; weak rectifying Figure 5 shows the inhibitory action of ATP on the unitaryK/ current recorded from dispersed SMC of the rabbitK/ channel) and G protein-gated K/ channels (IKACh;

GIRK1) (419, 433, 612). However, classical inward rectify- portal vein.Glyburide (glibenclamide) and other sulfonylurea de-ing, ATP-regulated, and G protein-gated K/ channels have

not yet been identified in VSMC. rivatives are selective blockers of KATP channels in pancre-atic b-, neuronal, cardiac, and SM cells. However, cloningof KATP channels from cardiac and pancreatic islet cells5. ATP-sensitive K/ channelsshowed that the subunits forming the channel pore hadno sulfonylurea binding site (35, 468). In fact, the sulfonyl-The ATP-sensitive K/ channels were identified in car-

diac muscle by Noma (827), and the channels have since urea receptor has been identified as a peptide with 13transmembrane domains (TM13) and two nucleotide bind-been found in various tissues, such as pancreatic b-cells,

neurons, cardiac and skeletal muscle cells, and SMC, ining (folds) sites, and it is thought that a combination ofinward rectifier K/ channel (composed of 2 transmem-cluding visceral tissues. Recent cloning of ATP-sensitive

K/ channels from the rat heart (cKATP; Kir 6.1) and pancre- brane segment, TM2; Kir 6.1, Kir 6.2) and sulfonylureareceptor (SUR1, SUR2) forms the KATP. It is reported thatatic islets (uKATP; Kir 6.2) has revealed that this channel

belongs to the superfamily of inward rectifying K/ chan- a combination of Kir 6.2 and SUR1 forms pancreatic KATP

and that a combination of Kir 6.2 and either SUR2A ornels, since it has two transmembrane domains with an H5

region and a weak rectifying property. The cKATP shows SUR2B forms the cardiac or SM-type KATP. It is of interestthat a combination of Kir 6.1 and SUR2 produces a GDP-a high degree of homology to other subfamilies (73% ho-

mology to GIRK, 70% to IRK, 64% to ROMK), but uKATP activated channel (359, 493, 1015, 1193). Figure 6 showsthe topography of KATP channels (A: KATP is composed ofshowed a lower level of homology (43–46%) (35, 468). By

Northern blotting of the mRNA of both KATP channels, it sulfonylurea receptor and KIR channel; B: the structure ofsulfonylurea receptors, Ref. 1018).has been found 1) that cKATP is highly expressed in heart,

kidney, spleen, submaxillary gland, thymus, hypothala- The KATP channels open under pathophysiologicalconditions, such as cardiac ischemia, but they may alsomus, and hippocampus, but not in skeletal muscle or SM,

including that of the small intestine and uterus (35); but open under physiological conditions via receptor activa-tion. This channel is also pharmacologically important as2) that uKATP is expressed in a wide variety of tissues,

including skeletal muscle and SM (stomach, small intes- a target for K/ channel openers. So far, activation of re-ceptors by Isop, CGRP, adenosine, vasopressin, somato-tine, and colon) as well as heart, kidney, liver, brain, testis,

adrenals, pancreatic islets, and ovary (468). However, the statin, and galanin has been reported to open KATP chan-nels, whereas ACh and vasopressin block the channelssame authors noted that uKATP was not expressed in an

insulin-secreting cell line, in which an ATP-sensitive K/ found in various tissues, including VSMC (Isop, Ref. 798;CGRP, Refs. 110, 910; adenosine, Ref. 234; somatostatin,channel has been recorded, or in other cell lines (PC12,

GH3, AtT-20, and endothelial cell lines), suggesting the Ref. 524; galanin, Ref. 67; ACh, Ref. 816; vasopressin, Ref.1143). The mechanism underlying the activation of KATPpresence of another type of KATP channel in these cells

(468). Both types of KATP channel had inward rectifying channels by CGRP and Isop has been said to involvecAMP-PKA stimulation, since forskolin, cAMP, and PKAproperties when Mg2/ was in the cytosol, and they had

similar unitary conductances (68 pS, cKATP; 70 pS, uKATP; directly open the channels, whereas okadaic acid, a phos-phatase inhibitor, prevents deactivation of the channelboth in symmetrical 140 mM K/ solutions). The value of



FIG. 5. Inhibitory actions of ATP on the 15-pS K/ channels of the smooth muscle cells (SMC) of rabbit portal veinat a holding potential of 010 mV. The 15-pS K/ channels were activated using physiological salt solution containingpinacidil (100 mM) with charbybotoxin (100 nM) in the pipette and with high-K/ solution containing GDP (1 mM)superfused in the bath. ATP was applied as Na2ATP. A: traces of 15-pS K/ channel in presence of 1 mM GDP alone andwith ATP. These traces were recorded from same patch membrane. Dashed lines indicated 1-min intervals. In A, tracestaken at a faster speed are also shown. B: open-time histograms obtained in presence of 1 mM GDP alone or in presenceof GDP with 10 or 100 mM ATP. Histograms were fitted with a single exponential curve with time constant (topen) notedin each histogram. Histograms were drawn from records for 4 min (no ATP) or 2 min (10 or 100 mM ATP), and all wereobtained from same prepartaion. [From Kajioka et al. (533).]

(1226). In the case of adenosine-induced activation, Dart bis-indolylmaleimide) or guanosine 5*-O-(2-thiodiphos-phate) (GDPbS). Because vasopressin is known to alsoand Standen (234) reported that the adenosine receptor

coupled with the KATP channel was the A1, and not the A2 stimulate PLC and thus synthesize InsP3 and DAG, vaso-pressin might inhibit KATP channels through PKC.receptor. They therefore concluded that the cAMP-PKA

system did not contribute to the activation of the KATP In SMC, it has been known for some time that severaltypes of drugs, such as nicorandil, cromakalim, minoxidil,channel by adenosine. Rather, they considered that a Gi

protein was involved in the adenosine-induced channel and pinacidil, hyperpolarize the membrane or relax thetissue (for review, see Refs. 28, 130, 263, 579, 626, 1176).opening, since direct introduction of the Gia subunit into

the cytosol activated the channel through a membrane- Now, it is well accepted that K/ channel openers directlyopen KATP channels in various cells; this has been showndelimited pathway.

With regard to the channel’s inhibition by vasopressin by means of single-channel current recordings from iso-lated cells and because cloned KATP channels could bein insulin-secreting and vascular cells, a direct blocking

action has been suggested on the basis that vasopressin opened by K/ channel openers (35, 468). On the otherhand, Helevinsky et al. (396) considered that KATP channelinhibits the KATP channel in the outside-out patch, but not

in the cell-attached patch configuration (698, 1143). On openers act as a potent Cl0 channel inhibitor in vascularSMC (hyperpolarization of the membrane).the other hand, Bonev and Nelson (109) showed that inhi-

bition of KATP channels by ACh (through muscarinic recep- Activation of KATP channels results in membrane hy-perpolarization, which causes suppression of action po-tor activation) could be mimicked by the application of

phorbol esters (phorbol 12-myristate 13-acetate) or diacyl- tential generation. In canine colonic SMC, cromakalimreduced the amplitude, duration, and maximum rate ofglycerol analogs (e.g., 1-oloyl-2-acetyl-sn-glycerol; OAG),

and attenuated by either PKC inhibitors (calphostin C and rise of the slow wave, without affecting its frequency (286,



FIG. 6. A: topology of ATP-sensitive K/ (KATP) channelthat is composed of 2 subunits. One subunit is composed ofsulfonylurea receptor [SU; an ATP-binding cassette (ABC)family] that contains 2 nucleotide-binding folds (NBF) andpossesses 13 transmembrane segments (TM13). AnotherKATP subunit is Kir and is composed of 2 transmembranesegments (TM2). B: structures of SU receptors (3 isoforms:pancreatic type, r-SUR1; cardiac type, m-SUR2A; smoothmuscle type, m-SUR2B). /, NH2-terminal glycolation sites;TM, transmembrane regions; n, phosphorylation sites ofprotein kinase A; m, phosphorylation sites of protein ki-nase C. (From Y. Kurachi, personal communication.)

898). Although cromakalim does have direct inhibitory The activity of KATP channels is modulated by cyto-solic factor(s), since channel activity disappears or de-actions on L-type Ca2/ channels (854, 898), the inhibition

of action potentials or of slow waves by K/ channel open- clines (‘‘rundown’’) on membrane excision in rabbit portalvein and guinea pig ventricular cells. Mg-ATP is one ofers are considered to be indirect actions that are second-

ary to membrane hyperpolarization. Neither muscle relax- this channel’s modulators, because this combination pro-duces a reappearance of channel opening by phosphoryla-ation nor contraction, whether induced by inhibitory

nerves, b-adrenoceptor, or muscarinic receptor stimulation. On the other hand, the rundown phenomenon wasnot seen in the KATP channels of the rat portal vein (534,tion, were modified by glyburide in the canine colon, indi-

cating that KATP channels do not participate in nerve-medi- 1225). The diphosphates of various nucleotides (e.g., ADP,GDP, UDP, IDP, and CDP) augmented KATP channel activ-ated responses in this tissue (929). Similarly, although

neurotransmission was inhibited by K/ channel openers ity and also reactivated the channels after the occurrenceof complete channel rundown. Actually, Mg2/ are nor-indirectly through membrane hyperpolarization, it was

not affected directly via inhibition of transmitter release mally thought to be essential for the reactivation of KATP

channels by ATP and other nucleotides, and for the pre-(in guinea pig and rabbit mesenteric arteries, Refs. 797,804). However, there is substantial evidence that the KATP vention of channel rundown (61, 62, 535, 976, 1093). How-

ever, in the rabbit portal vein, the application of GDPchannel does contribute to nerve-mediated responses inguinea pig small intestine and trachea and rat CA3 neu- without Mg2/ could open the channel even after rundown,

according to Beech et al. (61) and Tung and Kurachirons (67, 453, 714, 1236). In the guinea pig ureter, electricalfield stimulation evoked a TTX-sensitive transient hyper- (1110). In contrast, in the studies of Kajioka et al. (533)

and Kamouchi and Kitamura (538), GDP with or withoutpolarization (IJP, Ref. 958). Because these IJP were capsa-icin and glibenclamide sensitive, and because exogenous Mg2/ failed to reactivate the KATP channels in the rabbit

portal vein. The reason for this discrepancy is not clear,application of CGRP hyperpolarized the membrane, it wassuggested that CGRP might activate KATP channels in this but the presence of such a discrepancy may be an indica-

tion that other cytosolic modulating mechanisms, in addi-tissue (685, 958).



tion to GDP binding, are needed for channel reactivation. cells, no report has yet been published concerning itsexistence in SMC. Because cardiac Na/ channels have aChannel phosphorylation might be such a mechanism; in-

deed, in cardiac cells, nucleotide diphosphates (NDPs) sensitivity to TTX and STX that is low by comparisonwith that seen in neuronal cells, the TTX-resistant Na/proved able to reactivate the dephosphorylated channel,

indicating that either phosphorylation or NDP binding channels observed in some SMC might be classified ascardiac-type Na/ channels (type h1). On the basis of datamay be required for channel reactivation (975). Recently,

Furukawa et al. (326) demonstrated that F-actin, but not from blot hybridization analysis of the brain types I-IIIand glial-type Na/ channels, only type III channels areG-actin, prevented rundown in cardiac cells and that chan-

nel phosphorylation (by Mg-ATP) did not reactivate the present in intestinal SMC (345, 1058). In the rat azygosvein, the TTX sensitivity of the fast Na/ current was lowerKATP channel after the rundown induced by cytochalasin

D or long exposure to high Ca2/. They speculated that than the TTX sensitivity of those in cardiac and gastricSMC, so these vascular cells may have a novel Na/ chan-stabilization of the channels in the membrane by F-actin

is essential for maintaining their activity. Although the nel, like the epithelial Na/ channel (1044, 1119). Differ-ences in TTX sensitivity seem to be determined by differ-nature of any such interaction between KATP channels and

F-actin is not clear, this could indicate the presence of ences in amino acid sequence, with variation occurring inone amino acid in the SS2 region in domain I (395, 1082).additional endogenous mechanisms for channel reactiva-

tion. Other toxins that modify Na/ channel gating have not yetbeen tested on SM Na/ currents. Moreover, chloramine-The K/ channel openers failed to open KATP channels

after rundown in guinea pig cardiac and rabbit vascular T inhibited the inactivation of the TTX-sensitive Na/ cur-rent in the rabbit pulmonary artery (852). As far as thesecells; however, these drugs did open the same channels

in the cell-attached condition, or in the presence of NDP effects were concerned, the pharmacological propertiesof the voltage-dependent Na/ channels in SMC were theor Mg-ATP under cell-free conditions, in guinea pig ven-

tricular muscle and the rabbit portal vein (61, 533, 538, same as those seen in neuronal and cardiac cells.Kao and McCullough (543) first reported, in rat uterine975). Consequently, Shen et al. (975) and Kamouchi and

Kitamura (538) proposed the idea that KATP channels have and guinea pig taenia coli, that a part of the action potentialis accounted for by a Na/ current. Because the pioneeringtwo states, switchable by endogenous modulator(s), that

is, operative and inoperative states. They considered that experiments were carried out using the multicellular su-crose-gap method, the early consensus about the nature ofK/ channel openers could open the channel only when it

was in the operative state. the ionic currents making up the action potential in VSMCwas that it was due solely to activation of the VOCC. It isIn the guinea pig urinary bladder, KATP channels can

be continuously recorded in outside-out patches, and K/ now clear that many VSMC generate both Na/ and Ca2/

currents (rat azygos vein, rabbit main pulmonary artery,channel openers augmented the channel’s activity as theydid in the rat portal vein (108, 109, 534, 1225). Because rat portal vein, rat aorta, and rat vena cava, for review, see

Ref. 626; rat ileum, Ref. 997; rat and human colons, Ref.GDP and other NDP activated the KATP channel under cell-free conditions, other mechanism(s) might be present for 786; guinea pig taenia coli, Ref. 1202; rat stomach fundus

and guinea pig ureter, Ref. 1607; rat stomach, Ref. 1201;the prevention of the rundown phenomenon, other thanthose involving NDP or channel phosphorylation (by Mg- rat uterus, Refs. 24, 543, 848, 1019, 1021, 1022, 1217). The

Na/ current is activated within 1–2 ms, inactivated withinATP).10 ms, and is highly sensitive to extracellular Na/ concen-tration. From such evidence, we can deduce the presence

B. Na/ Channelsof a TTX-resistant Na/ current component in the actionpotential in a given tissue, i.e., some tissues are highly

1. TTX-sensitive and -resistant Na/ channelssensitive to TTX (an IC50 of 10–200 nM and found in ratportal vein, Ref. 854; rabbit pulmonary artery, Ref. 855;In excitable cells, the present classification of Na/

channels recognizes the following subtypes: I, II (IIA), III, guinea pig ureter, Ref. 786; rat and human colons, Ref.1187; rat portal vein, Ref. 854; rat vena cava, Ref. 746).ml, and h1 subtypes (on the basis of their cDNA cloning)

(174, 565). These have been described in rat neurons, These currents are similar to, or the same as, that observedin nerve fibers (presumably type II). Other tissues are resis-skeletal muscle, and heart cells. Saxitoxin (STX), TTX,

and m-conotoxin are the pharmacological tools used for tant or less sensitive to TTX (an IC50 of the micromolarorder; rat uterus, Refs. 25, 848; rat stomach, Refs. 786, 1200;their separation. Epithelial cells have a further type of

Na/ channel, which is sensitive to amiloride, a diuretic, rat azygous vein, Ref. 1244). Furthermore, a third type ofNa/ channel has been identified in cultured A7r5 cells andand which has a channel structure that is different from

that of the classical voltage-dependent Na/channels, such in rat aortic and portal vein cells (2341); this epithelial-likeNa/ channel had a voltage-independent property and wasas type I. Although the amiloride-sensitive Na/ channel

has been identified in rabbit colon and lung epithelial insensitive to TTX and amiloride, but sensitive to phenamil.



TABLE 2. TTX sensitivity of voltage-dependent Na/ channel in visceral smooth muscle cells

Cells KD (TTX) Vactivation, mV Vinactivation, mV Reference No.

RatGastric fundus 870 nM 786, 1200Uterine leiomyosarcoma 47 nM 015 068 635

68 nM 018 073 635Pregnant uterine 27 nM 848Pregnant uterine 2 mM (binding) 25Colon 130 nM 036.1 074.5 1187Ileum 4.5 nM 021.8 060 998Azygous vein 30 mM 060 1044

DDT MF2 (vas deferens) 200 nM 040 766A7r5 (aortae) ú100 mM 1119Human colon 14 nM 069.5 1187Rabbit pulmonary artery 8.7 nM 065 855

KD, dissociation constant; TTX, tetrodotoxin; Vactivation, activation potential; Vinactivation, inactivation potential.

In rat uterine SMC, the current density of the TTX- and leiomyosarcoma cells (0.6–0.7 and 3–4 ms, Refs. 78,635); however, the sensitivity to TTX was similar for bothsensitive Na/ channel increased during pregnancy (489,

697), possibly enhanced by factor(s) in the serum. In this components in a given tissue (IC50 Å 2.3 and 7.8 nM innode of Ranvier; 47.1 and 67.5 nM in leiomyosarcomatissue, Sperelakis et al. (1019, 1020) demonstrated that

the number of TTX-sensitive fast Na/ channels increased cells). In many Na/ channels, such as those in rat brainand cardiac muscle, b1- and/or b2-subunits are associatedduring gestation, the average current density (in pA/pF)

increasing linearly from 0 on day 5, to 0.19 on day 9, to with the a-subunit, and the b1-subunit has been found toaccelerate the inactivation kinetics of brain and skeletal0.56 on day 14, to 0.90 on day 18, then stabilizing at 0.86

on day 21. Kusaka and Sperelakis (635) speculated that muscle Na/ channels. However, a b-subunit was not al-ways found with an a-subunit in the voltage-dependentnerve growth factor (NGF) and fibroblast growth factor

(FGF) might play key roles in regulating Na/ channel ex- Na/ channel (for example, rat skeletal rNaSk2 channel,Ref. 1160), and expression of the a1-subunit would bepression during pregnancy, and possibly to some extent

in leiomyosarcoma cells. In a study in which they used a sufficient to produce a functioning channel capable ofshowing current inactivation. Because the b1-subunit hasconventional microelectrode technique, Inoue et al. (487)

found that superfusion with Na/-deficient solution elimi- been identified in the human uterus, the two inactivationkinetics observed in leiomyosarcoma cells may indicatenated the active responses (small action potentials) ob-

served in Ca2/-free solution in the human pregnant myo- the presence of two types of Na/ channels, one with andone without a b-subunit. However, Moran et al. (771) re-metrium, although they did not test the TTX sensitivity of

such active responses. The evidence suggests that a few ported that expression of the type II Na/ channel in Xeno-

pus oocytes produced a Na/ current with fast and slowto 10% of the population of Na/ channels is available atthe resting membrane potential of050 mV in the pregnant inactivation properties, suggesting that a single type of

Na/ channel might possess two different gating mecha-myometrium (1019–1021) and in other SMC (intestine,Ref. 1187; gastric fundus, Ref. 786). This ‘‘window current’’ nisms. Indeed, PKC slowed the current inactivation in rat

ventricular cells (831, 909), and forskolin produced a verymay contribute to the triggering of the Ca2/ action poten-tial at the resting membrane potential and to the depolar- slow inactivating component by a direct action on the

channel (858). There has been no report, so far, to indicateization phase just after membrane hyperpolarization.However, this interpretation will be inapplicable to the whether or not Na/ channels in SMC are modulated by

intracellular messengers.rabbit pulmonary artery, in which the TTX-sensitive Na/

channel was first described, because this tissue does not The site producing inactivation of Na/ channels hasproduce action potentials under normal conditions (855). been said to be in the linker region between domains IIIMore data are needed to clarify the physiological impor- and IV, because partial deletion of the amino acids in thistance of the Na/ channels found in SMC, particularly how region was found to eliminate the fast inactivation of theand where this channel current contributes to the genera- Na/ channel (rat brain type II channel, Refs. 890, 1171;tion of spontaneous activity (the TTX sensitivity of Na/ human heart channel, Ref. 386). On the other hand, Moranchannels, their Kd values, and activation and inactivation et al. (771) and Freig et al. (302) reported that proline orvoltages in VSMC are indicated in Table 2). lysine residues in the S4 region of domain II were im-

portant for the slow inactivation of the Na/ current. Be-From their inactivation time constants, fast and slowcomponents have been identified in frog node of Ranvier cause mutations in these positions do not alter the chan-



nel’s activation kinetics, activation and inactivation sites and 3) rapid inactivation (guinea pig taenia coli, Ref. 1213;human myometrium, Ref. 487). In guinea pig portal veincan be presumed to be distinctly separated, but multiple

sites may contribute to channel inactivation. and rat sensory neurons, the presence has been reportedof DHP-sensitive, low voltage-activated Ca2/ channels(294, 498); however, no such Ca2/ channel has been identi-

C. Ca2/ Channelsfied in VSMC.

In general, the L-type channel is thought to represent1. Subtypes of voltage-dependent Ca2/ channels and

the main pathway for Ca2/ entry into SMC after the gener-their specific characteristics

ation of action potentials, whereas the role of the T-typeor low-voltage threshold Ca2/ channel is uncertain be-The main ionic current for action potential generation

in SMC is the Ca2/ current that passes through VOCC. As cause this channel is normally inactivated at the restingmembrane potential seen in a variety of SMC. Recently,deduced from a number of pieces of evidence obtained

in microelectrode experiments with Ca2/ antagonists, the a low voltage-gated, DHP-sensitive Ca2/ channel was re-ported to be activated during the silent phase betweenmain Ca2/ channel distributed in the SM cell membrane

is the L-type channel. In VSMC, the Ca2/ current is a major spikes in rat dorsal root ganglion (DRG) neurons (294).The open probability of the 10-pS DHP-sensitive channelelement in the formation of the rising phase of the action

potential. Voltage-operated Ca2/ channels in VSMC are showed a reverse voltage dependence, when a brief butstrong depolarizing pulse was applied just before the stim-classified as either L type (long lasting; or high voltage

activated, HVA; 20- to 28-pS channel conductance) or T ulation. On the basis of simultaneous recordings of unitaryand whole cell currents, these authors concluded that thistype (transient; or low voltage activated, LVA; 7- to 15-pS

channel conductance), with the N type (neither L nor T) 10-pS channel did not contribute to the action potential,but instead participated in the modulation of spike fre-being absent. In some SMC, the T-type channel has been

identified (guinea pig taenia coli, Ref. 1214; human myo- quency. This idea might be tested in these SMC that pos-sess T-type channels.metrium, Ref. 487; guinea pig ileal circular muscle, Ref.

258; SM cell line, Ref. 303). On the other hand, the L-type Calcium channels possess heteromultimeric subunits(e.g., a1-, a2-, b-, g-, and d-subunits in the case of the L-channel alone has been found in other places. Such an

identification has been carried out 1) in vascular SMC type channel, Refs. 156, 1030). Similarly, a functional N-type Ca2/ channel in rabbit brain was found to be com-(rat mesenteric artery, rabbit mesenteric artery, rat portal

vein, rabbit ear artery, dog saphenous vein, guinea pig posed of a1-, a2-, b-, and d-subunits, as well as a 95-kDasubunit that was three times larger than the g-subunitportal vein, guinea pig coronary artery, rabbit coronary

artery, human cystic artery, human mesenteric artery, rab- (1177). The present classification of the channel pore-forming subunit (a1) recognizes a1s , a1A , a1B , a1C , a1D ,bit portal vein, and rabbit basilar artery, see review in

Ref. 1276), 2) in GI tract (intestine, Refs. 1, 341, 342, 490, and a1E types. Of these a1-subunits, three types (a1S , a1C ,and a1D) have been found to help form DHP-sensitive Ca2/644, 645, 652; stomach, Refs. 554, 761, 985, 1130, 1132,

1133), 3) in airway SMC (dog, Ref. 608; guinea pig, Ref. channels in skeletal muscle, brain, heart, pancreatic isletcells, and SMC. In three types of a1-subunit in DHP-sensi-418; rat and human bronchus, Refs. 696, 1179), and 4) in

urinary bladder (336, 337). Furthermore, three types of tive Ca2/ channels in SMC, an a1C-subunit has been identi-fied (rat aorta, Refs. 598, 599; rabbit lung, Ref. 87; canineCa2/ channels have been identified on the basis of mea-

surements of unitary current conductance in guinea pig colonic SM cell, Ref. 928). In the rat aorta and rabbitlung, the full length of the amino acid sequence of the a1-intestinal SMC (1214): two of them are thought to be T-

and L-type channels, but the third type has yet to be identi- subunit has been deduced; it shows a 65% homology withthat of rabbit skeletal muscle and 93% or higher homologyfied. Smirnov and Aaronson (998) and Bkaily (90) also

suggested that other types of Ca2/ channel might be pres- with that of the rabbit cardiac channel (87, 598). Northernblotting analysis has shown that this a1-subunit (a1C) isent in rat ileum and aorta, viz., N and R type channels;

however, this has yet to be confirmed. Characteristics of also present in uterus, stomach, lung, small intestine, andlarge intestine (598). The rat brain C-type a1-subunit hasvoltage-dependent Ca2/ channels in VSMC are shown in

Table 3 (except for vascular SMC). been further divided into to rbC-I and rbC-II subtypes(alternative splicing variants), the major splice in the caseL-type channels in VSMC have the same properties

as those found in cardiac and vascular SMC. Thus the of canine colonic SMC being the rbC-II, whereas that ofthe rabbit aorta, rabbit heart, rat heart, rabbit lung, andchannel 1) is activated at a relatively high membrane po-

tential, 2) permeates the Ba2/ better than Ca2/, 3) is human brain was of the rbC-I type (291, 928, 1009). Inrabbit intestine and trachea and rat A7r5 cells, the twoblocked by Ca2/ channel blockers, and 4) is inactivated

by both membrane depolarization and intracellular Ca2/. types of variant were equally transcribed (291). Other sub-types of the a1-subunit have been given the names P typeThe T-type channels found in visceral cells exhibited 1)

low voltage activation, 2) resistance to DHP derivatives, (a1A) and N type (a1B) according to the pharmacological



TABLE 3. Voltage-dependent Ca2/ channels in visceral smooth muscle cells

UnitaryConductance, DHP

Classification Cells Type Structure pS mRNA in SMC Sensitivity Reference No.

L type Canine colon a1C 12 kb (RNA) 21 Colon, heart, brain Sensitive 9288 kb (RNA) 8 Sensitive 928

Rabbit lung 2,166 aa; 242 kDa Sensitive 87Rat aortae 6.5 kb, 8.6 kb (RNA) Aortae, brain, Sensitive 599

skeletal muscleNewt stomach 12 Sensitive 156Guinea pig

taenia coli 30 Sensitive H. Tokuno, personalcommunication

Urinary bladder 25 Sensitive 1214Rabbit ileum 25 Sensitive 490Human uterus 29 Sensitive 487

T type Guinea pigtaenia coli 7 Insensitive 1214

Urinary bladder 8 Insensitive H. Tokuno, personalcommunication

Human uterus 12 Insensitive 407Intermediate type Guinea pig

taenia coli 12 1214

DHP, dihydropyridine.

and biophysical properties of their expressed channel (for and results with a specific antibody. There are two alterna-tive spliced forms of the d-subunit (skeletal muscle typereview, see Ref. 1030). An a1E-type subunit has been iden-

tified in the rat brain and classified as a low voltage-acti- da and brain type db). In cardiac cells, the presence of aa2-subunit has been confirmed, as it has in skeletal musclevated channel, but the inactivation rate of the current

recorded from the expressed channels showed different and brain (1177); however, this subunit has not yet beenidentified in SMC.kinetics from that of the previously described T-type chan-

nel (1016). Pore formation by the Ca2/ channel a1-subunit is welldocumented (177), and now the S5-S6 region of each do-The b-subunit is considered to be located beneath

the a1-subunit. There are four genes for the b-subunit, main is recognized as forming the channel wall. Othersubunits, when associated with the a1-subunit, producenamely, b1 , b2 , b3 , and b4 . Alternative spliced forms of

b1 and b2 (b1a , b1b , b1c , b2a , and b2b) also exist. Collin et modulation of the channel’s properties. Experiments per-formed after coexpression of various subunits with a1al. (207) identified a b3-subtype in the colon, small intes-

tine, and lung in humans as well as in brain and ovary, have shown that they positively modulate the channel’skinetics and incorporation into the membrane (171,but not in the heart, liver, or kidney. On the other hand,

Hullin et al. (445) demonstrated that b2- and b3-isoforms 172, 208, 376, 673). Itagaki et al. (494) reported that theamplitude of the Ba2/ current through the aortic a1-sub-could be identified in trachea and aorta. However, b1- and

b4-isoforms have not yet been detected in SMC (54, 900). unit (a1C type) was higher when it was coinjected withthe skeletal muscle b-subunit, but that this did not changeA further subunit, the g-subunit, has been identified in

skeletal muscle and lung, but not in brain, heart, kidney, the inactivation time constant. Welling et al. (1169) alsoreported that coexpression of an SM a1C-subunit with ei-liver, or stomach (115, 515, 899). This subunit is thought

to have four transmembrane regions and is expected to ther a skeletal b1- or an SM b3-subunit enhanced the Ca2/

current in the Chinese hamster ovary membrane, the b1-associate with an a1-subunit. Tissue-specific expressionof g-subunit RNA has not been tested for in other SM subunit inducing a much greater effect than the b3-sub-

unit. On the other hand, the b3-subunit moderately ortissues. Apparently, a2- and d-subunits are derived froma single gene and bound covalently by a disulfide linkage markedly enhanced the endogenous Ca2/ channels in Xen-

opus oocytes, which had DHP-insensitive Ca2/ channels(177, 272, 516). Two transmembrane regions in the a2-subunit and a single transmembrane region in the d-sub- or expressed cardiac a1-subunits (171, 207, 673). Further-

more, the skeletal muscle b-subunit reduced the ampli-unit are thought to be present (177). Recently, Gurnettet al. (376) proposed a different idea for the topological tude of the Ba2/ current passed by the skeletal muscle

a1-subunit but increased the current passed by the cardiaclocation of the a2d-subunit; in their scheme, the a2d-sub-unit was considered to have a single transmembrane re- a1-subunit (673, 1123). These pieces of evidence suggest

that all types of b-subunit probably modulate the activitygion in the d-area, to judge from the N-glycosylation sites



of the a1-subunit and that a given channel’s particular at which phosphorylation can be induced by PKA andPKC (564). In aortic and lung a1-subunits, there are said tocombination of a1- and b-subunits would be very im-

portant in determining its potency. be, respectively, five or two putative PKA phosphorylationsites (87, 598) located in the COOH-terminal regionBecause coexpression of a g-subunit with a cardiac

a1-subunit produced no effect on the channel current (S1574, S1626, S1699, S1847, and S1927 in rat aorta; S1622,S1923 in rabbit lung; in the lung, other serine residues(1166), the g-subunit is thought to contribute to the stable

expression of the a1-subtype (515). However, Wei et al. were also present in positions corresponding to those inthe aortic subunit). In cardiac and skeletal muscles, it has(1166) also reported that, when coexpressed, g-subunit

and a1- (cardiac type) and b1a-subunits (skeletal muscle been deduced that there are four or six predicted PKAphosphorylation sites in the COOH-terminal region andtype) acted cooperatively to change the Ca2/ current gen-

erated by an a1b-subunit. Because the g-subunit has been one in the NH2-terminal region or in the linker regionbetween domains II and III (175, 741, 938, 939). However,cloned only from skeletal muscle, and this type of g-sub-

unit is not present in the stomach (515, 899), determina- all these predicted sites may not always be phosphory-lated by PKA; in fact, in skeletal muscle, the most im-tion of the physiological relevance of the g-subunit to

SMC needs further investigation. portant serine residue is believed to be S1854 (639, 939).Furthermore, Hell and co-workers (397, 398) noted thatLike the b-subunit, the a2-subunit has been reported

to enhance the Ca2/ current without inducing a change there were two different-length forms of the neuronal a1C-and a1D-subunits (210 and 247 kDa vs. 190 and 187 kDa)in kinetics (494). On the other hand, Gurnett et al. (376)

found that an a2- or d-subunit alone induced no augmen- and that PKA only phosphorylated the longer subunit ofa1C . They considered that the shorter a1C-subunit might betation of the current passing through a1A- to b4-subunits.

They also reported that no synergistic action was seen created when the long form was truncated by proteolysis,whereas the shorter sizes of the other classes of a1-sub-even when a2- and d-subunits were independently ex-

pressed in the same cell from their separate cRNA, units (a1A , a1B , a1D , and a1E) might be expressed by alter-native splicings (397). Thus cAMP-dependent phosphory-whereas current augmentation was induced after coex-

pression of an a2d-subunit from the same cRNA. lation sites could be eliminated by posttranslational trun-cation of the a1C-subunit (1237).In the rat myometrium during pregnancy, Tezuka et

al. (1082) investigated the mRNA levels for the a1- and b- On the basis of electrophysiological experiments,cAMP-dependent and/or b-adrenergic modulation of thesubunits of the L-type VOCC to determine whether alter-

ations are associated with term or preterm labor. They L-type Ca2/ channel has been well documented in cardiacmuscle cells. However, the purified a1-subunit of cardiacconcluded that mRNA levels for the VOCC subunits in-

crease before both term and preterm labor but decline cells is not phosphorylated by PKA (967, 1209), suggestingthat the phosphorylation site for PKA in the cardiac L-during periods when VOCC are likely to be at their peaks.

The increase in the levels of mRNA for VOCC most likely type Ca2/ channel is on the b-subunit rather than the a1-subunit. In SMC, injection of cAMP or superfusion withreflects a change in the expression of VOCC during term

and preterm labor that may facilitate the increase in uter- membrane-permeable cAMP (dibutyryl cAMP) either hadno effect on the L-type current (guinea pig urinary blad-ine contractility required for this process. The so-called

‘‘progesterone withdrawal’’ or ‘‘progesterone blockade’’ der, Ref. 588; rabbit intestine, Ref. 846) or increased theCa2/ current (pig coronary artery, Ref. 310; trachea, Ref.appears to be responsible for regulating the levels of

mRNA for VOCC in the myometrium in preparation for 1168). Moreover, in guinea pig taenia coli, both resultshave been reported to be induced by Isop and cAMP appli-labor.cation (784, 1190). In pig coronary artery, b-adrenergicstimulation and forskolin both enhanced the Ca2/ current2. Modulation of Ca2/ channels by agonists andthrough a cAMP-dependent process (310), whereas in thesecond messengerstrachea and guinea pig taenia coli, enhancement of theCa2/ current by b-adrenoceptor activation did not involveLike the known regulatory actions of various receptor

agonists on Ca2/ mobilization, such as activation of ligand- a cAMP-dependent pathway (784, 1168). Recent molecularbiological studies have suggested that the cAMP-depen-gated ion channels and modulation of intracellular Ca2/

store sites, the direct and indirect regulation of voltage- dent phosphorylation site on the b-subunit is Thr-165 inskeletal muscle and neuronal b1- and b2a-subunits, a resi-dependent Ca2/ channels by receptor agonists has been

extensively investigated. Channel modulation by agonists due which is not present in the SM b3-subunit (1237).Therefore, coexpression of different types of b-subunitcan occur through one of two different types of mecha-

nism, viz., direct modulation via G proteins or indirect may be a critical factor for cAMP-dependent phosphoryla-tion of L-type SM Ca2/ channels. Interleukin-1b, tumormodulation via either second messengers or channel

phosphorylation. There is molecular biological evidence necrosis factor-a, and lipopolysaccharide also enhancedvoltage-dependent Ca2/ channels in the SMC of the ratfor the presence in the a1- and b-subunits of various sites



tail artery. However, these enhancements were not modu- alternative-spliced variants of the a1C-subunit might beaffected by PKC.lated by PGs but inhibited by dibutyryl cGMP (1173). The

underlying mechanisms have not yet been clarified (1186 It is interesting that all types of Ca2/ channel a1-subunits possess an EF-hand sequence in the COOH-ter-in vascular SMC). A nonreceptor tyrosine kinase (pp60c)

injected into the SM cells of the rabbit ear artery enhanced minal region; Ca2/ binding here might affect the channel’smodulation (37). Study of the first-order structures of thethe VOCC and, moreover, peptide A, tyrphostin-23, and

genistein (inhibitors of pp60c) inhibited the Ca2/ inward L-type Ca2/ channel (a1C-, a1S-, and a1D-subunits) hasshown that there is only one amino acid difference be-current (1172). These authors concluded that this modula-

tion of the voltage-dependent Ca2/ current was dependent tween them in the 29-amino acid sequence of EF hand,whereas 12-amino acid differences were present betweenon tyrosine phosphorylation, but not on activation of PKC.

It is well known that the coupling of various receptor L- and non-L-type channels. Because L-type channelsshow strong inactivating properties on exposure to highagonists with the Gq protein-PLC system causes augmen-

tation of the L-type channel in visceral and vascular SMC. intracellular Ca2/ (Ca2/-induced inactivation of the Ca2/

current, Ref. 846), differences in the Ca2/-binding se-Current augmentation is thought to occur through PKCactivation and/or by direct activation via PTX-sensitive quences of L- and non-L-type Ca2/ channels might be im-

portant for channel inactivation by ion flux or depolariza-and -insensitive G proteins, but not through InsP3/inositoltetrakisphosphate (InsP4) formation (850, 851, 1129, tion (37).1184). Direct application of DAG or PDBu, a phorbol ester,has been shown to produce augmentation of the Ca2/

3. Actions of Ca2/ agonists and antagonistscurrent, and H-7 or staurosporine prevented the currentaugmentation induced by PDBu (671, 851, 1129). How- From the structure of the channels and the actions

of Ca2/ antagonists, the Ca2/ channels in SM have beenever, Oike et al. (851) reported that histamine-inducedaugmentation of the L-type current was not affected by identified as L-type and a1C-type channels (87, 598, 599,

925, 1009). Exceptionally, Smirnov and Aaronson (998)such protein kinase inhibitors in rabbit vascular cells, sug-gesting that PKC-mediated current activation is not the classed as N type the Ca2/ channels in rat ileal SMC,

from their low sensitivity to DHP. The actions of Ca2/main process producing agonist stimulation. In guinea pigtaenia coli, calyculin A (a phosphatase type 1 and type antagonists and agonists fit well with the modulated re-

ceptor hypothesis proposed by Hille (411), and with the2A inhibitor) augmented the L-type current, and this effectwas inhibited by H-7 (1113). It is now thought that there modal model of Hess et al. (404). The Ca2/ antagonist

binding sites have been determined by physiological, bio-are several PKC phosphorylation consensus regions in thea1- and b-subunits (445, 564, 1030). In the case of the chemical, and molecular biological methods in neuronal

and cardiac Ca2/ channels (177, 619, 796, 921, 1018, 1041,neuronal a1C-subtype, different sizes of the protein (190and 210 kDa) have been reported (398), the smaller form 1076). Concerning the DHP-binding site of the L-type Ca2/

channel, Regulla et al. (923) reported that 10 amino acidsbeing the result of truncation by proteolysis. AlthoughPKA phosphorylation sites are deleted by such proteolysis near the EF-hand structure in the COOH-terminal region

formed the binding site for DHP antagonists in the skeletalat the COOH-terminal region, phosphorylation sites forPKC, CaM-dependent protein kinase, and PKG were pre- muscle a1S-subunit. Later, it was proposed that the major

functional interaction between DHP derivatives and theserved in both sizes of the subunit (377, 398). Stea et al.(1029) reported that, whereas the Ca2/ current passed by a1S-subunit occurred in its external region between S5

and S6 of domain IV (799, 1076). Electrophysiological evi-a1B- and a1E-subunits coexpressed with b1b was enhancedby PMA, that passed by a1A- and a1C-subunits was not dence tends to confirm this idea, since DHP derivatives

are effective in inhibiting the Ca2/ current only when ap-(1030). The PKC-dependent modulating site was deter-mined, by using a chimeric a1B/A-subunit, to be in a cyto- plied externally. There are big differences between sub-

units that are DHP sensitive (a1S-, a1C-, and a1D-subunits)plasmic region between domains I and II. These resultsindicate 1) that PKC phosphorylation sites in the b1b-sub- and those that are DHP insensitive (a1A-, a1B-, and a1E-

subunits) in terms of their amino acid sequence in theunit have no role in the current augmentation induced byPKC and 2) that PKC phosphorylation sites in the a1- SS1 region of domain IV, suggesting that a region near

SS1 of domain IV (S5–6) may be a DHP-binding site. Insubunit do not always modulate the channel’s activities.As the presence of a1C , but not of other a1-subunits, has SMC of the rabbit intestine and portal vein, D-600 has

been reported to inhibit Ca2/ channels from the extracel-been deduced in SMC, current augmentation by stimula-tion of PLC-coupled receptors may not directly involve lular side (649, 845). In contrast, in porcine coronary ar-

tery, as well as in neocortical neurons and cardiac ventric-regulation via the a1- and/or b-subunits. That being thecase, other subunits, such as b3 and/or a2 , may affect the ular cells, D-890 (a completely charged form of D-600)

inhibited the Ca2/ current from the inside of the mem-channel’s activity during PKC-mediated channel modula-tion. However, the possibility cannot be excluded that brane (239, 403, 589). The binding site for D-600 has been



predicted to be at the junction between the extracellular tion (156, 488), as also observed in rat DRG neurons (294).The latter property distinguished this channel from theand intracellular regions of S6 in domain IV (1041). Be-

cause the COOH-terminal region near S6 in domain IV is other T-like currents recorded in SM and cardiac cellsand other neurons. Although high concentrations of DHPwell conserved in various a1-subunits, including those of

the N- and P-type channels, and because there is no differ- derivatives inhibited other ionic currents (447, 1079,1208), inhibition of the above small conductive channelence between the amino acid sequences of cardiac and SM

a1-subunits, it is hard to explain the electrophysiological by DHP derivatives occurred at relatively low concentra-tions. Because there is as yet not a clear understandingdiscrepancy between the findings in these tissues in terms

of the structure of the phenylalkylamine-binding site of of the structure of DHP-resistant channels (T type) or ofthe DHP-sensitive, low-conductive channel, we mustthe a1-subunit. Consequently, modulation by other regions

of the a1-subunit or by other subunits should be consid- await further data before drawing firm conclusions aboutthe differences between these channels.ered. The effects of synthesized calciseptine (found natu-

rally in the venom of the black mamba) on voltage-depen-dent Ca2/ channels was observed by Teramoto et al.

D. Cl0 Channels(1080). Calciseptine voltage dependently and concentra-tion dependently inhibited the inward current and shiftedto the left the steady-state inactivation curve. Calciseptine Ion-sensitive microelectrode measurements put intra-

cellular Cl0 concentration at 40–50 mM, and the calcu-blocked the open probability of the 25- and 12-pS Ca2/

channels (more potently in the 25-pS channel) without lated Cl0 equilibrium potential from these values is 035to 020 mV (guinea pig vas deferens, cecum, and ureter,affecting the amplitude of the single-channel conduc-

tance. Thus they concluded that the actions of calcisep- Refs. 14–17). These results indicate a contribution by anactive Cl0 transport system to the accumulation of Cl0 intine are similar to those of DHP derivatives and that it

acts from the outside of the membrane. Figure 7 shows these SM cells, as well as accumulation by passive trans-port through Cl0 channels. In patch-clamp experiments, athe effects of nifedipine on the L- and T-type voltage-

dependent Ca2/ channel recorded from the guinea pig Ca2/-dependent Cl0 current has been identified in severalSMC (rabbit portal vein, Refs. 424, 1148, 1150, 1151; rabbitportal vein.

Unitary current analysis has shown that Ca2/ antago- colon, Refs. 1048, 1049; rat intestine, Ref. 841; rabbit pul-monary artery, Refs. 423, 424; rabbit esophagus, Ref. 18;nists shift the channel to mode 0 (no channel opening)

from mode 1 (short channel opening), a so-called reduc- A10 cell line, Ref. 425; guinea pig trachea, Ref. 515; pigtrachea, Ref. 668).tion of ‘‘channel availability,’’ whereas Ca2/ agonists shift

the channel to mode 2 (long channel opening) without a In SMC, genes expressing two types of Cl0 channelhave been identified by Northern blotting analysis,change in the mean open time (404, 517). In the rabbit

ileum, Inoue et al. (490) reported that DHP modified both namely, the ClC-2-type channel and the ICln channel (492,1085). The ClC-2-type Cl0 channels were first identified inchannel availability and mean open time; this was similar

to an observation made in guinea pig ventricular cells by rat heart and brain, although they have since been foundin various tissues including stomach, intestine, and A10Lacerda and Brown (637). A transition from mode 1 to

mode 2 was observed on application of b-adrenoceptor cell lines derived from the rat aorta (1085). These authorsshowed that the expressed ClC-2 channels were markedly,agonists, cAMP, or PKA in cardiac cells (404), suggesting

that channel phosphorylation by PKA changed the chan- but slowly, activated by membrane hyperpolarization; inthis, they resembled the hyperpolarization-activated cur-nel state (mode). In rat ventricular cells, a very strong

depolarization alters the channel mode without cAMP or rent. Like ClC-1, which is the major skeletal muscle Cl0

channel (1033, 1034), the ClC-2 channel usually showsb-adrenoceptor stimulation (896). Moreover, in the samestudy, such long channel opening was occasionally re- inwardly rectifying properties, although some ClC-2 chan-

nels have been found with slight outward rectifying prop-corded under normal conditions. In rabbit ileal SMC, In-oue et al. (490) also reported that long channel opening erties (324). The amino acid sequences have been deter-

mined for ClC channels, including ClC-0 (the Torpedocould be observed with a modest level of membrane depo-larization at low frequencies of stimulation, suggesting the marmorata Cl0 channel, Ref. 522), ClC-K1, ClC-K2 (the

specific kidney Cl0 channels, Refs. 6, 1112), and ClC-3 (apossible existence of a third mechanism, other than strongmembrane depolarization and channel phosphorylation. ClC-K1-like Cl0 channel, Ref. 556). They have 12 putative

transmembrane-spanning regions among 13 strongly hy-There are two conductive channels (large and smallunitary conductances) that are blocked by DHP in SMC drophobic sites, suggesting a very unique channel struc-

ture, a so-called ‘‘double-barreled’’ structure (743). These(156, 478, 1178). The large conductive channel has charac-teristics similar to those of the typical L-type channel, ClC channels have several PKA and PKC phosphorylation

sites. Recently, Furukawa et al. (324) found that a trun-whereas the small conductive channel has a low thresholdfor channel activation but shows little channel inactiva- cated form of the ClC-2 type Cl0 channel (ClC-2b; an alter-



native spliced form of ClC-2a) in the rabbit heart and current is activated at hyperpolarized potentials that aremore negative than 060 mV and in a time-dependent fash-cerebellum had poor volume dependency. Moreover, al-

though ClC-2a could be expressed in colon and cerebel- ion. This current is thought to contribute to some extentto the quick recovery from the afterhyperpolarizationlum, ClC-2b did not express in the colon (324). Another

volume-dependent Cl0 channel, named the ICln channel, back to the resting membrane potential and to the slowdepolarization that acts as a trigger for action potentialhas been identified in various epithelia and in nonepithe-

lial cells, including SMC (492, 611, 891). This ICln has nei- generation.ther PKA and PKC phosphorylation sites nor a long hy-drophobic amino acid chain forming the common trans-

V. RECEPTOR-OPERATED ION CHANNELS INmembrane structure, but is believed to form a channel

VISCERAL SMOOTH MUSCLE CELLSby dimerization (301). The expressed ICln Cl0 channelsin Xenopus oocytes showed marked outward-rectifying

It has long been known that, in many types of VSM,properties and Ca2/ insensitivity. One of the specific char-

neurotransmitters released from peripheral autonomicacteristics of ICln is that this channel has a nucleotide-

nerves elicit contractions accompanied by a transient de-binding domain with the same sequence as the cystic fi-

polarization that subsequently evokes Ca2/-spike dis-brosis transmembrane conductance regulator (CFTR)

charges, although in some cases the opposite change, hy-channel, suggesting cAMP or ATP modulation of channel

perpolarization, is observed. In other types of SMC, onactivity (891).

the other hand, locally produced bioactive substancesThe physiological role of Cl0 channels in VSMC is

(such as those produced by inflammation, mechanicalone that is related to changes in cell volume, as suggested

stresses, hypoxia, and metabolic perturbations), and evenfor other cells. Recent work on epithelial cells has indi-

some circulating hormones secreted from distant organs,cated that stabilization of F-actin prevented, whereas de-

can produce long-lasting membrane depolarizations andstruction of F-actin activated the large-conductance Cl0

contractions (106). These depolarizations are attributedchannel (971). This indicates that actin and other cytoskel-

mainly to induction of cationic (Na/ and Ca2/) or anioniceton proteins link closely to Cl0 channels and help regu-

(usually Cl0) conductances, and in some cases to suppres-late the channel’s activity. With regard to the role of Cl0

sion of K/ conductance, thereby increasing the rate ofchannels in determining membrane excitability, it has

Ca2/ entry into the cell through voltage-dependent andbeen noted that Cl0 channel blockade by many drugs pro-

-independent pathways. Subsequent attempts to charac-duces an inhibition of channel activity and hyperpolarizes

terize the electrophysiological nature of the above eventsthe membrane. In skeletal muscle, blockade of the ClC-1

using the patch-clamp technique have revealed that thechannel produces muscle stiffness, and lack of the Clc-1

pathways involved could lead to the opening of receptor-gene leads to a failure to express functional Cl0 channels

operated cation channels (ROCC), Ca2/-dependent Cl0in myotonic mice (1033), suggesting that Cl0 channels

channels, or the closing of the M channel (M current),have an important role in muscle relaxation. On the other

with the subsequent activation of VOCC.hand, Sun et al. (1047) reported that the PTX-sensitive, G

The ROCC so far identified in SMC are quite diverseprotein-coupled Cl0 channel in rabbit colonic SMC was

in their mode of activation, biophysics, pharmacology,activated by substance P, a neurokinin-1 (NK-1) receptor

and perhaps physiological functions. On the basis of theseagonist. They speculated that activation of this Cl0 chan-

differences, they can be further subdivided into at leastnel might participate in the initial membrane depolariza-

four distinct families (i.e., purinoceptor-coupled cationtion induced by NK-1 receptor stimulation and thus acti-

channels, G protein-coupled voltage-dependent cationvate voltage-dependent Ca2/ channels.

channels, G protein-coupled voltage-independent cationchannels, and Ca2/-activated cation channels; see Tables4–6).E. Nonselective Cation Channels

There are two major ionic currents in vascular SMC A. Purinoceptor-Coupled Channels

that permeate several cations nonselectively. One is a re-ceptor-operated nonselective channel, and the other is Purinoceptors can be subdivided into two distinct

classes, i.e., metabotropic and ionotropic types. The me-a hyperpolarization-activated channel. Receptor-operatedand G protein-coupled or intracellular second messenger- tabotropic purinoceptors (P2t , P2u , P2y , and uridine nucle-

otide-sensitive receptors; 7 transmembrane segment type)mediated channels are discussed in detail in section V. Inthis section, we merely mention the hyperpolarization- are linked to G proteins such as Gi/o and Gq/11 . Of these,

Gq/11 is coupled with the activated P2u and P2y receptors;activated channels, the presence of which has been re-ported in several SMC with spontaneous excitability (rab- it is capable of synthesizing InsP3 by hydrolysis of phos-

phatidylinositol 4,5-bisphosphate through activation ofbit jejunum, Ref. 73; rabbit portal vein, Ref. 539). This



PLC. Gi/o is coupled with the activated P2t receptor; it developing and desensitizing cationic current with a mod-erate inward-rectifying property and a reversal potentialreduces the amount of cAMP through an inhibition of

adenylate cyclase (818). In contrast, ionotropic purino- close to 0 mV. In activating this current, the order ofpotency was b,g-methyleneadenosine 5*-triphosphateceptors (P1 family type; 2 transmembrane segment type)

are directly coupled with cation channels without media- (AMP-PCP)Å ATPú ADPúú adenosine, suggesting thatthe receptor responsible was of the P2x subtype. The singletion of G proteins. Genes expressing such ionotropic re-

ceptors have recently been identified and termed P2x1-P2x6 channels underlying the ATP-activated cationic currentare nonselective cation channels (NSCCATP) that discrimi-and P2x7 (previously termed P2z receptor) (97, 125, 133,

187, 209, 1053, 1115). These receptors may consist of nate poorly among cations but are not permeable foranions (however, see the alternative view of Thomas andmultiunit subtypes (552). In addition, Chang et al. (185)

isolated the cDNA encoding a novel P2 receptor from the Hume, Ref. 1087). Calculations from reversal potentialsusing a modified Goldman-Hodgkin-Katz equation sug-rat aortic SMC library and functionally characterized it.

They found it to be a member of the G protein-coupled gested that NSCCATP have a severalfold higher selectivityfor Ca2/ and Ba2/ over the monovalent cations Na/, K/,receptor family that includes P2u and P2y receptors; this

cloned P2 receptor was found to be coupled to PLC, and and Cs/ (e.g., the permeability ratio PCa/PNa is Ç3; forother examples see Table 4). This further gave an estima-not to adenylate cyclase, in C6 rat ganglioma cells trans-

fected with the cloned P2 expression vector. The rank tion that Ç6% of the ATP-induced cationic current wouldbe carried by Ca2/ under physiological conditions. How-order of agonist potency, as judged by intracellular Ca2/

mobilization responses, was UTP ú ADP Å 2-methylthio- ever, the unitary conductance of NSCCATP was muchsmaller with Ca2/ as the sole charge carrier (Ç5 pS withATP ú adenosine 5*-O-(2-thiodiphosphate) ú ATP Å

adenosine 5*-O-(3-thiotriphosphate) (ATPgS). These re- 110 mM Ca2/) than it was in physiological saline (Ç20 pS,with 130 mM Na/ plus 1.5 mM Ca2/). These two seeminglysults indicate that the novel metabotropic P2 receptor has

pharmacological characteristics distinct from any of the paradoxical observations can be interpreted as indicatingthe presence of a binding site (or sites) inside the NSCCATPP2 receptor subtypes thus far identified and suggest the

existence of a novel regulatory system by which extracel- pore with a higher affinity for Ca2/ than for Na/; suchsite(s) would preferentially trap Ca2/ and slow its perme-lular nucleotides may modulate potential difference. As

agonists for the P1 receptor, ATP, a,b-methylene-ATP, and ation, as has been suggested for high-threshold VOCC (56,75). Organic and inorganic blockers of VOCC, such as2-methylthio-ATP are commonly used, whereas the antag-

onists used are suramin, FPL-67156 (mainly for P2x), reac- nifedipine (10 mM) and Cd2/ (500 mM), on the other hand,were ineffective at inhibiting the current representingtive 2 (low-affinity binding site for the P2x and high-affinity

binding site for the P2y), as well as EPL-66096 and EPL- NSCCATP (note that Cd2/ was later shown to reduce itsamplitude, as were Zn2/, Mn2/, and La3/, Refs. 448, 800).67085 (for the P2t receptor). In the rest of this section, weNakazawa and Matsuki (801) also found in the rat vasfocus mainly on receptor-operated ionotropic responses.deferens an ATP-induced inward current that desensitizesrapidly and is not affected by presence of nicardipine or1. Cation channels involved in the response toCs/. The current conductance measured using the cell-purinoceptor activation (purinoceptor-operatedattached patch-clamp procedure was 20 pS, and it re-channel)versed its polarity at Ç0 mV. These results were exciting,

Benham’s group (72, 75) was one of the first to clearly because they seemed the first direct proof of the hypothe-demonstrate a molecular entity to which such purinocep- sis of receptor-operated Ca2/ channels (106).tors link. They showed, using the patch-clamp technique Subsequent exploration has revealed that, despite

some differences (Table 4), ATP-activated currents (orin rabbit ear artery SMC, that ATP activates a rapidly

FIG. 7. Features of L- and T-type voltage-dependent Ca2/ channels of guinea pig portal vein. I, A: unitary Ba2/

currents observed at a holding potential of 080 mV (Aa) and 060 mV (Ab). Pipette was filled with 50 mM Ba2/ solution,and bath was superfused with high-K/ solution. Depolarization pulses of 150 ms in duration to various membranepotentials were applied. Open and solid circles indicate typical channel openings of 12- and 22-pS channels, respectively.Data of Aa and Ab were obtained from same cell. B: current-voltage relationship in 50 mM Ba2/ solution of 2 types ofunitary Ba2/ channel current, with unitary slope conductance of 11.5 pS (s) and 21.8 pS (l). II: effects of nifedipineon activity of 2 types of unitary Ba2/ channel current. A: unitary Ba2/ current evoked by a depolarizing pulse (duration150 ms) to 020 mV from holding potential of 080 mV in absence (Aa) and presence (Ab) of 0.3 mM nifedipine.Depolarization pulses were applied every 10 s, and sequential traces were recorded. Data were obtained from samecell. Open and solid circles indicate typical channel openings of 12- and 22-pS channel, respectively. B: relationshipbetween open probability of 2 types of unitary Ba2/ channel and concentration of nifedipine. Open probability at eachconcentration of nifedipine was calculated from total sweeps. Data were obtained only from cells that showed activityof both types of unitary Ba2/ channel during a depolarization pulse of 020 mV at a holding potential of 080 mV incontrol condition (nÅ 3–5). [From Inoue et al. (488).]



TABLE 4. ATP-activated cation channels in visceral smooth muscle cells

VoltageUnitary Ionic Selectivity or Dependence/

Cell Type Nucleotide Sensitivity Conductance, pS Permeant Ions [Ca2/]i Dependence Other Properties Reference No.

Rabbit ear artery AMP-PCP Å ATP ú ADP Ç20 (130 Na/130 Na) PCa/PNa Å 3.3 Inward Rapid activation and 23, 69, 72, 75,úú adenosine Ç5 (110 Ba or 110 rectification/— desensitization 926

Ca/130 Na) Coagonists: 5-HT,angiotensin II

Blockers: DIDS (IC50

õ 0.1 mM),furosemide (IC50 Å0.5–1 mM)

Nifedipine (5 mM): IECd2/ (0.5 mM): IE

Rat vas deferens ATP ú ADP, AMP-CPP, Ç20 (PSS/130 K) Na, K, Mg Inward Rapid activation and 801AMP-PNP ú AMP-PCP rectification/— desensitization[ED50 (ATP) Å 4.6 mM]

Guinea pig urinary ATP ¢ AMP-PCP ¢ NT PCa/PNa Å 1.0 Inward Rapid activation and 472, 476,bladder AMP-CPP ú ADP úú rectification/— desensitization 966

AMP [ED50 (ATP) Å 2.3 Q10 Å 1.25mM] D-600: IE

Rat myometrium ATP ú ATPgS ú AMP-CPP NT Monovalent cation —/— Rapid activation, no 429aselective desensitization

Downregulation byestrogen

Inhibition by divalentcations (see text)

Cloned P2x receptor MeS-ATP ¢ ATP ú Ç19 (150 Na / PCa/PNa Å 4.8 Inward Rapid activation and 1115(rat vas deferens) AMP-CPP úú ADP 2 Ca/115 K) rectification/— desensitization

[ED50 (ATP) õ 1 mM] Blockers: suramine(3–100 mM),PPADS (10–30mM)

Amiloride: IERabbit portal vein ATP ú AMP-PNP ú NT Na, Ca, Ba —/— Slow activation, no 1185

AMP-CPP ú ADP desensitizationPTX sensitive, G

protein mediated

NT, not tested; IE, ineffective; MeS-ATP, 2-methylthio-ATP; PPADS, pyridoxalphosphate-6-axophenyl-2*,4*-disulfonic acid; PTX, pertussis toxin; 5-HT, 5-hydroxy-tryptamine; PSS, physiological saline solution.

channels) with much the same properties are ubiquitous Recently, a cDNA encoding the P2x receptor has beencloned from rat vas deferens (1115) and human urinaryamong other types of SMC (including rat vas deferens,

guinea pig urinary bladder, rabbit portal vein, guinea pig bladder (282). Expression of such a clone in Xenopus

oocytes was alone sufficient to reproduce the full profileileum, and rat aorta; Refs. 304, 476, 385, 801, 966). Furthercharacteritics of NSCCATP were elucidated in these stud- of the native NSCCATP, such as agonist selectivity, single-

channel conductance, kinetics, ionic selectivity, and phar-ies: 1) activation of NSCCATP can occur on a millisecondscale (latency for activation is shorter than 100 ms, Ref. macological properties. This confirmed the previous idea

that the P2x receptor is a macromolecular complex con-304; as short as 18 ms, Ref. 476); 2) binding of at leasttwo ATP molecules seems necessary to open NSCCATP sisting of receptor and channel domains. Another striking

prediction from this study was that the cloned channel has(i.e., positive cooperativity), with the half-effective con-centration being of the micromolar order (4.6 mM, Ref. only two membrane-spanning domains, as in the inward

rectifier K/ channel family. Furthermore, the putative641; 2.3 mM, Ref. 476; 1.1 mM, Ref. 1115); and 3) the tem-perature dependence of NSCCATP is low, with a Q10 value pore, which is likely to be a part of the second transmem-

brane domain, contains a sequence (Thr-Met-Thr-Thr-Ile-comparable to that estimated for passive diffusion inaqueous media (1.25, Ref. 966). These properties are remi- Ile-Gly-Ser-Gly) that closely resembles a well-conserved

motif of the pore region of the voltage-gated K/ channel.niscent of those of directly ligand-gated channels, sug-gesting that NSCCATP is a member of a channel family These results strongly suggest that the cloned P2x receptor

is entirely different from the so-far cloned heteromultim-that requires neither G proteins nor intracellular secondmessengers for activation (56, 75). It is consistent with eric ligand-gated channels that are activated in a mem-

brane-delimited fashion (e.g., nicotinic, GABAergic, gly-this view that the activity of NSCCATP could be recordedfrom outside-out membrane patches with high concentra- cine, and glutamate receptors). Unexpectedly, the charac-

teristic architecture of the P2x receptor shows sometions of Ca2/ buffers and no added nucleotides on thecytoplasmic side (90, 800). resemblance to the mechanosensitive channels found in



Caenorhabditis elegans and also to the epithelial amilo- erably larger than when a depolarization of similar magni-tude to that induced by ATP was generated electrically.ride-sensitive Na/ channels, although these show no ho-

mology in terms of primary amino acid sequence (see Despite such supporting evidence, a contrary conclu-sion has been drawn from contractile studies in otherRef. 1052).

The concept of significant Ca2/ permeability through SMC, in which ATP-mediated transmission does seem toplay a physiological role. Blakeley et al. (91) reportedNSCCATP (based on reversal potential measurements) in-

volves a number of not inconsiderable problems. It relied that in the rat vas deferens, where ATP is the excitatorytransmitter generating the fast EJP, nifedipine selectivelyon the unrealistic assumption that it is possible to ignore

any interaction between permeating ions (i.e., the inde- abolished the fast component of the nerve-evokedtwitchlike contraction by inhibiting the superimposedpendence principle for ion permeation). Furthermore,

junction potentials, particularly those that arise between spike activity without affecting the amplitude of the fastEJP. Katsuragi et al. (551) demonstrated that both thethe cytosol and the patch pipette, and which change time

dependently, were not seriously considered; this may have ATP-induced [Ca2/]i increment and the concomitant ten-sion development in guinea pig urinary bladder can beled to erroneous estimations of permeability ratios (805).

To avoid these complications, a more straightforward ap- completely blocked by pretreatment with nifedipine ornitrendipine, although they are unaffected by v-cono-proach has been attempted, involving simultaneous mea-

surements of the ATP-induced current and the concomi- toxin, a blocker of presynaptic VOCC. Furthermore, a mildinhibition of ATP-induced responses caused by use of atant rise in [Ca2/]i using the fluorescent Ca2/ indicator

indo 1 (69, 966). In rabbit ear artery SMC, it was found lower concentration of nifedipine was reversed by thedihydropyridine agonist BAY K 8644. These findings arethat exogenously applied ATP (1–10 mM) is as potent as

NE (10 mM) or caffeine (10 mM) in elevating [Ca2/]i (by difficult to reconcile with the notion of an important directCa2/ entry through NSCCATP and instead favor the ideaup toÇ500 nM) at membrane potentials at which voltage-

dependent Ca2/ entry is unlikely (143). This rise in [Ca2/]i that the primary action of ATP is to depolarize the mem-brane and thus activate VOCC, thereby inducing contrac-seems likely to result from direct Ca2/ entry through

NSCCATP rather than from a liberation of stored Ca2/, tion. We do not fully understand the background behindthese disparate findings made in either single cells or insince store depletion with NE or caffeine failed to affect

it, whereas elimination of external Ca2/ or the use of whole tissue experiments. Recently, Katsuragi et al. (550)observed the effects of ATP on guinea pig ileal longitudi-highly depolarized conditions (which should reduce the

Ca2/ driving force) abolished it. Nevertheless, the ability nal muscle segments; they concluded that the release ofATP by receptor stimulation may result mainly from anof this ATP-induced current to elevate [Ca2/]i appears to

be quite low. Inspection of the relationship between activation of PLC, coupled with a PTX-insensitive G pro-tein, and the subsequent accumulation of InsP3 in SMC.[Ca2/]i increment and the charge transported through

NSCCATP indicates that a 1-pC entry produces only a 0.5 Another interesting effect of ATP-induced Ca2/ entrythrough NSCCATP has recently been reported from the ratnM increment in [Ca2/]i in ear artery SMC. This is much

smaller than the value typically reported for VOCC, which portal and human saphenous veins (669, 872). In theseSMC, the magnitude of the ATP-induced [Ca2/]i rise waspass Ca2/ highly selectively (e.g., 3 nM/pC in guinea pig

ureter, Ref. 2). A similar conclusion was reached for the greatly decreased after the use of procedures that inhibitCICR, such as pretreatment with caffeine, ryanodine, orurinary bladder by comparing the relative abilities to ele-

vate [Ca2/]i shown by ATP-induced and voltage-dependent tetracaine, whereas the inhibitor for the InsP3 receptor,heparin sodium, was found to be ineffective. These resultsCa2/ currents (VOCC) in one and the same preparation;

the former involved a cation entry Ç19 times larger than give rise to the idea that a modest Ca2/ entry via thereceptor-gated pathway could serve to initiate a subse-in the latter (966). These calculations can be further trans-

lated to indicate that Ç10% of the ATP-induced current is quent large Ca2/ release from the ryanodine-sensitivestores through the CICR mechanism. Such an amplifica-carried by Ca2/ under physiological conditions. Moreover,

direct Ca2/ entry through NSCCATP may be significantly tion (by way of internally stored Ca2/) of the effect on[Ca2/]i of Ca2/ entry is an attractive idea; such a mecha-attenuated when the membrane is allowed to depolarize

under unclamped conditions, and in contrast, the role of nism may be more generally involved in agonist-inducedCa2/ mobilization than presently envisaged. A furthervoltage-dependent Ca2/ entry would then become more

important. However, this is unlikely to be the case in the complication may need to be added to the story of theeffects mediated by ATP; in the rabbit pulmonary artery,guinea pig urinary bladder. In this preparation, Schneider

et al. (968) measured the increment in [Ca2/]i resulting pretreatment with ATP at a concentration too low to acti-vate any detectable membrane currents dramatically po-from ATP-induced Ca2/ entry and found that, under cur-

rent-clamped conditions, the ATP-induced current is just tentiated the Ca2/-dependent Cl0 current activated by a1-adrenoceptor-mediated Ca2/ release (400). The mecha-as effective in elevating [Ca2/]i as under a voltage clamp.

The size of this increase in [Ca2/]i was found to be consid- nism underlying this synergistic effect remains unclear,



but an examination of the possible involvement of second may imply the presence of a heterogeneity among SM P2x

receptors. To date, seven subtypes (P2x1-P2x7) have beenmessengers such as arachidonic acid or cADP ribosemight prove rewarding. cloned from rat vas deferens, PC12 pheochromocytoma

cells, rat dorsal root sensory neurons, and autonomic gan-The purinoceptors distributed in SM cells may not belimited to the P2x subtype. Thus, in pig cultured aortic glia. Although both P2x1 and P2x3 are activated by submi-

cromolar concentrations of ATP or a,b-methylene-ATPSMC, exogenously applied ATP causes a large Ca2/ re-lease from the internal stores, thereby activating a Ca2/- and desensitize rapidly, the P2x2 subtype is insensitive to

a,b-methylene-ATP or b,g-methylene-ATP, requires muchdependent Cl0 conductance, but not a cationic conduc-tance (251). Although no pharmacological characteriza- higher concentrations of ATP than the former two sub-

types for its activation, and shows only slow desensitiza-tion was attempted in this study, it is conceivable thatthe Ca2/ release might result from a stimulation of the tion (similar features have also been found for P2x4 , P2x5 ,

and P2x6 subtypes). Furthermore, the possibility has re-production of InsP3 via a G protein-coupled subtype of theP2 receptor, the structure of which is completely different cently been suggested that P2x2 and P2x3 subunits may

form a heteropolymer and generate a cationic conduc-from that of P2x (e.g., P2y or P2u; Refs. 255, 384). Similarobservations have been reported in studies of rat cultured tance with hybrid properties, namely, activation by a,b-

methylene-ATP and slow desensitization kinetics (562,aorta (1137) and in studies of freshly isolated SMC fromthe rabbit pulmonary artery (400) and rat aorta (876). 563). Thus it seems that molecular characterization of

SM P2x receptors will be an intriguing subject for futureInterestingly, the latter study suggested that expressionof the P2 receptors linked to cationic channels (P2x) and investigation.

Exogenously applied to rabbit portal vein SMC, ATPInsP3 production (P2y) may vary during the course of cellculture; freshly isolated cells may express the former ex- can activate, in addition to the typical NSCCATP, a long-

sustained cationic current (1184). This long-sustained cat-clusively, but the latter gradually comes to predominatewith repeated passages of reseeding and culturing. In con- ionic current was activated only weakly by a,b-methyl-

ene-ATP, and it did not desensitize appreciably, as showntrast, the Ca2/ release from the internal stores in this cell,caused by P2u receptor stimulation (UTP being used as the by the response to subsequently applied ATP. Activation

of this current was completely blocked by pretreatmentagonist), remained constant, regardless of the progress ofcell culture. It is an important implication of this study with PTX, and its amplitude was significantly reduced by

internal dialysis with GDPbS. This suggests that a PTX-that those SMC with higher excitability might preferen-tially express the effectors participating in fast Ca2/ mobi- sensitive G protein is involved in its activation. A similar

PTX-sensitive G protein-mediated potentiation of VOCClization (such as VOCC, ryanodine-sensitive stores, anddirectly ligand-gated channels with fast kinetics, like could be demonstrated in the same preparation.

In summary, the purinoceptors found in SMC exhibitNSCCATP) but that these might be replaced in cells in aless excitable or undifferentiated state by InsP3-producing considerable heterogeneity and, accordingly, their related

functions may be divergent. Even if we look just at thereceptors and InsP3-sensitive stores (704, 876).Finally, we should turn to the presence of ATP-activa- cation channels, their roles in Ca2/ mobilization seem to

differ considerably depending on the type of SMC in whichtable cationic conductances, the properties of which areclearly different from those so far described. The ATP- they are found, presumably because the type and density

of Ca2/ stores as well as of Ca2/-permeable channels mayinduced cationic conductance recorded from rat myome-trium is selective for monovalent cations and exhibits no differ.discernible desensitization in the continued presence ofATP. Even though activation of this current is thought to 2. K/ channels involved in the response toinvolve the P2x receptor (on the grounds that a,b-methyl- purinoceptor activationene-ATP is effective, but adenosine and ADP are not) andnot to require second messengers, the receptor involved Adenosine 5*-triphosphate also activates other types

of purinoceptors that participate in inhibitory responsesmay be different from the ear artery P2x receptor (i.e.,NSCCATP). The evidence for this is that only the free form in SMC. For example, in the GI tract of many species,

bulk application of ATP into the bath or iontophoreticof ATP, in submillimolar concentrations, seems able toinduce this conductance; millimolar concentrations of di- application of ATP evoked a sustained or a rapidly devel-

oped hyperpolarization, respectively, each being mainlyvalent cations such as Co2/, Mg2/, Ca2/, and Ba2/ de-pressed the current severely in a manner predictable from due to an increase in K/ conductance. Yamanaka et al.

(1203) compared the effects of ATP and nicorandil [a K/the chelation of ATP by these metals. Another noteworthyproperty is that the current is significantly downregulated channel (KATP) opener and nitro compound] on circular

SMC of the guinea pig small intestine and found that bothby treatment with estrogen or after gestation, suggestingthat it has a functional significance during the nonpreg- agents hyperpolarized the membrane and increased ionic

conductance. In the presence of a maximally hyperpolar-nant period as well as in the menstrual cycle. These results



izing concentration of nicorandil, the membrane was still eral, the phasic type of muscle (e.g., intestinal SM) isspontaneously active in terms of the firing of action poten-further hyperpolarized by ATP. However, these hyperpo-

larizing actions seem to have different underlying mecha- tials and contracts to spasmogenic stimuli in a rapid andtransient manner. In contrast, tonic muscle, such as thatnisms: 1) that induced by nicorandil was Ca2/ dependent,

but this was not the case for ATP; 2) the hyperpolarization found in large blood vessels, is electrically quiescent orless excitable (showing a graded electrical response) andinduced by nicorandil was not prevented by apamin, but

that induced by ATP was; and 3) local anesthetics inhib- contracts with a slow and maintained time course. Themolecular basis of these differences could be explainedited the hyperpolarization induced by either agent, but

with different potencies. Moreover, the IJP evoked by field by a differential contribution by VOCC (or action poten-tials), or by two internal stores, or by distinct propertiesstimulation were blocked by apamin but, in the presence

of ATP, the amplitude of the IJP was markedly reduced, of the contractile system in the two types (1014). We havenoticed, in addition, that the distribution of G protein-whereas in the presence of nicorandil it was only slightly

reduced. Thus exogenously applied ATP and nicorandil coupled ROCC may also differ between these two muscletypes; there is a preferential distribution of voltage-depen-(KATP) may well activate different K/ channels. Zagorod-

nyuk et al. (1222) investigated the effects of ATP on iso- dent cation-permeable channels in phasic muscle, but ofvoltage-independent ones in tonic muscle. As describedlated SMC prepared from the human intestine; they con-

cluded that an increase in K/ conductance is responsible below, these channels also appear to differ in their otherproperties, such as [Ca2/]i sensitivity and, presumably,for the generation of NANC-IJP, and that is due to an

enhancement of the Ca2/-dependent K/ channel (see also Ca2/ permeability (Tables 5 and 6).Briefly, G proteins are composed of a- and b,g-sub-in urinary bladder). However, the IJP and ATP-induced

hyperpolarization were both insensitive to TEA and to 4- units. The former come from the Gs family (Gas-1 toGas-4 and Golfa), the Gi family (Gagust , Gat-1 , Gat-2 , Gai-1AP, whereas apamin slightly decreased the amplitude of

both the IJP and the ATP-induced hyperpolarization. Fur- to Gai-3 , Gao-1 , Gao-2 , and Gaz), the Gq family (Gaq ,Ga11 , Ga14–16), or the G12 family (Ga12 and Ga13). Thether detailed investigations are required to help us better

understand both the ATP-induced hyperpolarization and b,g-subunit is composed of b1–5 and g1–10 (for reviews,see Refs. 1899, 2449). There are more than 50 low-molecu-the ionic features of IJP in the intestine. However, if these

hyperpolarizations are due to activation of the Ca2/-de- lar-weight soluble G proteins (mol wt 20,000–30,000);these proteins do not contain subunits and are placed intopendent K/ channel, it is tempting to postulate that, in

these tissues, ATP may activate metabotropic purinocep- ras, rho, rab, and other families. It is now clear that a-and b,g-subunits, by their association and dissociation,tors (via Gq/11 activation) and that an increase in cytosolic

Ca2/, caused by release of Ca2/ from the SR, may activate not only modify the metabotropic process (via activa-tion of phospholipase A2, PLC, and others) but also di-these channels, as reported in many other tissues. On the

other hand, Dart and Standen (234) reported that adeno- rectly and indirectly regulate ion channel activity (Ca2/,K/, Na/, Cl0, or nonselective receptor-operated ionsine activated K/ channels in the pig coronary artery

through activation of the A1 receptor. This K/ channel channels).Many subtypes of the G protein constituent subunitspossessed a small current amplitude (ú2 pA, at 060 mV

in 143 mM K/) and was blocked by inhibitors of K/ chan- are present in SMC, and a number of subtypes of therelevant agonist receptors are also distributed in excitablenel (KATP) openers, but not by charybdotoxin or apamin.

Therefore, they suggested that activation of the A1 purino- and nonexcitable cells. Thus it is reasonable to supposethat a large variety of receptor-G protein-coupled ionicceptor may induce an activation of KATP in this tissue.responses, produced either directly or indirectly throughthe synthesis of second messengers, may underlie the

B. G Protein-Coupled Cation Channels range of VSMC responses. For example, among musca-rinic receptors, M1, M3, and M5 receptors are coupled withthe Gq/11 family and synthesize InsP3, whereas M2 and M4In comparison with fast ligand-gated NSCC

(NSCCATP), the majority of agonist-activatable cation receptors are coupled with the Gi/o family and induce anincrease or decrease in K/ channel activity and a decreasechannels (ROCC) in SMC have much slower kinetics of

activation and desensitization, and they are thus likely in Ca2/ channel activity, as well as a decrease in theamount of cAMP. If we look at adrenoceptors, we findto be associated with G protein-coupled receptors. The

properties of these ROCC are diverse, apparently in line that the a1-adrenoceptor subtypes (a1A , a1B , and a1D) arecoupled with Gq/11 , whereas the a2-adrenoceptor subtypeswith the variety of target organs in which they are distrib-

uted. Smooth muscles have been divided into functionally (a2A , a2B , and a2C) are coupled with Gi/o , and the b-adreno-ceptor subtypes (b1 , b2 , and b3) are coupled with Gs (acti-different groups, depending on the time course and pat-

tern of their contractile responses (termed ‘‘phasic’’ and vation of adenylate cyclase). Furthermore, among the 5-HT receptor subtypes, 5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and‘‘tonic’’ muscle by Somlyo and Somlyo, Ref. 1010). In gen-



TABLE 5. GTP-binding protein-coupled cation channels (voltage-dependent) in visceral smooth muscle cells

VoltageDependence/

Agonists or Unitary Ionic Selectivity [Ca2/]i

Cell Type Receptors Conductance, pS or Permeant Ions Dependence G Protein Other Properties Reference No.

Rabbit jejunum Muscarinic NT Cations /// Yes? 74, 663Guinea pig Muscarinic histamine Ç25 (PSS/138 K PCa/PCs Å 2.03 /// PTX sensitive Blockers: quinine 190, 473,

ileum / 10 Na) PNa/PCs Å 1.06 (quinidine) (IC50 Å Ç1 478–480mM), flufenamate (32mM), divalent cations(see text)

Guinea pig Muscarinic NT Na, Ca /// NT 870jejunum

Canine stomach Muscarinic Ç30 (PSS/140 Cs) PK/PNa Å 1.7 /// NT Coagonist: caffeine? 1134(pyrolus)

Canine stomach Muscarinic NT Na (other cations, /// NT 984(corpus) not tested)

Canine colon Muscarinic substance NT Na (other cations, /// NT Blockers: quinine (0.5 mM), 651, 652aP neurokinin A not tested) Ni or Cd (50–100 mM)

Heparin: IE.Canine stomach Carbachol NT PK/PCs Å 1.1 /// Yes Blockers: quinine (IC50 Å 568, 569

(antrum) PNa/PCs Å 0.98 0.25mM), calmodulinprevents rundown

Rabbit portal a1-Adrenergic Ç25 (PSS/140 CS) PBa/PNa Å 4.6 ///? (see text) Yes? Blockers: Cd 153, 391, 482,vein muscarinic Opener: fenamates 1150, 1192

Ca has dual effectsCanine trachea Muscarinic NT Na, Li, Mn, Ba //NT NT 512, Inoue,

unpublisheddata

Definitions are as in Table 4.

5-HT1F are coupled with Gi/o , whereas 5-HT2A, 5-HT2B, and or decrease in the amount of cAMP induced via the ac-tions of Gs or Gi/o (e.g., K/ channels, Ca2/ channels). Sec-5-HT2C are coupled with Gq/11 . Moreover, 5-HT4, 5-HT6,

and 5-HT7 are coupled with Gs, but 5-HT3 is directly cou- tion VB3A includes a discussion of the G protein-coupledprocesses described in section VB1.pled with NSCC. Among purinoceptors, a variety of rela-

tionships between individual receptor types and coupled In relation to G protein-coupled responses, there arethree recognized classes of PLC isozymes, referred to asG proteins have been described (1184). Thus activation

of a given receptor modifies an ionic channel through b, g, and d (654). Sternweis and Smrcka (1034) and Nohet al. (826) reviewed that a variety of studies have shownactivation of the receptor’s coupled G protein. This may

involve 1) direct regulation of ion channels without the that the b-isozymes (PLC-b1, -b2, -b3, and -b4) are acti-vated by the a- or b,g-subunit of heterotrimeric G pro-synthesis of second messengers (e.g., ROCC), 2) regula-

tion of ion channels through an increase in the Ca2/ con- teins. Thus ligands, such as AT II, that bind to seven trans-membrane receptors are thought to activate the PLC-bcentration in the cytosol via the actions of InsP3 and initi-

ated by activation of Gq/11 (e.g., K/ channels, Cl0 chan- isozymes. In contrast, the PLC-g isozymes (PLC-g1 and-g2) appear to be activated by tyrosine phosphorylation.nels), or 3) regulation of ion channels through an increase

TABLE 6. GTP-binding protein-coupled cation channels (voltage-independent) in visceral smooth muscle cells

Agonists or Ionic Selectivity or Voltage Dependence/ ReferenceCell Type Receptors Permeant Ions [Ca2/]i Dependence G Protein Other Properties No.

A7r5 (cultured aortic ET AVP Cations 0/0 NT (/)PN200-100: IE 1120–SMC) (PCa/PCs ú 17)* Blockers: Ni, Cd, Co, 1122

Mn, LaMesenteric artery ET-1 safratoxin Na, Li, Cs 0/? NT Nifedipine: IE 186

S6b AVPLtk0 cells expressed ET-1 Na, Ca 0/0 NT Blockers: mefenamic 276

with cloned acid (300 mM)human ETA Nifedipine: IE.receptor

A7r5 AVP Cations 0/0 PTX insensitive Blockers: LOE 908 612a(IC50 Å 0.56 mM)

Gd, La, Cd

NT, not tested; IE, ineffective; AVP, arginine vasopressin; ET, endothelin * Current contains a component selective for Ca2/.



These forms of PLC play a major role in the InsP3 genera- lar type of muscarinic Icat channel (or channels) was foundin the canine pyloric sphincter (1134); in gastric (984),tion of growth factor receptors, such as the receptor for

FGF or PDGF. Growth factor receptors contain intrinsic colonic (651), and guinea pig gastric SMC (569); and (un-expectedly) in chromaffin cells as well (471). These find-tyrosine kinase activity that leads to PLC-g activations.

The activation of the d-isozymes of PLC is, at present, not ings suggest that this type of NSCC may be ubiquitousthroughout the whole gut (Table 5).well understood.

It was originally reported that, in rat anococcygeusmuscle, sympathetic stimulation or iontophoretically ap-1. Voltage-dependent, [Ca2/]i-sensitive receptor-plied NE can depolarize the membrane through two dis-operated nonselective cation channelstinct ionic mechanisms (111, 112). The initial fast depolar-ization was attributed to Ca2/-activated Cl0 conductanceMost of the ROCC that may fall into this group have

been discovered in spontaneously active SMC, such as (112); this was later confirmed in single-cell experiments.Subsequently, it was reported that a similar response canthose of the GI tract and portal vein (Table 5). Arguably,

tracheal ROCC from several species might be somehow be recorded in vascular SMC, such as those of the guineapig pulmonary and rat mesenteric arteries (153). The sec-related to this group, if we use a looser definition.

The phasic contractile activity (or spontaneous con- ond component of the NE-induced depolarization is nowthought to be induced by activation of cationic currentstractions) of gut SM is thought to be closely associated

with the amplitude and frequency of the spike discharges via the a1-adrenoceptor (a1-adrenergic Icat) (rabbit portalvein, Refs. 153, 1150; rabbit ear artery, Refs. 22, 23, 1149,superimposed on slow membrane potential oscillations

(slow waves) (135, 136). This is because the spike activity 1150). The NSCC underlying the a1-adrenergic Icat hasbeen found to have a unitary conductance of Ç25 pSreflects the rate of voltage-dependent Ca2/ entry into the

cell through high-threshold VOCC, which itself results in a (in saline), to be modestly dependent on the membranepotential, and to be also activated by muscarinic agonistsperiodic increase in [Ca2/]i and the resulting contractions

(e.g., Ref. 414). Spasmogens capable of augmenting phasic (417, 482). These properties imply that some similaritiesmay exist between the muscarinic Icat and the a1-adrener-activities are often able to depolarize the membrane,

thereby altering the frequency and pattern both of the gic Icat (482).A muscarinic receptor-inducible cationic component,slow waves and of the spike activity (269, 440). One of

the major electrophysiological changes underlying such which was unfortunately not clearly separated from thecoexisting Cl0 conductance, has been reported in caninedepolarizing actions in SMC is increased cation entry

(Na/, Ca2/) via receptor-gated pathways, as postulated and guinea pig tracheal SMC (512). Although its detailsremain unclarified, this current component may be volt-from reversal potential and radioactive ion flux measure-

ments in whole tissue studies (106, 107). age-dependent (512) and permeable to divalent cations(see below), and it thus could be placed in the same groupThe first clear proof for the presence of a receptor-

inducible cationic conductance was provided by a patch- as the muscarinic Icat and a1-adrenergic Icat .In the following sections, more details are given ofclamp experiment in rabbit jejunal longitudinal SMC. In

these SMC, iontophoretic application of ACh to single the properties of these Icat .A) INVOLVEMENT OF G PROTEINS IN ACTIVATION. Activa-cells was found to depolarize the membrane to close to

0 mV, with an associated decrease in membrane resis- tion of the gut muscarinic Icat occurred with a latency ofÇ500 ms, required several seconds to reach maximum,tance, and to elicit spike discharges and contractions; all

these features were consistent with the results of previous and desensitized only slowly (over several tens of sec-onds) despite continued receptor stimulation (74, 474,microelectrode experiments (105). Switching to the volt-

age-clamp mode revealed that ACh elicited an inward cur- 475). A similar temporal profile of activation has beendescribed for the a1-adrenergic Icat in rabbit portal veinrent passing cations nonspecifically (as deduced from the

reversal potential) in a potential-dependent manner (Icat). (latency Ç900 ms, rise time Ç4 s, half decay time 11 s;Ref. 1150). These kinetics are much slower than thoseAn Icat with almost identical properties was later recorded

from guinea pig ileal SMC (481) in a study in which single- typically reported for directly ligand-gated ion channels,such as NSCCATP, but may be faster than the responseschannel recordings were made under whole cell clamp

conditions. The results of this study suggested that the mediated by second messengers, such as InsP3 and cAMP.An analog of this type of activation kinetics is seen in themacroscopic inward current activated via muscarinic re-

ceptors is not a mixture of individual Na/-, K/-, or Ca2/- muscarinic K/ channel (KACh) that is thought to coupledirectly to a PTX-sensitive trimeric G protein (Gk) in aselective currents but probably reflects simultaneous

openings of 20- to 30-pS nonselective cation channels membrane-delimited manner (620). In general, occupationof G protein-coupled receptors by agonist results in an(NSCCs) having nonspecific cationic permeability, as well

as voltage-dependent kinetics that would account for the increased rate of GDP release from the a-subunit, towhich GTP binds in exchange, thus promoting the dissoci-kinetics of the macroscopic current. Subsequently, a simi-



ation of the GTP-bound form of the a-subunit from the be a few times more permeable than monovalent cationsthrough this channel (70, 474). Moreover, the rapid re-b,g-subunit. Although subsequent interaction of activated

G proteins with target effectors was initially believed to moval of external Na/ from the bath reduced the ampli-tude of the muscarinic Icat by 90%, suggesting that Ç10%be mediated solely by the GTP-bound form of the a-sub-

unit, it is now known that the b,g-subunit can also regu- of the current would be carried by Ca2/ under physiologi-cal ionic conditions (474, 475). However, these conclu-late a number of cellular effectors, including KACh (919;

see sect. III). sions may now be invalid, since the assumption of inde-pendent ion permeation is unlikely to be tenable. In addi-The possible involvement of G proteins in the musca-

rinic activation of Icat has been examined in the light of tion, [Ca2/]i measurements made using fluorescent Ca2/

indicators have clearly revealed that, after store depletion,such G protein kinetics, using strategies similar to thoseemployed to investigate KACh (477, 478, 604, 606). The muscarinic agonists are unable to elevate [Ca2/]i by more

than a few tens of nanomolar (869). These observationsfollowing list summarizes several supporting observationsmade in these studies. 1) Intracellular perfusion with strongly suggest that any direct contribution made by Ca2/

entry through muscarinic NSCC to the initiation of con-GDPbS, a nonhydrolyzable inactive form of GTP, or withpreactivated PTX incubated with NAD/ and the sulfhydryl tractions may be only minor. Instead, membrane depolar-

ization resulting from stimulated Na/ entry through thereagent, dithiothreitol, completely inhibited the activationof Icat , when the muscarinic receptor was repeatedly stim- NSCC channel, which would in turn increase Ca2/ influx

through VOCC, may have more physiological importance.ulated. 2) Noisy and sustained cationic currents (IGTPgS)could be induced by continuously dialyzing GTPgS, an The data from whole tissue experiments are indeed con-

sistent with this view. It has been shown that the contrac-active nonhydrolyzable form of GTP, into the cell. Activa-tion of IGTPgS was greatly promoted by application of mus- tile responses induced by muscarinic stimulation are ex-

clusively sensitive to blockade by organic Ca2/ antago-carinic agonists. 3) Detailed comparison has revealed thatIGTPgS and the muscarinic Icat are almost identical in terms nists, in most gut SMC (124). However, the possibility

cannot entirely be excluded that local increases in [Ca2/]i ,of many of their pharmacological and biophysical proper-ties (170, 484). 4) Second messengers such as InsP3, caused by Ca2/ entering through the NSCC and undetect-

able by global [Ca2/]i measurements, might play an im-cAMP, and Ca2/ were ineffective at activating Icat (478).These four results are compatible with the idea that the portant role if they occur in close proximity to the NSCC.

Such local roles might include regulating local membranemuscarinic activation of Icat occurs via a PTX-sensitive Gprotein, probably without mediation by second messen- conductances (808) or possibly the activities of pumps,

carriers, and enzymes. In this regard, the use of mem-gers, as postulated for KACh . However, the possibility can-not entirely be excluded that cell membrane-localized pro- brane-specific fluorescent dyes (e.g., C18-fura 2, Ref. 279)

may help future workers explore the cellular destinationsduction of unknown signaling molecules (e.g., arachidonicacid or fatty acids) may play the fast second messenger of the Ca2/ that enters through muscarinic NSCC, as well

as enabling a more accurate estimation of the degree ofrole in activating Icat , as reported for stretch-activated K/

channels (859). Indeed, in guinea pig ileal SMC, it has Ca2/ permeability.A relatively high selectivity for Ca2/ over monovalentbeen reported that muscarinic activation stimulates the

production of arachidonic acid, thereby increasing Ca2/ cations (PBa/PNa Å 4.6) has been suggested for the a1-adrenergic Icat (1150). However, no [Ca2/]i measurementsinflux into the cell (569). Thus it will be necessary in

the future to reexamine the precise role of G proteins in have yet been made to test this notion. A divalent cationpermeability is likely to exist, too, in the canine trachealmuscarinic Icat activation on a more fundamental basis,

using, for example, single-channel recording or molecular muscarinic Icat (Fig. 8A), and agonist-stimulated Ca2/ en-try through a pathway distinct from VOCC or Na//Ca2/characterization of muscarinic NSCC.

As for the Icat in other SMC, no experiments have exchange has been suggested by fluorescence measure-ments (791). The significance of these observations re-been conducted to test explicitly the involvement of G

protein. However, some recent studies have indicated mains to be determined.C) VOLTAGE DEPENDENCE. As originally found in thethat, in some tissues at least, GTPgS can activate cationic

currents that closely resemble an agonist-induced Icat rabbit jejunum, the chord conductance of the muscarinicIcat declines considerably as the membrane is hyperpolar-(guinea pig stomach, Ref. 1172; rabbit portal vein,

Ref. 1185). ized. This gives a typical U-shaped current-voltage rela-tionship, with an apparent inward peak at around 060 toB) CA2/ PERMEABILITY. Originally, the Ca2/ permeabil-

ity of muscarinic NSCC was thought to be high, since 020 mV (74, 433, 569, 651, 984). Detailed analysis of thevoltage-dependent inactivation of Icat in the guinea pigsizable inward currents flow during the application of

muscarinic agonists with just Ca2/, Ba2/, or Mn2/ in the ileum has suggested that the availability of muscarinicNSCC could be determined by the position of an equilib-bath (474, 475, 479). The relative permeability ratios calcu-

lated from the reversal potentials suggested that Ca2/ may rium between at least two distinct states: one open (A-



FIG. 8. Characteristics of muscarinic cation currentsin canine tracheal SMC. Nystatin-perforated recording (ce-sium aspartate in pipette). A: 50 mM ACh activated inwardcurrents at a holding potential of 060 mV, with physiologi-cal saline (left) or its isosmotic substitution of BaCl2 (103mM; right) in the bath. B: current-voltage relationship ofACh-evoked inward current evaluated by a 2-s-long risingramp (0120 to 80 mV). (From R. Inoue, unpublished obser-vations.)

R*) state and one closed (A-R) state. In this scheme, the tor [(RT/F)/k represents the number of voltage-sensingcharges, R, F, and T having their usual meanings]. Typicalrate of closing [i.e., the transition from the open to the

closed states (a)] might more critically govern the overall Vh values (i.e., from the steepest part of the Boltzmanncurve) found for rabbit jejunal and guinea pig ileal SMCvoltage dependence than the rate of opening (b)(060 to 050 mV) match closely the resting membranepotentials (Vrest) measured in whole tissue experiments.

A / R s

k/1

k01A 0 R(closed) s A 0 R

b

a

* (open) (scheme 1)This suggests that small excursions from Vrest could leadto profound potentiation or attenuation of the depolariz-ing ability of ACh. In support of this view, Inoue andThis interpretation is compatible with single-channel

data demonstrating that the open lifetime becomes longer Isenberg (479) observed that a slight electrical membranehyperpolarization (Ç10 mV) greatly retarded the rate of(481) or the channel open probability increases as a result

of membrane depolarization (481, 1134). The steady-state ACh-induced depolarization, which was, however, dra-matically accelerated upon termination of the electricalactivation curve for Icat (or open probability for NSCC)

obtained with maximal receptor stimulation can be well hyperpolarization. A similar effect was also observedwhen the membrane was chemically hyperpolarized withdescribed by a Boltzmann-type equationthe K/ channel opener pinacidil (Fig. 1A in Ref. 485).These results suggest that the physiological importanceI Å Imax/{1 / exp[(Vm 0 Vh)/k]}of the voltage-dependent property of the muscarinic Icat

may lie in its involvement in positive- and negative-feed-where Vm, Vh , and k, respectively, denote the membranepotential, half-maximal activation potential, and slope fac- back mechanisms. It may serve as a self-reinforcing mech-



anism for cholinergic EJP by accelerating and prolonging with no obvious nadir, a significant deviation from thelinearity expected from the independence principle. Thisthe rising and maintained phases, respectively. It may also

serve to finely shape the time course and magnitude of nonlinear relationship is suggestive of interactions be-tween permeating cations inside the channel pore byACh-induced depolarization in concert with spike dis-

charges, slow membrane oscillations (slow waves), or b- which the voltage dependence of Icat might be affected. Itis now becoming increasingly probable that an interactionadrenoceptor-mediated hyperpolarizations (138); in this,

it might act together with [Ca2/]i-dependent potentiation. between permeant cations is a common feature of nonse-lective cation channels, such as the InsP3 receptor, ryano-One such example has been suggested in canine colonic

SMC, where the enhanced amplitude and prolonged du- dine receptor RyR, NSCCATP, and vasopressin-activatedNSCC (see below). This being so, cation-to-cation interac-ration of the slow waves induced by ACh have been at-

tributed to the potentiation of a small Icat through volt- tion in muscarinic NSCC deserves further, detailed inves-tigation.age-dependent as well as [Ca2/]i-dependent mechanisms

(651). Some modulatory effects of divalent cations on thevoltage dependence of Icat have also been noted. CalciumThe molecular mechanism responsible for voltage-

dependent gating of the muscarinic Icat is unlikely to in- was found to inhibit a Cs/-carrying Icat in the guinea pigileum in a fashion not predictable from the surface-chargevolve a permeation block by external Mg2/ (cf. N-methyl-

D-aspartate-activated NSCC; Refs. 711, 830), since removal neutralizing effects (1231; see below).The a1-adrenergic Icat was originally thought to beof external Mg2/ does not affect this property (74, 1231).

However, it must be conceded that a thorough chelation less voltage dependent (although some inward rectifica-tion has been noted in the ear artery: Amedee et al., Ref.of trace amounts of Mg2/ with EDTA was not attempted

in these studies. Taken at face value, these studies suggest 23). However, a clear voltage dependence has been de-tected by selective recording of cationic conductance inthat voltage dependency is probably an intrinsic property

of NSCC. the rabbit portal vein, using either tail-current analysis(482) or slow-ramp protocols (399). As was the case forIt is worth noting, however, that at least two factors

can profoundly affect the voltage dependency of Icat . the muscarinic Icat , the voltage dependence of the a1-ad-renergic Icat followed a Boltzmann-type sigmoid curveZholos and Bolton (1230) have found that higher intensity

muscarinic receptor stimulation produces a marked nega- (399). However, as noted by Inoue and Kuriyama (482),the Vh values are variable, due presumably to the condi-tive shift of the steady-state activation curve for Icat with-

out affecting the slope factor, whereas desensitization tion of the individual cells examined. It is conceivablethat metabolic conditions in the cell, such as the levelproduces the opposite effect. These observations, to-

gether with the finding that photolytically released GTP of phosphorylation of NSCC, might contribute to such avariability in voltage dependence.and GDPbS mimic the effects of more intensive musca-

rinic activation and desensitization, respectively, led the Figure 8B illustrates our unpublished preliminarydata on the voltage dependence of the ACh-induced Icat ,authors to propose that the concentration of active G

protein a-subunits (i.e., of the GTP-bound form) might evaluated by the use of a slow-rising ramp voltage in thecanine trachea; a clear suppression of the current-voltagecritically determine the state of voltage dependence of

the channel. Although no molecular information is yet curve at more negative potentials can be seen.D) [CA2/]I DEPENDENCE. The magnitude of the muscarinicavailable, it would be intriguing to examine whether such

GTP dependence could result from the multimeric nature Icat decreases greatly when high concentrations of Ca2/ buff-ers, such as EGTA or BAPTA, are loaded into the cell (480,of a G protein/NSCC complex, as has been proposed for

the cardiac KACh channel (620). Another important factor 651, 671). This does not mean that Ca2/ is the primary activa-tor for muscarinic NSCC, since maneuvers that elevatethat might affect the voltage dependence of Icat is the

possible interaction of cations in the course of perme- [Ca2/]i cannot activate Icat in the absence of muscarinic ago-nists. For example, Ca2/ entry through VOCC, which doesation. Although not definitely determined in the previous

studies, it is likely that Cs/ is the most potently conductive not in itself induce a discernible Icat , can cause a markedaugmentation of the amplitude of Icat , when accompaniedcation through the muscarinic NSCC in the guinea pig

ileum (1153, 1231). The magnitude of Icat with Cs/ as the by muscarinic receptor stimulation (480, 569, 651, 663). Therelationship between the degree of this potentiation and thesole charge-carrying cation is, at 060 to 050 mV, sever-

alfold larger than that seen with Na/, which is always Ca2/ charge transported through VOCC indicates that half-maximal potentiation would occur at 2–4 pC (480). Thisaccompanied by a marked negative shift in the channel’s

voltage dependence. We investigated this property in value is comparable to the charge carried by a single actionpotential (2.5–3 pC, assuming a cell input capacitance of 50some detail by changing the molar ratio of Na/ to Cs/

while keeping the total cationic concentration constant pF and a spike amplitude of 50–60 mV). A potentiation ofIcat was also observed when [Ca2/]i was elevated by using(Fig. 9). The amplitude of Icat measured with various con-

centrations of Na/ and Cs/ shows a concave relationship caffeine to release stored Ca2/. Conversely, the amplitude



FIG. 9. Influence of molar ratio ofexternal cations on muscarinic cationcurrent in guinea pig ileal SMC. Pipettecontained cesium aspartate internal solu-tion. A: at a holding potential of 050 mV,molar ratio of 2 external cations (Na/ andCs/) was varied to values marked on fig-ure, by keeping total concentration ofcations constant (140 mM). Other cationsthan Na/ and Cs/ were omitted. To mini-mize desensitization of cation current,it was activated by internal perfusionof guanosine 5*-O-(3-thiotriphosphate)(GTPgS; 50 mM) from patch pipette. B:relative amplitude of cation current isplotted with respect to ratio of Na/ toCs/. For better comparison, current am-plitude is normalized to that with ratioof Na/ to Cs/ of 10:0. Symbols and barsindicate means { SE from 3 measure-ments. [From Inoue et al. (486).]

of Icat was depressed, both when the store was depleted by ment with caffeine, and then resembles the currentevoked by caffeine in both magnitude and appearance.pretreatment with caffeine or ryanodine (480, 870) and when

InsP3-dependent Ca2/ release, which occurs concomitantly Such a tight link to the internal store implies that releaseof Ca2/, rather than a G protein-mediated pathway, mightwith the activation of Icat , was prevented by intracellular

application of heparin sodium (651, 870). These observations play a primary role in activating Icat in this muscle.point strongly to the physiological importance of Ca2/ entry Quantification of the steady-state [Ca2/]i dependencethrough spike discharges and Ca2/ release from internal of the muscarinic Icat has been attempted by dialyzing intostores as steps in a positive-feedback mechanism, the effect the cell various ratios of EGTA to Ca2/ (guinea pig ileum,of which is to increase the rate and prolong the duration of Ref. 470). The relationship between Icat conductance andthe ACh-induced depolarization. Presumably, the prolonga- [Ca2/]i constructed in this way could be described by ation of action potentials or slow waves by ACh (651) or the Michaelis-Menten-type curve with half-maximal and maxi-occurrence of inward current oscillations during muscarinic mal levels of [Ca2/]i of 100–200 nM and 1 mM, respectively.activation (605) may result, at least in part, from this mecha- These concentrations more or less cover the physiologi-nism. cally attainable [Ca2/]i range, thus confirming that [Ca2/]i-

mediated Icat potentiation may indeed occur under physio-A more obligatory role for Ca2/ has been proposedlogical conditions.in respect of muscarinic NSCC activation in canine gastric

SMC (984), on the basis that the Icat evoked by ACh is The [Ca2/]i dependence of the a1-adrenergic Icat wasoriginally thought to be negligible, since inclusion of evengreatly diminished in Ca2/-free solution or after pretreat-



high concentrations of EGTA cannot prevent the activa- strated in a similar type of tissue (intestinal SM of theguinea pig taenia coli). These inhibitory effects were ex-tion of this current (26, 482). However, it was later shown

that buffering of the Ca2/ in the pipette to a level lower erted on carbachol-induced contractions, which dependexclusively on external Ca2/ (124) and are accompaniedthan the physiological [Ca2/]i (14 nM) results in a larger

and more sustained Icat (399). Conversely, in Ca2/-free by an increased level of phosphotyrosine (276). Theseobservations suggest that tyrosine kinase activity may besolution, the a1-adrenergic Icat was first potentiated but

eventually abolished after prolonged exposure (1150). The of great functional importance in regulating muscarinicreceptor-stimulated Ca2/ influx, or muscarinic NSCC, atexact background behind these phenomena is as yet un-

known. least in intestinal SM. In addition, the converse of theeffect exerted by tyrosine kinase inhibitors [i.e., activationE) DEPENDENCE ON HIGH-ENERGY PHOSPHATES. It is gen-

erally recognized that the amplitude of ionic currents de- of a large-conductance NSCC (139 pS with 150 mM Na/)]has been reported from cultured coronary arterial SMC,creases gradually over time, during continued cell dialysis

from the patch pipette. This phenomenon is called run- but the physiological relevance of this channel, and there-fore of such activation, is unclear (745).down and is thought to reflect the washout of diffusible

intracellular constituents needed to maintain the chan- The mechanism underlying the rundown of the mus-carinic Icat may be related, in part, to the washout ofnel’s activity (806). A relevant example is the decline in

the amplitude of Icat found to begin on switching from diffusible Ca2/-binding proteins such as CaM, since theintroduction of this protein into the patch pipette greatlynystatin-perforated to conventional whole cell recordings,

with the muscarinic receptor being stimulated repeatedly reduced the rundown of Icat in guinea pig gastric SMC(569). This preventive action of CaM may be linked tightly(479). The rate of decline is appreciably accelerated when

there is no inclusion of high-energy nucleotides, but con- to Ca2/ influx across the plasma membrane, since Icat wasinhibited dose-dependently when the external Ca2/ con-siderably slowed by the addition of millimolar Mg-ATP to

the solution in the patch pipette (483). This suggests a centration was reduced in a stepwise manner. Further-more, a CaM antagonist, W-7, produced a marked suppres-critical requirement for high-energy phosphates in the

maintenance of Icat . Recently, Bakhramov (38) made an sion of Icat , although it was almost ineffective when Icat

was activated in a way that bypassed the receptor (viz.,extensive screening of compounds that would be ex-pected to affect the intracellular energy state, using a 5- by internally perfusing GTPgS). The most common modes

of action of CaM involve either a direct interaction withmin intracellular dialysis protocol. Interestingly, creatinephosphate was found to be highly effective in upregulating the target or an alteration in the phosphorylation/dephos-

phorylation balance of the target proteins via stimulationIcat , whereas creatine was ineffective. A somewhat puz-zling observation was that millimolar Mg2/ added in the of Ca2//CaM-dependent protein kinase II (126) and of a

phosphatase (i.e., calcineurin). In this context, the resultspipette did not facilitate this upregulation, but rather sup-pressed it significantly. On the other hand, dialysis of ATP of this study (569) may most simplistically be interpreted

to indicate that a certain concentration of the Ca2//CaMat concentrations lower than 1 mM resulted in an Icat ofincreased amplitude, whereas concentrations higher than complex is required to maintain the activity of muscarinic

NSCC and that this maintenance occurs in both phosphor-1 mM caused a dose-dependent inhibition. The inhibitoryeffect of ATP apparently counteracted the potentiating ylation-dependent and -independent ways. If correct, this

interpretation may help to partly explain the moleculareffect of creatinine phosphate but could not be mimickedby either of the nonhydrolyzable analogs AMP-PNP and basis of [Ca2/]i dependence.

F) MECHANOSENSITIVE MODULATION. The muscarinic IcatATPgS. These results can be best taken to indicate thepresence of at least three regulatory sites for Icat in the has recently been reported to be subject to mechanosensi-

tive modulation, i.e., hypotonic exposure augments thecell: two inhibitory sites (for Mg2/ and ATP) and onefacilitatory site (for creatine phosphate). The latter two amplitude of Icat in parallel with cell swelling (518, 1153).

This potentiating effect is unlikely to be the consequencemight involve some phosphorylation/dephosphorylationreactions, but the actual details remain unclear. of an indirect effect of hypotonic swelling, such as dilution

of cellular constituents, since an increased rate of superfu-With regard to the possible role of phosphorylation inmaintaining the activity of the muscarinic NSCC, a recent sion can produce a similar potentiating effect (Fig. 10).

Although the details of this potentiation still remain un-study of the guinea pig ileum is worth mentioning. Inthese cells, tyrosine kinase inhibitors (such as genistein, clear, it may be a more ubiquitous type of regulation of

receptor-operated, as well as voltage-gated channels (e.g.,tyrphostin A25, and lavendustin) suppressed the musca-rinic Icat dose-dependently (IC50 Å Ç5 mg/ml or 18.5 mM maxi-K/ channel, Ref. 570) than has been previously en-

visaged (771). In fact, in some preliminary experiments,for genistein), whereas a tyrosine phosphatase inhibitor,orthovanadate (30 mM), augmented it (483, 486). Inhibi- we have observed that the a1-adrenergic Icat is also po-

tentiated by hypotonic exposure (Y. Waniishi and R. In-tory effects of almost the same magnitude as those ex-erted by tyrosine kinase inhibitors have been demon- oue, unpublished data).



FIG. 10. Flow rate affects musca-rinic cation current in guinea pig ilealSMC. Bath and pipette contained physio-logical saline and cesium aspartate inter-nal solution, respectively. At dashed bars,flow rate was raised by Ç10-fold. Right

bottom panel indicates current-voltagerelationship of cation current with fastand slow superfusion rates. Cation cur-rent was activated by internal perfusionof 50 mM GTPgS. (From R. Inoue, unpub-lished observations.)

G) PHARMACOLOGY, INCLUDING BLOCKADE BY INORGANIC tion or role of ROCC with the aid of these pharmacologicaltools.CATIONS. Some detailed information has been recently pro-

vided about the pharmacological profile of the muscarinic Fenamates, such as mefenamic acid and flufenamicIcat (170, 568, 651). Many frequently used K/ channel acid, are derivatives of diphenylamine-2-carboxylateblockers (TEA, 4-AP, procaine, quinine, and quinidine), (DPC) and have been proposed as potent inhibitors ofVOCC blockers (verapamil, nicardipine, Cd2/, and Ni2/), the Ca2/-activated NSCC in rat exocrine pancreatic cells.Ca2/-releasing agonists or inhibitors (caffeine, procaine), Chen et al. (190) investigated the effects of these com-and Cl0 transport (or channel) blockers (fenamates, pounds, together with those of other DPC derivatives, onDIDS) have all been found to be effective at inhibiting the the muscarinic Icat in the guinea pig ileum. Among them,muscarinic Icat . The site of inhibition is unlikely to reside flufenamic acid was found to be relatively selective foron the muscarinic receptor, since the GTPgS-induced Icat Icat (IC50 Å 32 mM). With regard to its inhibitory efficacies,exhibits similar pharmacological sensitivities (170, 568). the ratio for flufenamic acid (muscarinic Icat over voltage-Among these blockers, quinine and its stereoisomer quini- dependent Ca2/ current) was found to be 7.9 at a concen-dine seem to be relatively selective for the muscarinic Icat , tration that causes aú80% reduction in this Icat (100 mM).since their IC50 values (0.25–1 mM) are significantly lower In contrast, the ratio for quinine (at 10 mM) was found tothan the typical values reported for their actions on other be slightly inferior at 4.5. Interestingly, a recent investiga-classes of channel (214). In contrast, the IC50 values for tion in the rabbit portal vein revealed that fenamates exertthe other blockers coincide almost exactly with the con- potentiating effects on the a1-adrenergic Icat (1192). Thecentrations in which they are normally used. For example, possession of such dual actions by fenamates suggeststhe concentrations of TEA, 4-AP, procaine, and caffeine that DPC derivatives might be useful as a starting pointthat effectively inhibit Icat fall within the range 1–10 mM, in the design of more selective antagonists and agonistsand those of verapamil and nicardipine fall in the micro- for the voltage-dependent type NSCC in SM.molar range. These results suggest either that the inhibi- In addition to those of organic blockers, complex

effects of divalent cations on voltage-dependent Icat havetory actions of standard K/ and Ca2/ channel blockersmay not reflect a strict discrimination between different been reported. In the guinea pig ileum, standard VOCC

blockers, such as Zn2/, Cd2/, Ni2/, Co2/, and Mn2/, havechannel structures, or alternatively that the basic designof the channel pore region might not differ greatly among been found to inhibit the muscarinic Icat with IC50 values

of 38 (Inoue, unpublished data), 98, 131, 700, and 1,000the different types of cation-permeable channels. In thiscontext, the reader should be reminded that the amino mM, respectively (473, 474, 1153). This inhibition is likely

to occur through occupation of a single binding site, prob-acid sequence of the putative pore of NSCCATP shows astriking similarity to the common motif for the pore region ably located almost outside the transmembrane electrical

field (i.e., Hill coefficient ofÇ1.0, almost voltage-indepen-of the voltage-gated K/ channel (1115). In any case, spe-cial care should be taken when examining the contribu- dent inhibition). A similar type of inhibition has been re-



ported for the muscarinic Icat in the canine colon (Cd2/, sustained phases (548, 983, 1141). The transient [Ca2/]i

rise is thought to reflect mainly liberation of stored Ca2/Ni2/, Ç100 mM; Ref. 651), and for the a1-adrenergic Icat inrabbit portal vein (Cd2/, 100 mM; Ref. 482). In contrast, via production of InsP3 , whereas the long-sustained rise

in [Ca2/]i reflects a Ca2/ entry from the extracellularthe effects of Ca2/ are more variable. It was originallyreported by Inoue (473) that, in Na/-rich external solution, space that is partly sensitive to dihydropyridine inhibi-

tion (407, 944).millimolar concentrations of Ca2/ potentiate the musca-rinic Icat in the guinea pig ileum in a way that is probably Van Rentergem and co-workers (1120, 1121) explored

the mechanisms underlying the contractile and [Ca2/]i re-independent of [Ca2/]i . However, this observation waslater questioned by the finding that the effect of Ca2/ on sponses to these vasoconstrictors by means of the patch-

clamp technique in an established culture line of vascularthe muscarinic Icat in Cs/-rich external solution is solelyinhibitory (1231). Similar conflicting results have very re- SMC, namely, A7r5 cells. In addition to the inhibitory ef-

fects on VOCC, two common electrophysiologicalcently been presented for the hydrogen ion (479, 1232).A study of the literature reveals that potentiating and in- changes were observed in response to the ET agonist ET-1

or AVP. These changes were 1) an early hyperpolarizationhibitory effects of Ca2/ may occur in neuronal-type andmuscle-type nicotinic NSCC, respectively. The most plau- accompanied by cessation of spontaneous spike activities,

and 2) a subsequent slow depolarization with superim-sible mechanisms to account for these effects could wellbe 1) a permeation block caused by competition for occu- posed increased spike discharges. The hyperpolarization

probably reflects a secondary activation of Ca2/-depen-pancy of the channel (pore) between Ca2/ and monova-lent cations and 2) a direct facilitatory modulation of cat- dent K/ channels due to liberation of stored Ca2/ via the

InsP3-dependent mechanism, whereas the depolarizationionic conductance by Ca2/ (229, 1127). A similar situationmay exist for the muscarinic Icat , since significant interac- appears to be associated with an increased permeability

to monovalent as well as divalent cations through NSCC.tion seems to occur between permeant cations in theNSCC pore. It is consistent with this idea that a recent The AVP-induced cationic current could still be recorded

when the cell was dialyzed with 10 mM EGTA, but it waspatch-clamp study in the rabbit portal vein has revealedthat the a1-adrenergic Icat is indeed dually regulated by not activated by the Ca2/ ionophore A23187 or by InsP3

alone. This suggests that activation of AVP-activatedexternal Ca2/; micromolar Ca2/ augmented the current,whereas millimolar Ca2/ inhibited it (399). Obviously, NSCC is unlikely to be mediated by an elevation in [Ca2/]i

due to InsP3-mediated Ca2/ release, although such Ca2/more information will be needed before we can com-pletely untangle such complex aspects of ion permeation insensitivity has not been tested in the case of the chan-

nels activated by ET-1. The activity of the ET-1-inducedthrough the muscarinic as well as the a1-adrenergic NSCC.In summary, the Ca2/ permeability of voltage-de- cationic current seems to be independent of the mem-

brane potential, since the reported current-voltage rela-pendent NSCC may not be particularly important in di-rectly providing Ca2/ for the contractile system. In- tionship is almost linear. These results suggest that the

NSCC activated by ET-1 or AVP may constitute a newstead, their enhancement of voltage-dependent Ca2/ en-try through their depolarizing actions seems likely to family of ROCC, capable of passing divalent cations and

independent of voltage or [Ca2/]i .have a major functional importance. Because the mem-brane potential, the [Ca2/]i , and the mechanical load Similar actions of ET-1, namely, depolarizing the

membrane and inducing inward currents, have been re-are all changing dynamically in spontaneously activeSM, the presence of immediate positive-feedback con- ported from rat aortic and mesenteric arterial SMC in

short-term primary culture. In these cells, it was foundtrols over the NSCC (voltage dependence, [Ca2/]i depen-dence, and mechanosensitivity) may be of great benefit that ET-1, as well as its homolog safratoxin S6b, and AVP

(but not phenylephrine) can activate nifedipine-insensi-for the effective tuning of their kinetics; this probablyworks in close cooperation with other Ca2/-handling tive cationic currents that pass Na/, Li/, and Cs/, and

which are inhibitable by submillimolar Ni2/ or Co2/ andeffectors, such as VOCC, Ca2/ stores, and perhaps Na//Ca2/ exchange (485). abolished by removal of external Ca2/. In this study, how-

ever, a possible contribution of a Ca2/-inducible conduc-tance (such as a Cl0 current) was not eliminated; the ET-2. Voltage-independent, [Ca2/]i-insensitive1-induced current showed a transient time course, ratherreceptor-operated NSCCthan a sustained one, and it was abolished by replacingexternal Ca2/ with Sr2/ or Ba2/, or by inclusion of 10 mMPotent vasoconstrictors such as endothelin (ET)

and arginine vasopressin (AVP) can produce a long- BAPTA in the pipette.The AVP-induced cationic currents in A7r5 cells canmaintained contraction of tonic SM, such as rat aortic

SMC, which appears to be associated with either a mo- be further divided into two distinct components: onehighly selective for Ca2/ (PCa/PCs ú17) and the other anophasic transient increase in [Ca2/]i (694, 1141) or a

biphasic rise in [Ca2/]i , consisting of transient and long- cationic one (1120). The Ca2/-selective component was



found to exhibit a high permeability to Sr2/ (but only would imply that selective inhibition of these channelsmight be of therapeutic importance for certain pathologi-poorly to pass Mn2/) and to be inhibited by multivalent

cations known potently to block Ca2/-selective channels cal conditions such as hypertension, where vasoconstric-tor peptides may play essential roles (852).such as VOCC, as well as ICRAC (see below). The observed

sequence of inhibitory efficacy was La3/ ú Cd2/ ú Co2/ ú The activation mechanism for vasoconstrictor pep-tide-activated NSCC remains unclear. Although it proba-Ni2/ Ç Mn2/. Another interesting property of this current

component is that the current amplitude tends to saturate bly does not involve [Ca2/]i or InsP3 directly, we cannotrule out the involvement of PKC (852) or some other un-at high external Ca2/ concentrations, in a manner that fits

with Michaelis-Menten kinetics (apparent Kd Å 9.7 mM). known intracellular mechanisms that transduce cell-sur-face signals to the channel proteins. For example, cou-This property is suggestive of the presence of one or more

high-affinity binding sites for Ca2/ in the channel pore, pling of G protein to these NSCC, which has already beenmentioned above, might also exist as a general rule, link-from which the high selectivity for Ca2/ might result. It

has been estimated that the rate of [Ca2/]i increase due ing G protein-coupled receptors and ROCC in vascularSMC. This view is supported by the observation that acti-to this current component under unbuffered conditions

would be as high as several hundreds of nanomolar per vation of AVP-activated NSCC in A7r5 cells is completelyprevented by dialyzing GDPbS into the cell, via a PTX-second at physiological concentrations of external Ca2/.

A recent study in which molecular techniques were insensitive mechanism.combined with patch-clamp and [Ca2/]i measurements ex-amined the effects of extremely low concentrations of

C. Cytosolic Ca2/-Activated Nonselective CationET-1 (õ0.1 nM) on mouse fibroblast cells overexpressedChannelswith recombinant human ETA receptors (Ltk0 cells) and

on freshly dissociated SMC from the rabbit aorta (276).These low concentrations of ET-1, which may be physio- Two types of NSCC have been reported to be directly

activated by an elevation in [Ca2/]i in SM. In rat portallogical, have been reported to produce membrane depo-larizations and vasoconstrictions that are partly insensi- vein SMC, various agents and events that can elevate

[Ca2/]i , such as NE, ACh, caffeine, Ca2/ influx throughtive to DHP, without stimulating InsP3 production (702).The novelty of this approach lies in the overexpression VOCC, and the slow continuous leakage of stored Ca2/

caused by ryanodine, all induced resolvable openings oftechnique, which achieves an amplification of responseslinked to ETA receptors that are normally very tiny and NSCC with an extremely large conductance (Ç200 pS in

saline) under whole cell recording conditions (670). Thesenot suitable for electrophysiological analysis. The resultsclearly indicated that, in Ltk0 cells overexpressed with channel openings occur concomitantly with the main con-

ductance activated during agonist-mediated Ca2/ mobili-ETA receptors, a maintained [Ca2/]i rise can be elicitedby ET-1 at concentrations as low as 0.1 nM, whereas at zation in this preparation, namely, Ca2/-dependent Cl0

currents (875), and are completely abolished when [Ca2/]ihigher concentrations (1–10 nM), the same agent can alsoelicit a rapid [Ca2/]i rise, which probably reflects Ca2/ elevation is prevented by the inclusion of a high concen-

tration of EGTA in the pipette or by replacing externalrelease. Correspondingly, in voltage-clamp experiments,0.1 nM ET-1 induced a sustained Ca2/-permeable cationic Ca2/ with Ba2/. The most noteworthy feature of this NSCC

is its extraordinarily high selectivity for Ca2/: the PCa/current insensitive to voltage or [Ca2/]i . The ET-1-inducedcationic current and concomitant rise in [Ca2/]i in trans- PNa calculated from reversal potential measurements is as

high as 21, whereas the unitary conductance measured infected Ltk0 cells was not affected by the VOCC blockernifedipine (10 mM) but was almost completely suppressed high-Ca2/ containing solution (91 mM outside; Ç75 pS) is

substantially smaller than that measured in saline. Suchby a general blocker for NSCC, mefenamic acid (300 mM).A cationic current exhibiting similar pharmacological and an apparent discrepancy between high Ca2/ selectivity

and low Ca2/ conductance is suggestive of the preferentialbiophysical properties could be recorded from freshly dis-sociated aortic SMC. These results, taken collectively, sojourn of Ca2/ inside the cation channel pore, a phenom-

enon already mentioned above. However, the physiologi-suggest that activation of voltage- and [Ca2/]i-indepen-dent, highly Ca2/-permeable NSCC may serve as a key cal significance of the activation of this NSCC is uncertain,

since its opening appears to occur with an extremely lowCa2/-entry pathway for inducing persistent vasoconstric-tion under physiological conditions. This would be in frequency, even with maximum intensity agonist stimula-

tion (no more than triple simultaneous openings havesharp contrast to the voltage-dependent, [Ca2/]i-sensitiveNSCC found in spontaneously active SM (however, note been observed in the whole cell preparation). As a possi-

ble function, a role has been proposed for this channelthat another important effect of ET is to sensitize thecontractile machinery, see sect. VC). as a route for refilling the internal stores after agonist

stimulation (670).This presumed physiological function of vasocon-strictor-activated NSCC, namely, as a Ca2/-entry route, Another type of Ca2/-dependent NSCC has been re-



ported to exist in ear artery SMC (1149). This channel the pipette, but not by application of ACh or by depolariz-ing pulses that elicit comparable elevations in [Ca2/]i . Aexhibits a basal activity that is further enhanced by the

Ca2/-releasing agents caffeine and NE or by the Ca2/ iono- relatively high Ca2/ permeability (Ç20% of the currentbeing carried by Ca2/) has been deduced from a compari-phore ionomycin. The unitary conductance of this NSCC

is low (28 pS in saline), and its Ca2/ permeability seems son of the charge transported through the channel andthe [Ca2/]i rise measured in the presence of ryanodine,extremely low (no discernible current flows with 89 mM

Ca2/ alone). These properties are in sharp contrast to after correcting for endogenous buffering and removalcapacities (375). Activation of this channel is unlikely tothose of the Ca2/-dependent NSCC found in the rat portal

vein and may be closer to those of channels found in involve the intracellular accumulation of cAMP due tophosphodiesterase inhibition, since a stable and mem-tissues other than SM, including cardiac myocytes, neuro-

blastoma cells, pancreatic acinar cells, and lacrimal glan- brane-permeable analog of cAMP, 8-bromo-cAMP, failedto mimic the effects of caffeine.dular cells (1064). The physiological role of such ear ar-

tery NSCC may not in essence differ from the generallypresumed function of depolarizing the membrane and in-

E. Muscarinic Receptor-Inactivated K/ Current:creasing voltage-dependent Ca2/ entry. However, the in-M Currentteresting question has been raised in this study (2403) as

to why the probability of recording Ca2/-activated NSCCin the ear artery seems higher with a low Cl0 concentra- The M current was first described in the bullfrog sym-

pathetic ganglion (129) as a novel K/ conductance thattion in the pipette than in Cl0-rich conditions. We madean analogous observation when studying the a1-adreno- was found to be inhibitable by muscarinic activation and

distinguishable from other voltage-dependent K/ conduc-ceptor-activated NSCC in the rabbit portal vein, namely,that the potentiating effects of fenamates on this type of tances. It was later identified in a broad range of tissues

including hippocampus, sympathetic neurons, a neuro-NSCC (see above) tend to decrease under Cl0-rich condi-tions (K. Yamada and R. Inoue, unpublished data). More- blastoma-glioma cell line, NG108–15, and SM (894). The

M current is activated at potentials more positive thanover, the importance of intracellular Cl0 activity in regu-lating ion channel activity has recently been recognized, 070 to 060 mV and exhibits a sigmoidal dependence on

the membrane potential. This property enables the currente.g., in KATP (719, 1072). This form of regulation will bean intriguing subject for future research on the NSCC to act as if a net ‘‘inward’’ current were flowing during

muscarinic suppression that would increase at more posi-in SM.Although their place in this section may seem ques- tive potentials and decrease at more negative potentials.

The best-characterized example of M current in SM istionable, since changes in [Ca2/]i would not be a primaryregulator, we should mention here that Ca2/-sensitive found in the toad stomach (987), and a similar K/ current

component has been detected in guinea pig stomach SMCNSCC having a conductance (211 pS in symmetrical K/

saline) as large as the conductance of those in the rat (641). The reversal potential of the toad stomach M cur-rent was found to accord closely with the K/ equilibriumportal vein have been identified in the rat cerebral artery

(719). These NSCC are spontaneously active and only potential when external K/ concentrations were varied,thus suggesting its strict selectivity for K/ over otherweakly dependent on [Ca2/]i as well as on the membrane

potential, and they are poorly permeable to Ca2/, and not monovalent cations (987). The steady-state activationcurve for this current is well fitted by a Boltzmann-typestretch activatable. The functional significance of these

channels may lie not in depolarizing the membrane, but sigmoid relationship with a half-maximum activation volt-age of 049 mV and a slope factor of 9 mV (987).rather in passing outward currents at membrane poten-

tials more positive than approximately040 mV (the rever- Voltage-jump experiments have revealed that the cur-rent appears to activate or inactivate upon depolarizationsal potential is about 042 mV), thereby limiting excessive

membrane depolarization during action potential activity. or hyperpolarization, respectively, to a new level appro-priate for the absolute level of the membrane potential,with time constants ranging from tens to hundreds of

D. Unclassified Cation Channels milliseconds (10, 986, 990). This kind of gating behavioris reminiscent of other types of ligand-gated channels,such as nicotinic NSCC at the end plate and muscarinicConductances observable during caffeine application

are generally thought to be triggered by the release of Ca2/ K/ channels in the heart (11), and shows a striking quanti-tative similarity to the voltage-dependent NSCC (e.g., Ref.from the internal stores and thus to be Ca2/ dependent.

However, Guerrero et al. (374) have discovered a novel 479). Indeed, a current-clamp experiment in the toadstomach (987) revealed that ACh is able to depolarizeclass of 80-pS NSCC that open in direct response to caf-

feine (20 mM) via a [Ca2/]i-independent mechanism. the membrane rapidly, with a time course that cannot bedistinguished from that caused by NSCC activation inThese channels can be activated with 10 mM BAPTA in



other SMC (74, 479, 569, 651, 1134). However, the activity ribose) imitates, while streptozotocin (which reduces thelevel of NAD/) attenuates, the inhibitory effect of AChof the toad stomach M current was not affected by re-

moval of Ca2/ from the bath or inclusion of high concen- on single-channel activities representing the M current,suggesting a possible second messenger role for cADPtrations of EGTA in the pipette (986). This means that the

M current is not affected by changes in [Ca2/]i , which ribose in M current (channel) inhibition (406).contrasts with a remarkable dependence of voltage-de-pendent NSCC on [Ca2/]i . In contrast, the presence of a

VI. MOBILIZATION OF CALCIUM ANDmillimolar concentration of Ba2/ in the bath, which is

MAINTENANCE OF CALCIUM IONalso known to affect a number of voltage-dependent K/

HOMEOSTASIS IN VISCERAL SMOOTHcurrents (214), strongly inhibits this current. It is now

MUSCLE CELLSgenerally agreed that a variety of neurohormones, such assubstance P and luteinizing hormone-releasing hormone,

Basically, the induction of a contraction (activationmodulate the M current through their own receptors. Like-

of cross bridges between contractile proteins) is initiatedwise, the toad stomach M current has also been found to

by the formation of a Ca2/-CaM complex triggered by anbe suppressed by tachykinins (substance P, substance K)

increased Ca2/ concentration in the cytosol. In this sec-via receptors distinct from the muscarinic ones (989). In

tion, we review the ways in which Ca2/ homeostasis isaddition to such inhibitory actions, it has been found that

maintained in VSMC; that is, by the influx and efflux ofthe toad stomach M current has a unique property: it can

Ca2/ across the sarcolemmal membrane (SL) and by thebe augmented by the b-adrenergic agonist Isop (986, 988).

release from and uptake of Ca2/ into the cytosolic Ca2/This upregulating effect of b-adrenoceptor activation is

store sites (namely, the SR). In VSMC, the total surfaceprobably due to increased cAMP production through stim-

area of the SR is thought to be the equivalent of a fewulated adenylate cyclase activity, since forskolin or the

percent of the total SL surface, a value much smaller thanmembrane-permeant analog 8-bromo-cAMP is also effec-

that for skeletal muscle (327). Some of the SR is distrib-tive in augmenting the current. The physiological signifi-

uted just beneath the SL (in close contact with caveolescance of this mechanism is unclear but may lie in b-adre-

or the SL), and some is deep in the cytosol. A usefulnoceptor-induced relaxation of this muscle, since in-

review of Ca2/ homeostasis in VSMC was published bycreased M-current activity may lead to membrane

Himpens and Missiaen (413).hyperpolarization, thereby lessening the membrane excit-ability and reducing the likelihood of the generation ofspikes. A. Ca2/ Concentration in the Cytosol of VSMC

The molecular mechanism underlying the muscarinicsuppression of the M current remains unclear, although In VSMC, in studies using the luminescent Ca2/ indi-

cator aequorin, the [Ca2/]i was first measured by Fay etan involvement of a PTX-insensitive G protein has beensuggested (259, 691). To date, modulatory effects on this al. (287) in toad stomach, and later by Morgan and Morgan

(775). Since those pioneering studies, many investigatorscurrent exerted by InsP3 and a PKC activator, phorbolester, have been demonstrated (201, 202, 259, 688, 692). using various dyes have measured the [Ca2/]i from differ-

ent tissues and species. In resting and active tissues, Ca2/However, neither of these has been proved to be obliga-tory (130). A recent cell-attached recording of single M- concentrations are mainly measured using aequorin, quin

2, fura 2, furaptra, fluo 3, rhod 2, or indo 1 (96, 373, 1105,channel activity (three distinct conductances of 19, 12,and 7 pS have been reported; Ref. 981) has revealed that 1107, 1142). Under resting conditions, an average value

for [Ca2/]i is thought to be 100–150 nM. However, thisthe channel activity can be suppressed even when musca-rinic agonists are applied outside the patch, suggesting a value is affected by various factors (e.g., temperature,

stretch, cytosolic and external ionic environments, andcontribution to muscarinic suppression by one or moreremote signal(s), presumably diffusible second messen- experimental protocol), and it also differs according to

the tissue and species (335, 336, 776). Bukoski et al. (134)gers. Clapp et al. (202) observed a modulation of the ac-tions of ACh on the M current by a synthetic DAG, 1,2- estimated that, in resistance arteries, the [Ca2/]i was 79

nM in the resting state (measured using fura 2), whereasdioctanoyl-sn-glycerol (DiC68). They concluded that AChand DiC8 each suppressed both the endogenous and Isop- Jensen et al. (519), using the same fura 2 procedure, re-

ported a value of 114 nM in Wistar-Kyoto rat mesentericinduced M currents without altering the time course of Mcurrent deactivation. This suggested that these agents act artery at the resting membrane potential of 061.2 mV. In

the guinea pig coronary artery, Ganitkevich and Isenbergby decreasing the number of channels available to beopened, thus providing evidence that muscarinic regula- (338, 339) found a resting [Ca2/]i of 155 nM, when the cell

was voltage-clamped near the resting membrane poten-tion of M currents is mediated by DAG. Interestingly, avery recent study in neuroblastoma/glioma NG108–15 tial. In the guinea pig urinary bladder, it has been reported

that, at a holding potential of 062 mV, a short (1 s) caf-cells has demonstrated that a NAD/ metabolite (cADP



feine application (10 mM) increased [Ca2/]i within 1 s is or is not distributed homogeneously during active statesin SMC. It should be also mentioned here that it is ques-from the resting 118 to 1,490 nM, but upon washout of

caffeine, the [Ca2/]i did not return immediately to the tionable whether [Ca2/]i measured by dyes accurately re-flects the concentrations in the cytosol and, furthermore,resting level; in fact, it was reduced to 47 nM (this phe-

nomenon is termed ‘‘undershoot’’; Ref. 336). According to whether or not the SR contains similar densities of theryanodine and InsP3 receptors. In this context, an interest-Itoh et al. (501), in the rabbit mesenteric artery the resting

value was Ç110 nM. In Ca2/-free EGTA-containing solu- ing notion was put forward by Yamaguchi et al. (1196).They argued that when the peripheral layer of the cytosoltion, this value fell to 70–80 nM, and the addition of a K/

channel opener caused a further reduction (to 50–60 nM; has a high [Ca2/]i , its fluorescence can sometimes con-found attempts to measure [Ca2/]i with conventional op-Refs. 508, 1056). In rat portal vein SMC, exogenously ap-

plied ATP (10 mM) increased the cytosolic Ca2/ from the tics. Thus a given level of mechanical response and ofelectrical activity in the SL induced by mobilization ofresting value of 92 to 557 nM; however, when NE (10

mM) was given as pretreatment, only 23.6% of the control Ca2/ in the cytosol may not give rise to consistent mea-surements due to nonhomogeneous distributions of Ca2/increase (i.e., that seen with ATP alone) was induced by

subsequently applied ATP (872). during the active state of the cells. Yamaguchi et al. (1196)measured the Ca2/ distribution in airway SMC cytosolsThe amount of [Ca2/]i required to activate contractile

proteins may also be estimated; this is done by measuring after the application of Isop or caged cAMP, using fura2 with confocal microscopy. They concluded that Isopthe contraction evoked by Ca2/ in MC permeabilized

(‘‘skinned’’) by saponin, a-toxin (a Staphylococcus exo- decreased [Ca2/]i in the inner cytosol but increased it inthe peripheral cytosol by mechanisms that depended ontoxin), or b-escin (a saponin ester). Saponin is commonly

used to prepare a completely permeabilized cell because both extracellular SL and SR Ca2/ release channels. Ifcorrect, this means that a heterogeneous distribution ofit forms small pores through the cell membrane (SL), and

the latter two agents are used to only partially permeabil- CICR- and InsP3-induced Ca2/ release (IICR) receptorsin SMC presumably exists. Furthermore, when Remboldize the SL (smaller pores through which substances of

more than 1,000 mol wt cannot penetrate) without damag- (924) studied Ca2/ movements in arterial cells from mea-surements of the intensities of aequorin- and fura 2-Ca2/ing agonist receptors (326, 462). Using these permeabiliza-

tion procedures, many investigators have studied the part transients, as well as myosin light chain phosphorylationand force, they concluded 1) that the focal increase inplayed by Ca2/ in mechanical responses. According to

such studies, the minimum concentration of Ca2/ that [Ca2/]i induced by histamine occurred in different cellularregions from those in which Ca2/ restoration took place,will produce a contraction is Ç200 nM, and a maximum

contraction was evoked by 0.8–1.0 mM. Between these and 2) caffeine inhibited SM contraction by localizing theincrease in [Ca2/]i to a region distant from the contractileextremes, the mechanical response evoked is in propor-

tion to the concentration of Ca2/. Comparison with the apparatus. They further suggested that there can be tran-sient and sustained focal increases in [Ca2/]i . Aequorinvalues given earlier shows that the Ca2/ concentration

required to reach the threshold for contraction is just was more accurate than fura 2 at detecting increased[Ca2/]i in a small region of the cytoplasm during its releaseabove the resting cytosolic concentration. In a combined

study, Jensen et al. (520) measured [Ca2/]i from the rat after refilling of the Ca2/ stores and on caffeine stimula-tion. Etter et al. (280) applied a new Ca2/ indicator, FFP18,mesenteric artery, using both the Ca2/ electrode (1194,

1195) and fura 2. They found that, at the resting membrane which is more water soluble than previously used indica-tors, and reported that images of the intracellular distribu-potential (057 mV), the value given by the Ca2/ electrode

was 115 nM and that obtained with fura 2 was 129 nM. tion of FFP18 show that more than 65% is located on ornear the plasma membrane. Calcium transients measuredUnder treatment with 1 mM NE, the membrane potential

was 036 mV and the Ca2/ concentration measured using using FFP18 during membrane depolarization-inducedCa2/ influx revealed that near-membrane [Ca2/]i risesthe above two procedures was 708 or 537 nM, respec-

tively. This result sits well with the opinion of Fay et quickly and reaches millimolar levels at early times (riseswithin 20 ms, peaks at 50–100 ms at near-membraneal. (288) that, in vivo, the response to a wide range of

physiological stimuli would involve a rise in [Ca2/]i to sites). In contrast, levels of only a few hundred nanomolarare measured by fura 2. They therefore postulated that theno more than 600–800 nM, because of the existence of

powerful feedback mechanisms that resist further change existence of such large, rapid increases in [Ca2/]i directlybeneath the surface membrane may explain how numer-beyond this level (57, 1175, 1191). Thus, between the rest-

ing and active states, [Ca2/]i may vary from 100–150 nM ous Ca2/-sensitive membrane processes are activated attimes when the changes in bulk cytoplasmic Ca2/ concen-to 600–800 nM.

Although the [Ca2/]i has been estimated during rest- tration are too small to activate them. A region just be-neath the SL is called a ‘‘fussy’’ zone, because it maying and active states, and such estimates are informative

and useful, it is also necessary to know whether the [Ca2/]i retain Ca2/ or it may form a barrier against diffusion.



Significantly, some SR vesicles are distributed just be- Ca2/ gradient, and 5) some agonists generate InsP3, whichthen short-circuits the SBB to increase the effectivenessneath the SL membrane. In fact, in a variety of SMC includ-

ing rabbit and guinea pig portal veins, spontaneous tran- of Ca2/ influx in raising [Ca2/]i , and consequently in-creases the SM contraction. More findings related to thesient outward currents (STOC), which are thought to

reflect a periodic local release of Ca2/ stored in the super- refilling of Ca2/ into the store sites after their depletionto the lumen are discussed below.ficially localized SR (involving bursts of mainly Ca2/-de-

pendent K/ currents; for details see below), were re- The main channels involved in Ca2/ release from theSR are normally classified as ryanodine- and InsP3-sensi-corded from both whole cell and cell-free patch mem-

branes (71, 808, 8247, 1185). tive Ca2/ channels. On the other hand, after consideringthe nonhomogeneous distributions of the ryanodine- andLesh et al. (657, 658) demonstrated, using an immuno-

histochemical procedure, that the ryanodine receptor, InsP3 receptors, Iino et al. (459) and Iino (456) put forwardthe idea that the Ca2/ compartments responsible for theRyR (a CICR-mechanism activator; see below) is mainly

distributed on the more superficial SR vesicles in the release of Ca2/ should be classified as either Sa (containsCICR and IICR receptors) or Sb (contains IICR alone) onguinea pig taenia coli, but not in the aorta. Xiong et al.

(1185) found, in the guinea pig portal vein, that SR vesicles the basis both of their modes of Ca2/ release and theresulting contractions observed in intact and skinnedwere attached to SL fragments that were sensitive to caf-

feine (which stimulates Ca2/ release via the CICR path in muscle tissues. They estimated that Sa makes up 40% ofthe total Ca2/ components in the taenia coli, Ç50% in thethe SR) or heparin sodium (an inhibitor of IICR from the

SR). This was deduced from measurements of electrical pulmonary artery, õ20% in the portal vein, and õ10% inthe myometrium. However, these estimates were not com-currents using the cell-free patch-clamp procedure. They

further concluded that because caffeine was more effec- pletely supported by other investigators (750).It is of interest that caffeine, a releaser of Ca2/tive than heparin at blocking STOC or the increased open-

ings of K/ unitary currents in such a cell-free preparation, through the CICR mechanism, does not produce a con-traction in the myometrium. Indeed, Morgan et al. (774)the caffeine-sensitive Ca2/ channels (RyR) are presum-

ably preferentially distributed near the surface membrane. found that caffeine has no effect on resting [Ca2/]i incultured human myometrial cells. Moreover, Lynn et al.The functional connection between the SR and the

SL in VSMC has been discussed mostly to try to explain (677) found, in the same preparation, that the CICR mech-anism was accelerated by a combined application of ryan-SR refilling from the extracellular space. Thus it has been

proposed that there is a direct path between the SR and odine and caffeine, but not directly activated by caffeinealone. Similarly, Lynn and Gillespie (676), using 45Ca2/SL (117, 168), or a continuous vectoral release of Ca2/

from the SR lumen to the extracellular space (188, 189). technique in skinned muscle preparations from the humanuterine artery, found that the CICR mechanism in thisJanssen and Sims (513) observed Ca2/ mobilization in

canine tracheal myocytes during the ACh-induced inward tissue was sensitive to ryanodine and ruthenium red, butnot to caffeine. It thus would appear that there is a tissuecurrent and contraction and concluded that the current

and contraction are mediated via Ca2/ released from an specificity in respect of the CICR mechanism in VSMC.Concerning the actions of caffeine, Guerrero and co-work-internal store that can be depleted by prolonged removal

of extracellular Ca2/, or by repeated applications of either ers (374, 375) observed, using simultaneous measure-ments of the transmembrane ionic current and [Ca2/]i ,caffeine or the Ca2/-ATPase inhibitor CPA. At least two

Ca2/ influx pathways appear to contribute to the refilling that activation by caffeine of nonselective cation channelsin the SL was not a consequence of the emptying of inter-of the internal store: one is not activated by depolarization

or ACh, and the second involves direct contact with the nal Ca2/ stores. Rather, they proposed that caffeine acti-vates these SL channels either by direct interaction orSR and thus conducts extracellular Ca2/ directly into the

SR, bypassing the cytosol. Van Breemen et al. (1115) sum- alternatively by a linkage between the RyR on the SR andthe ROCC on the SL.marized their own view that, in SMC, the superficial SR

accumulates a portion of the Ca2/ that enters the cellthrough the SL, and thus functions as a buffer barrier to

B. Factors That Increase [Ca2/]i in VSMCCa2/ entry into the cytosol (‘‘superficial buffer barrier’’;SBB). Evidence for this notion is as follows: 1) contrac-

1. Influx of Ca2/ through the SLtion is related more to the rate than to the extent of Ca2/

entry, 2) refilling of the SR from the luminal space is In the rabbit coronary artery, Matsuda et al. (707)estimated that, with the average peak current at /20 mVmediated by Ca2/ influx and Ca2/ pumping by the SR Ca2/

pump, 3) the superficial SR unloads Ca2/ to the lumen by being 217 pA in 110 mM Ba2/, the probability of Ca2/

channel opening is 0.13, average cell surface area isa multistep process that involves the opening of Ca2/- andInsP3-sensitive channels followed by Ca2/ extrusion via 1.87 1 103 mm2, and the single-channel conductance is

0.53 pA at /20 mV. From the above values, the estimatedNa//Ca2/ exchange, 4) the SBB generates a peripheral



channel density is 1.71 channels/mm2. Nelson and co- electron microscopy (pinwheel-like structure, Ref. 170).3) In the cerebellum, micromolar concentrations of Ca2/workers (809, 810) made similar calculations using data

obtained by means of the patch-clamp procedure, after inhibit InsP3 binding to the receptor when a Ca2/-bindingprotein, calmedin, is present (232); however, the vas def-making various assumptions. Sumimoto et al. (1046) also

estimated the number of voltage-dependent, nifedipine- erens InsP3 receptor does not exhibit such inhibition,probably due to a lack of endogenous calmedin activitysensitive Ca2/ channels; after making various assump-

tions, they concluded that the density of the nifedipine- (779). 4) PKA, PKC, and CaM-dependent kinase II can allphosphorylate the cerebellar InsP3 but act on the receptorbinding sites was 6–14 sites/mm2. Of course, such figures

will not hold true for all tissues; some, such as the iris at different sites. The vas deferens InsP3 receptor wasinitially reported to be stoichiometrically phosphorylatedsphincter or dilator, appear to have a very sparse distribu-

tion of this channel (1062, 1216). by PKA (779). In support of the functional significance ofthis phosphorylation, it was later shown, using membranehomogenates, that inhibition of [3H]InsP3 binding can be2. Ca2/ release from the SRinduced by various cAMP-increasing agents (rabbit tra-cheal SMC, Ref. 968). However, the net consequence ofIn many cells, distributions of two types of receptor-

related store, i.e., InsP3-sensitive and ryanodine-sensitive cAMP-dependent phosphorylation may be quite complex,since this process may either increase the Ca2/ contentstores, have been postulated, and this is also true for

VSMC. It is well recognized that, when Ca2/-mobilizing of the stores by stimulating SR Ca2/-ATPase (500, 901) orinhibit the agonist-stimulated hydrolysis of phosphoinosi-receptors are stimulated, the [Ca2/]i of VSMC, as moni-

tored using Ca2/ indicators, shows changes with a com- tide (903). Thus cAMP-dependent phosphorylation couldproduce both enhancement (139) and inhibition (500, 603,plex time course. It is often found that a persistent eleva-

tion in [Ca2/]i during prolonged receptor stimulation is 843, 1050) of Ca2/ release.In phasic (e.g., vas deferens) and tonic (e.g., aorta)readily abolished on elimination of external Ca2/, whereas

an early transient rise in [Ca2/]i tends to last longer in the SM, the localization of InsP3 receptors was investigatedby Nixon et al. (823). They found that the InsP3 receptorabsence of external Ca2/. It is now agreed that the early

transient [Ca2/]i rise corresponds mainly to the release of and calsequesterin occurred together in both types ofSMC. In the vas deferens, InsP3 receptors were localizedstored Ca2/; this is mediated by the opening of InsP3-gated

Ca2/-release channels through stimulated phosphoinosi- at the cell periphery, whereas in the aorta they were pre-dominantly central. The ultrastructure of the SR, investi-tide breakdown via G protein-dependent PLC activation.

In addition, another type of Ca2/ release channel, which gated in stereo views of semi-thick and thin sections (os-mium ferricyanide stained) showed fenestrated sheetsopens in direct response to an elevation of [Ca2/]i , may

also contribute to the agonist-induced Ca2/ mobilization. and numerous regions of continuity between central andperipheral SR, suggesting that they formed a single com-Later, Itoh et al. (505) suggested that the CICR may oper-

ate physiologically in guinea pig mesenteric artery. They partment within these SMC. Regions of the SR wereclosely apposed to and often ensheathed mitochondria.came to this conclusion after comparing the effects of

caffeine and procaine (a blocker of the CICR). They concluded that InsP3 receptors are present in boththe central and peripheral SR of tonic and phasic muscle,In the following sections, the detailed features of

these two modes of Ca2/ release are described. consistent with the results of electron-probe analysisshowing Ca2/ release from both regions.A) INSP3 RECEPTOR. The receptor responsible for InsP3

binding was first purified, and found to be a 260-kDa pro- Ehrlich and Watras (268), working on canine aorticSMC, found an InsP3-activated Ca2/ channel in the SRtein, from the rat cerebellum (742, 1051). Subsequently,

proteins that possess essentially the same properties as using planar lipid bilayers. This channel had no voltagedependency, and it was activated by InsP3 but not bythose of the cerebellar InsP3 receptor were purified from

two or three types of SM, namely aorta (224 kDa, Ref. caffeine. Furthermore, heparin sodium blocked the chan-nel’s activity when applied from the cytoplasmic side. The170) and vas deferens (260 kDa, Ref. 779). The characteris-

tics of these InsP3 receptors are as follows: 1) the receptor unitary conductance of this channel was Ç10 pS(cis/trans Å Ç1007 M Ca2//53 mM Ca2/) only from thehas a single binding site for InsP3, with a very low Kd in

the nanomolar range (2.4 nM in aorta, Ref. 170; 10 nM in cytoplasmic side (not luminal), and this was greatly inhib-ited by 10 mg/ml heparin sodium. In contrast, both thevas deferens, Ref. 779); these values contrast with the

significantly higher values obtained from functional stud- agonist and the blocker for Ca2/ release in skeletal mus-cle, caffeine (1 mM) and ruthenium red (1–20 mM), re-ies (e.g., flash photolysis, Ref. 1013). 2) Compared with

the proteins mentioned above, the native InsP3 receptor spectively, were ineffective. Another interesting findingfrom this study was that a nonhydrolyzable analog of ATP,has a larger molecular mass (Ç1, 000 kDa, Ref. 779) and

thus is probably a homotetramer consisting of four InsP3- namely, AMP-PCP (100 mM), which by itself was ineffec-tive, enhanced the InsP3-induced channel activity signifi-binding subunits, as also suggested by negative-staining



cantly, in a manner independent of phosphorylation. This 455) clearly showed that the rate of InsP3-induced Ca2/

release increases with an elevation in [Ca2/]i up to 300property may be common among the various InsP3 recep-tors found in different tissues (83, 680). It was later shown nM, but at higher concentrations, this reverses to a de-

crease. This dual effect of [Ca2/]i is unlikely to result fromthat the direct effect of ATP on InsP3-gated channels isbiphasic; an augmentation of channel activity occurs with altered InsP3 affinity for the receptor, since quantitatively

similar degrees of augmentation or inhibition were causedATP in the submillimolar range, whereas a competitiveinhibition of InsP3 binding occurs at millimolar ATP con- by [Ca2/]i at different InsP3 concentrations (0.1–30 mM).

The importance of this conclusion was soon confirmedcentrations and attenuates InsP3-induced Ca2/ release (83,547). Inositol trisphosphate-activatable channels have by different experimental approaches such as measure-

ment of rapid 45Ca2/ flux (rat brain synaptosome, Ref.also been recorded from a near-purified InsP3 receptorobtained from bovine aortic SMC (713). Ion-substitution 295) and lipid bilayer single-channel recording (85). Inter-

estingly, the latter study further showed that a biphasicexperiments suggested that Ca2/ is about twofold morepermeable through the channel than monovalent cations (or bell-shaped) dependence on [Ca2/]i can occur not only

for the InsP3-gated channel but also for the RyR-Ca2/(PCa/PK Å Ç2), whereas Cl0 is almost impermeable. Acti-vation of InsP3-gated channels was specific for channel, suggesting some structural resemblance between

the two receptors. However, the peak of the bell-shapedIns(1,4,5)P3 , because Ins(1,3,4)P3 and Ins(1,3,4,5)P4 werealmost ineffective in inducing the channel’s activity (com- [Ca2/]i dependence occurs at Ç0.2 mM for the former,

which probably allows for both positive- and negative-pare with Ca2/ flux studies, Refs. 293, 762). The slopeunitary conductance calculated between050 and020 mV feedback control of Ca2/ release over the physiologically

attainable [Ca2/]i range, whereas that for the latter is(0.5 M K//50 mM Ca2/) was 32 pS, a value similar tothat obtained from the canine aorta after correction (a above 100 mM, which is far beyond the physiological maxi-

mum and thus may serve solely as an activating mecha-reevaluation of the current-voltage curve published byEhrlich and Watras between 050 and 020 mV gives Ç30 nism. Using flash photolysis of caged InsP3 and Ca2/, Iino

and Endo (458) have demonstrated that Ca2/-dependentpS, Ref. 713), and in good agreement with those reportedelsewhere (e.g., Ref. 680). It has, however, been reported feedback control can operate on a subsecond scale. Such

a fast Ca2/-dependent control may provide for an immedi-that the InsP3 receptor in brain neurons exhibits four sub-conductive states in the presence of different concentra- ate acceleration of the initial phase of IICR, but then soon

begin to act as a brake to prevent further, excessive, re-tions of InsP3 (65 and 80 pS), and the binding of four InsP3

molecules to the receptor is needed to induce the full lease. Furthermore, this property, together with the posi-tive cooperativity of IICR, which may serve to detect andconductance of 80 pS. This indicates that InsP3 would

need to act additively to open the channel (268, 1139, effectively amplify small changes in InsP3 concentration(458, 738), may partly contribute to the generation of rapid1163). These observations are reminiscent of the homotet-

ramer structure proposed for the InsP3 receptor (680). and transient changes, both temporal and spatial, in[Ca2/]i (i.e., [Ca2/]i oscillation and Ca2/ wave propagation;Recently, a detailed investigation was carried out on the

conductive property of InsP3-gated Ca2/ channels from Refs. 79, 458, 738).Although no time-dependent decline is evident forthe cerebellum (84). Surprisingly, although consistent

with the predictions of cloning studies, in this property the opening of InsP3-gated Ca2/ channels incorporatedinto an artificial lipid bilayer (e.g., Ref. 84), IICR doesthe InsP3-gated channel is essentially similar to the RyR-

Ca2/ channel; the conductance sequence is Ba2/ ú appear to fade with time if a constant concentration ofInsP3 is persistently present. Muallem et al. (780) observedSr2/ ú Ca2/ ú Mg2/, and the relationship between the

unitary amplitude and the divalent cation concentration that stimulation of pancreatic acinar cells with InsP3-pro-ducing agonists (carbachol and CCK) at submaximal con-follows Michaelis-Menten-type kinetics. The latter finding,

together with the absence of evidence for the anomalous centrations results in only a partial release of stored Ca2/,but a subsequent maximal agonist stimulation can evokemole fraction effect, strongly suggests that both InsP3-

gated and RyR channels may behave as saturable single- a further release of the Ca2/ remaining in the store. Thisphenomenon is designated ‘‘quantal release.’’ A subse-ion pores, and thus can clearly be distinguished from

VOCC. quent experiment using reconstituted vesicles containingpurified InsP3 receptors revealed that agonist-inducedAnother important property of InsP3-gated channels,

their biphasic dependence on Ca2/, was originally pro- quantal release is not the result of increased InsP3 degra-dation but is due to an inherent property of the InsP3posed after studies of permeabilized SMC from the guinea

pig taenia coli (454, 455). Iino (454, 455) carefully pre- receptor (292). The quantal nature of InsP3-induced Ca2/

release, which could be alternatively termed ‘‘incrementvented Ca2/ uptake into the SR or mitochondria and inhib-ited hydrolysis induced by InsP3 phosphatase, and also detection’’ (292, 739), has been accounted for by two dif-

ferent mechanisms. The first one involves the existencefunctionally eliminated the contribution of caffeine-sensi-tive stores by the use of ryanodine. As a result, Iino (454, of several subsets of stores having differential InsP3 sensi-



tivities (i.e., InsP3-binding affinity and/or channel-activat- presence of other factors, a contribution by InsP3 receptorheterogeneity (see above), loss of accessory proteinsing efficacy would differ); this is thought to underlie the

all-or-nothing behavior of IICR (880). The second explana- needed to sense luminal Ca2/ concentration (84), or aheterogeneity in the density of InsP3 receptor distributiontion emphasizes the importance of luminal control of

Ca2/-releasing activity; this has its origin in Irvine’s model (417), cannot be ruled out.B) RYR. The properties of a purified ryanodine-sensi-(491). However, two recent reports of experiments on

single SMC have suggested that different types of all-or- tive Ca2/ channel have been investigated using a planarlipid bilayer system. Thus the unitary conductance is 100–nothing Ca2/ release can occur at the whole cell level too

(guinea pig taenia coli, Ref. 460; rat ileum, Ref. 842). The 150 pS (e.g., 120 pS for the skeletal RyR1 and 100 pS forthe cardiac RyR2 when 50 mM Ca2/ was present on themechanism responsible for the second type of all-or-noth-

ing release is unlikely to involve a differential sensitivity to trans-side). These values are about four times larger thanthat reported for the InsP3 receptor channel (723). How-InsP3. Instead, this mechanism may involve an immediate

feedback potentiation triggered by the released Ca2/ re- ever, we should mention that Suarez-Isla et al. (1044) re-ported, in skeletal muscle, a small-conductance Ca2/maining in the vicinity of the InsP3-gated channel; this

may enable a rapid and regenerative growth of [Ca2/]i channel (5 pS) that was sensitive to ryanodine and alsoto caffeine. Detailed investigation of the skeletal RyR re-transients, which would thus be of almost identical shape

and amplitude (458, 460). vealed that the channel follows saturable kinetics for bothdivalent and monovalent cations (e.g., its Kd is Ç3 mMEvidence supporting the idea that the luminal Ca2/

content may control Ca2/-releasing activity was first ob- and its maximum conductance is 172 pS with millimolarCa2/), suggesting the presence of a single- or multi-iontained in permeabilized hepatocytes in the shape of the

finding that the releasing potency of InsP3 appears to re- pore structure (999). In the absence of Ca2/, the RyRchannel exhibits a broad selectivity for monovalent cat-cover gradually as spontaneously discharged stores refill

(749, 758). This hypothesis was further elaborated in per- ions and a larger conductivity than with Ca2/ (700–800pS with symmetrical 0.25 M K/). On the other hand, inmeabilized A7r5 SMC, by comparing the InsP3 sensitivity

of steady-state 45Ca2/ efflux under various store-loading the presence of Ca2/, the channel behaves as a pore thatconducts Ca2/ relatively selectively (e.g., PCa/PK Å 6, Ref.conditions (747, 748, 759, 832). In these studies, the ob-

served rate of 45Ca2/ efflux stimulated by a given concen- 999). The RyR channel is activated by micromolar Ca2/,acidic pH, millimolar ATP, and millimolar caffeine (how-tration of InsP3 became significantly smaller when the

Ca2/ content of the stores was reduced, whereas the oppo- ever, the transforming growth factor-b-induced RyR3-likereceptor is insensitive to caffeine, Ref. 349); it is inhibitedsite effect occurred on partially reloading the store. The

relevance of this hypothesis has recently been examined by millimolar Ca2/, millimolar Mg2/, and micromolar CaM,ryanodine, and ruthenium red (722, 724, 941, 1017). Com-using single-channel recordings of cerebellar InsP3 recep-

tor Ca2/ channels incorporated into a bilayer (84). In- pared with the skeletal muscle RyR channel, the cardiacRyR channel is activated by lower concentrations of Ca2/creased luminal Ca2/ concentration, however, resulted in

a monotonic decrease, rather than an increase, in the open but is less sensitive to Mg2/ inhibition and has a higheraffinity for ryanodine (725, 1017). Furthermore, cADP ri-probability of InsP3-gated channels, in disagreement with

the results of Missiaen et al. (747, 748). A similar piece of bose, a metabolite of NAD/ that has recently been sug-gested as an intrinsic agonist for CICR, potentiates theconflicting evidence (i.e., no dependence of InsP3-induced

release on luminal Ca2/ concentration) has also been pro- cardiac RyR (i.e., the RyR2 isoform) selectively (333, 334,653). An analysis of the cardiac RyR channel suggestedvided by a direct measurement of luminal Ca2/ concentra-

tion on a subhundred millisecond base, by use of the low- that cADP ribose may sensitize the channel to cytosolicCa2/ by acting at the adenine nucleotide binding site (991).affinity Ca2/ indicator Furapta (guinea pig portal vein

SMC, Ref. 417). One explanation that could reconcile The presence of the RyR channel in SMC was firstdemonstrated in canine and bovine aorta, in which thethese apparently conflicting results may lie in the idea

that the reduction in luminal Ca2/ concentration during ryanodine-sensitive store is thought to be rather less abun-dantly distributed (402, 812). The reconstituted channel,progressive Ca2/ release may lead to a diminished force

driving Ca2/ out of the stores (i.e., a reduced unitary am- which was identified as an [3H]ryanodine-bindable 30Sprotein, proved to be nonspecifically conductive to cat-plitude), thereby slowing down the rate of Ca2/ release.

This interpretation seems plausible because the product ions (but more selective for Ca2/, PCa/PK Å 3.8) and tohave a unitary conductance comparable to those of otherof the unitary amplitude and open probability of the InsP3-

gated channels, which should represent the rate of re- RyR channels with Ca2/ as the charge carrier (100–110pS with 100 mM trans Ca2/; however, with symmetricallease, would be maximal atÇ10 mM luminal Ca2/ concen-

tration but would fall drastically at lower luminal Ca2/ 0.25 M K/, this value became 367 pS, about one-half thatof the skeletal or cardiac RyR). It also proved to be acti-concentration levels, although the true luminal Ca2/ con-

centration might be different from this value (84). The vated by micromolar Ca2/ and by caffeine (2 mM) and



inhibited by millimolar Mg2/ and by ruthenium red (30 measurement of Ca2/ transients. It is possible to list theimportant features characteristic of CICR in VSMC as fol-mM). These properties suggest a close resemblance be-

tween aortic RyR and other purified RyR and are consis- lows (as pointed out in Ref. 456). 1) The rate of CICRincreases steeply with [Ca2/]i up to pCa 5.5, but the thresh-tent with many of the features observed for CICR in

skinned fiber experiments (see below). However, a num- old is not lower than a pCa of Ç6. 2) Increased Mg2/

concentration (0.5–5 mM) or reduced pH (7.3–6.7) inhibitber of notable differences have been found. Potentiationby ATP of the activity of the aortic RyR channel appeared the rate of CICR by shifting the relationship between CICR

and pCa to the right. 3) A nonhydrolyzable analog of ATP,to require prior channel activation by micromolar Ca2/.Furthermore, the effect of ryanodine on the channel’s ac- namely, AMP-PCP, potentiates CICR in a hyperbolic man-

ner with a half-maximal concentration of Ç0.3 mM. 4) Intivity was not bimodal, with complete channel closureoccurring at a concentration as high as 500 mM (a long- the presence of both AMP-PCP and 1.5 mM Mg2/, the pCa-

CICR curve becomes steeper, and the CICR significantlylived subconductive state at nano- to micromolar concen-trations and permanent closure at millimolar concentra- larger at pCa 5.5, than in their absence. 5) Millimolar

caffeine causes a dramatic shift to the left of the pCa-tions have been reported for skeletal and cardiac RyR,Refs. 638, 942). The latter observation may prove difficult CICR curve (i.e., to a lower [Ca2/]i), thereby enabling the

CICR to operate at a physiological [Ca2/]i .to reconcile with the results of experiments using skinnedfibers (456). The study by Becker et al. (57) was one of the first

to test the physiological importance of CICR in SMC. ThisThe second report of an SM RyR channel came froman example of a different type of SM tissue (i.e., phasic was achieved by evaluating the relationship between volt-

age-dependent Ca2/ entry and a subsequent elevation inmuscle), namely, the stomach. In this tissue, caffeine canelicit a large ryanodine-inhibitable Ca2/ release with an [Ca2/]i monitored with fluorescent Ca2/ indicators (using

toad stomach SMC, which are supposed to contain ryano-efficacy comparable to that of the InsP3-producing agentACh (1189). In this study, its biochemical profile sug- dine-sensitive stores). The time course of the [Ca2/]i

change induced by membrane depolarization in this cellgested that the stomach RyR is related to the cardiac RyR.Thus the former tissue showed the presence of relatively was found to be approximately linearly related to the

amount of Ca2/ entry through VOCC (calculated as thelarge-capacity (0.5 pmol [3H]ryanodine per mg protein; cf.0.1 pmol/mg in aorta, Ref. 402) and high-affinity binding time integral of the Ca2/ current) for the initial 100–200

ms of the depolarizing pulse. This suggests at most a smallsites for ryanodine (Kd Å 2 nM) and comigration andcross-reactivity with the cardiac RyR or its monoclonal amplification of Ca2/ entry by the CICR mechanism. Ganit-

kevitch and Isenberg (337) attempted a similar approach,antibody (but not with that against the skeletal RyR). Nev-ertheless, data from microsomal 45Ca2/ flux measure- but with a slightly elevated external Ca2/ concentration

(3.6 mM) and a physiological temperature of 367C inments and from lipid bilayer single-channel recordingshave suggested some essential differences, such as weak guinea pig urinary bladder SMC. Under these conditions,

depolarizations sufficient to activate VOCC induced bi-channel potentiation by millimolar ATP or caffeine and arequirement for high concentrations of Ca2/ for channel phasic changes in [Ca2/]i comprising an initial transient

peak, occurring within 300 ms, and a maintained elevationactivation (the maximal rate of 45Ca2/ flux being achievedat 1 mM). Interestingly, a recent gene-targeting study has underlying and outlasting the peak. The peak rise was

abolished in the continued presence of caffeine or by pre-demonstrated that after knock-out of the RyR1 gene inmurine skeletal muscle (after which only RyR3 would ex- treatment with ryanodine or thapsigargin and showed lit-

tle dependence on the external Ca2/ concentration andist), the pCa-Ca2/ release curve shifted to about a 10-foldhigher level of Ca2/ (1074). This finding, together with the little response to the VOCC agonist BAY K 8644. In con-

trast, the tonic rise was strongly dependent on externalfinding of an insensitivity to caffeine of the transforminggrowth factor-b-induced RyR in mink lung epithelial cells Ca2/ and was enhanced by BAY K 8644. The relative con-

tribution of the latter was estimated to be Ç30% of the(359), this being a receptor which shows a high degree ofhomology to RyR3 (94%), implies that in the stomach, total in the presence of ryanodine, and thus more than

50% of the initial [Ca2/]i rise induced by depolarizationand presumably in other types of SMC as well, the majorisoform may be the RyR3. Consistent with this view is a could be attributed to the secondary release of stored

Ca2/ via the CICR. It is still unclear from this study, how-very recent finding that 10 mM caffeine is unable to re-lease Ca2/ from the ryanodine-sensitive store in perme- ever, to what extent CICR would contribute to a rise in

[Ca2/]i induced by a single action potential, since the dura-abilized human myometrium, where RyR3 is thought tobe the dominant isoform (676). tion of the typical action potential in SMC would be

shorter than 100 ms (505). A somewhat different patternC) CICR AND IICR MECHANISMS. 1) CICR mechanism. InVSMC, the CICR phenomenon was first reported by Itoh of contribution by CICR has been reported in rat portal

vein SMC (366). In this cell, a large ryanodine-inhibitableand co-workers (499, 505), and subsequently by many in-vestigators, using skinned muscle preparations or the increase in [Ca2/]i developed persistently after the termi-



nation of a 1-s depolarization. The amplitude of this in- oscillations or wave propagation in nonexcitable cells(79). The functional importance of CICR in agonist-in-crease appeared to be proportionate to that of the voltage-

dependent Ca2/ current in that it showed a bell-shaped duced Ca2/ mobilization has recently been demonstratedin freshly isolated cells from both vascular and intestinalvoltage dependence, suggesting that a regenerative CICR

might occur after the termination of a voltage-dependent SM (615, 793, 872). Murthy and Maklhouf (794) found thatin the longitudinal muscle of the guinea pig ileum, a smallCa2/ influx. However, this interpretation was very recently

disputed by Kamishima and McCarron (536). Using a dou- InsP3-independent, methoxyverapamil-inhibitable Ca2/ in-flux, which is probably activated by the CCK-induced re-ble-step pulse protocol that first activates VOCC and then

jumps to a potential that would diminish the Ca2/ driving lease of arachidonic acid via G protein-coupled phospholi-pase A2, could trigger a large CICR, which might furtherforce (/120 mV), these authors (536) attributed the regen-

erative increase in [Ca2/]i after the termination of a depo- be potentiated by cADP ribose (614). Interestingly, theyfurther suggested that such a contribution of CICR tolarization to a Ca2/ influx (possibly through Ca2/-activated

cation channels), rather than to stored Ca2/ release via agonist-mediated Ca2/ mobilization might be specific forthe longitudinal muscle, the circular muscle apparentlyCICR. A similar assessment of CICR has also been carried

out in less excitable SMC (tonic muscle) such as those utilizing exclusively IICR to fulfill this role.The CICR mechanism in various excitable tissues hasfrom the guinea pig coronary artery and equine trachea

(299, 340), but no clear evidence was obtained for the been shown to be modified by various agents, such ascaffeine, procaine (456, 605), ryanodine, sphingomyelinphysiological operation of CICR. Interestingly, the study

in the coronary artery showed that the ryanodine-sensitive (717), 1,1*-dihepeptyl-4,4*-bipyridylium dibromide (541),hydralazine (377), and FK-506 (13). Ryanodine has a high[Ca2/]i rise, which normally occurs only during a pro-

longed depolarization as a slow creeping rise, could be affinity for the CICR-inducing channel and fixes the chan-nel in an open state (298, 722, 940, 942), but this actionpotentiated and accelerated by artificially increasing the

amplitude of the voltage-dependent Ca2/ current by an needs the presence of caffeine, which fully opens theCICR channel in the SR. Furthermore, CaM directlyelevated external Ca2/ concentration, by BAY K 8644, or

by trypsin dialysis. These results point to the importance blocked the Ca2/ release channel in swine skeletal muscleby direct binding to the RyR, thus causing an inactivationof the magnitude of voltage-dependent Ca2/ entry in the

activation of CICR. The size of the contribution made of SR Ca2/ release during excitation-contraction coupling.Chowdhury et al. (194) investigated the sustained contrac-by CICR to excitation-contraction coupling in SMC may

depend on more complex factors. Although, on scanning tion produced by caffeine after ryanodine treatment inthe circular muscle of the guinea pig gastric antrum andthe literature, the reader may gain the impression that

spontaneously active VSMC (phasic muscle) utilize CICR rabbit portal vein. They concluded that, in these tissues,caffeine produced a sustained contraction after applica-more than less excitable VSMC (tonic muscle), and are in

this respect more like cardiac SM, the information so far tion of ryanodine and generated Ca2/ influxes that wereinsensitive to Ca2/ channel blockers. They hypothesizedobtained is still scanty, and we are far from being able to

give a comprehensive explanation of the situation. that the SL membrane in these tissues is similar to theSR membrane, in that Ca2/ permeability can be increasedThe CICR mechanism has also been implicated in

some forms of agonist-mediated Ca2/ mobilization. Blatter almost irreversibly by a combination of caffeine and ryan-odine in the presence of the SL. Discussing the actions ofand Wier (93) observed that, in A7r5 cells, focal applica-

tion of AVP to one end of a cell resulted in an initial caffeine in relation to CICR, Steenbergen and Fay (1030)noted that Ca2/ release from its stores occurs in a gradedlocalized heparin-inhibitable increase in [Ca2/]i and a sub-

sequent propagation of Ca2/ waves toward the other end manner in response to increasing concentrations of InsP3

and caffeine. They concluded that the quantal nature ofat a constant velocity. Furthermore, Ca2/ waves showinga similar spatiotemporal pattern could be activated by the release of Ca2/ in response to caffeine in SMC is

largely due to the activity of the SERCA, which appearsfocally damaging the cell membrane with a sharp micropi-pette. The spreading velocity of the latter effect was not to return a portion of the released Ca2/ back to the SR,

even in the presence of ATP. Apparently, the SERCA isappreciably affected by the presence of the InsP3 antago-nist heparin, but it was significantly reduced after pro- fueled by ATP, which is either compartmentalized or

bound to the SR. Furthermore, it has also been reportedlonged treatment with caffeine. These observations ledthe authors to conclude that AVP-induced Ca2/ propaga- that activation of CICR was accelerated by caffeine, but

that caffeine inhibited IICR and that this inhibition wastion is most likely to result from CICR, i.e., a localizedInsP3-dependent Ca2/ release initiated by AVP near the prevented by ATP (754).

2) IICR mechanism. We turn now to the IICR mecha-plasma membrane triggers subsequent Ca2/ release fromadjacent caffeine-sensitive stores, and this in turn spreads nism in SMC, a mechanism first described by Suematsu

et al. (1045), and subsequently by other investigators (388,over the rest of cell in a regenerative fashion. A similarrole for CICR has been postulated in the genesis of Ca2/ 1011, 1199). The InsP3 receptor distributed in the brain



(also known as P400; Ref. 318) and peripheral tissues could be inhibited by Na/ and K/, but almost not at allby Ca2/ and Mg2/. It was also inhibited by sperminewas found to be inhibited by heparin, which thus became

known as an inhibitor of the IICR mechanism (1050, 1051). (a blocker of Ca2/-releasing channels which decreasedBmax without changing the Kd), but it was not affectedIn skinned SM tissues, heparin was found to inhibit the

release of Ca2/ from the SR that occurred via activation by caffeine, procaine, or ruthenium red. They thereforeconcluded that myotoxin-a binds to an important regula-of the InsP3 receptor, but to have no effect on caffeine-

induced Ca2/ release (410, 596, 1198). tory protein of Ca2/ release, which is not the RyR.O’Rourke et al. (860) reported that the Ca2/ release in-The IICR mechanism is also dependent on the cyto-

solic Ca2/ concentration (68). In fact, in the longitudinal duced by InsP3 was blocked by apamin, by TEA, and bya monoclonal antibody of a 63-kDa membrane protein,muscle layer of the small intestine, Zholos et al. (1233)

reported that IICR can be inhibited by a certain amount but not by charybdotoxin, 4-AP, or glyburide (also ablocker of KATP). Therefore, they thought that the produc-of Ca2/ at a time when the Ca2/ stores have refilled and

can be made to release Ca2/ by caffeine. Using permeabil- tion of optimal Ca2/ release by InsP3 from platelet mem-brane vesicles may require the function in tandem of aized A7r5 SMC, Missiaen et al. (751) demonstrated that

luminal Ca2/ could functionally substitute for cytosolic K/ channel. Other observations made recently are thatNO inhibited the IICR mechanism during bile canalicularCa2/ in triggering Ca2/ release in the presence of a con-

stant InsP3 concentration. Furthermore, the same group contraction (256) and that activation of the L-type Ca2/

channel may directly fill the InsP3-sensitive Ca2/ store (332).(888) observed that the quantal release of Ca2/ in perme-abilized A7r5 cells is not caused by intrinsic inactivations The kinetics of IICR in SMC were elegantly investi-

gated by Iino and Endo (458) using either flash photolysisof the InsP3 receptor. Missiaen and co-workers (755, 756,758) further observed that the InsP3 receptor can be intrin- of caged InsP3 or caged Ca2/. They concluded that Ca2/-

dependent immediate positive-feedback control is an im-sically inactivated, but that this inactivation is unable toprevent the slow release of Ca2/, which is especially pro- portant determinant of the time course of Ca2/ release.

The positive-feedback mechanism is also important fornounced when the Ca2/ pumps are active.In VSMC and cerebellar neurons, Ca2/ release by the loading dependence of IICR. Their observations also

support the idea that the operation of positive-feedbackInsP3 could be activated in the presence of 200–300 nMCa2/, but higher concentrations of Ca2/ inhibited the Ca2/ control augments the steep relationship between InsP3

concentration and Ca2/ release. These inherent propertiesrelease (85, 457, 1046). In cerebellar neurons, but not inVSMC, the presence of calmedin was thought to be re- of IICR may be expected to give rise to a temporally

abrupt and/or spatially confined Ca2/ release within thequired to produce the Ca2/ dependency (85, 232). In addi-tion to heparin, thimerosal, an antibacterial antifungal cell. Moreover, the existence has been postulated of a

negative-feedback control giving rise to Ca2/ oscillationsthiol reagent, also has the ability to modulate the InsP3

receptor. The latter agent releases Ca2/ from the SR and through activation of a Ca2/-dependent inhibition of theInsP3 receptor (80, 85, 93, 295, 300, 458, 527, 570, 659, 747,induces Ca2/ oscillations (79, 80) without the need for an

increase in the cytosolic InsP3 in HeLa cells, A7r5 SMC, 880, 943, 1228). Thus both RyR and InsP3 receptors, asreported by Chen et al. (188) and Chen and Van Breemenand pancreatic acinar cells. This agent sensitized the InsP3

receptor to the Ca2/ releasing effect of InsP3, and more- (189), may contribute to the IICR mechanism. The impli-cation of this conclusion is that the IICR- and CICR-in-over, a monoclonal antibody to the COOH terminus of the

InsP3 receptor inhibited the enhancing effects of thimero- duced processes do not exactly correspond to the effectsof activation of the InsP3R and RyR, respectively, but thatsol on InsP3-induced Ca2/ release in the intact hamster

egg (112, 113, 747, 758, 763). The thimerosal-induced Ca2/ they do act in a mutually related way to regulate releaseof the Ca2/ from the SR.oscillation was inhibited by heparin in pancreatic acinar

cells (1089), although, confusingly, the RyR has been said Hirose and Iino (417) carried out a further elegantinvestigation of the kinetics of Ca2/ mobilization in rela-to be the target receptor for thimerosal (185, 1065), and

this agent has been reported to inhibit the Ca2/-ATPase tion to the role of InsP3 in the guinea pig portal vein, usinga low-affinity Ca2/-sensitive fluorescent dye, Furaptra, inin the SR (112, 113). However, using permeabilized A7r5

cells, Missiaen and co-workers (752, 753) found that thi- permeabilized cells. They hoped to clarify the mechanismunderlying incremental defection (7392), since twomerosal, in concentrations from 0.1 to 1 mM, can sensitize

InsP3-induced Ca2/ release without inhibiting the Ca2/ hypotheses have been put forward to explain this phe-nomenon. The first supposes that Ca2/ stores consist ofpump and without increasing the passive Ca2/ permeabil-

ity of the SR. Ohkura et al. (837) reported that myotoxin- multiple compartments (quanta) with different sensitivi-ties to InsP3 (113, 292, 856, 879), whereas, in the second,a, isolated from the prairie rattlesnake (Crotalus viridis

viridis), is a powerful releaser of Ca2/ (via the CICR; Ref. the rate of Ca2/ release is supposed to be modulated bythe Ca2/ concentration in the lumen of the Ca2/ stores319) from the heavy fraction of the SR [Kd Å 0.4 mM,

maximum binding (Bmax) Å 6 nM/mg protein]. This effect themselves (491, 672, 747, 887). However, Hirose and Iino



(417) found that the results they obtained could not be ingly, the bulk of positive immunolabeling was localizedexclusively in surface membrane invaginations, (i.e., ca-explained by either of these hypotheses. Consequently,

they postulated that the Ca2/-sensitive channels on the veolae) where the plasma membrane forms close contactwith the sarcoplasmic reticulum and with an intercalatingCa2/ stores had complex kinetics as well as different sen-

sitivities to InsP3 and may have different functions in Ca2/ ridgelike structure (1012), which might represent the largecytosolic portion of the 240-kDa InsP3 receptor-like pro-mobilization. Faced with this new hypothesis, Missiaen

and co-workers (755, 756) were critical but later Hirose tein (306). These results tempt us to regard InsP3 as havingimportant roles in activating Ca2/ release and in activatingand Iino defended their comments (Nature 376: 300–301

and 301, 1995, respectively). plasmalemmal Ca2/ entry channels in receptor-mediatedCa2/ mobilization, both in nonmuscle cells and in SMC.Some findings consistent with this view, neomycin-inhibit-3. Refilling of Ca2/ stores and the associatedable AVP-induced 45Ca2/ influx and a heparin-sensitiveCa2/ entryMn2/-impermeable Ca2/ entry which is activated by NE(but not by ryanodine or caffeine), have recently beenIn general, stimulation of Ca2/-mobilizing receptors

leads to IICR from the internal stores, which are then made in two distinct types of rat SMC (respectively, inportal vein SMC, Ref. 607; aorta, Ref. 873).replenished by reuptake of Ca2/ from the cytosol, as well

as by promoted Ca2/ entry across the plasma membrane. An alternative mechanism that might be responsiblefor Ca2/ entry upon receptor stimulation is that proposedTo account for the latter mechanism, as described pre-

viously, several distinct pathways that are closely linked in the capacitative Ca2/ entry hypothesis, which was origi-nally formulated by Putney (907–909), Jacob (510), andto the internal stores have been envisaged. These involve

1) VOCC, 2) a direct connection between plasma mem- Bird and Putney (88) on the basis of data obtained fromnonexcitable cells. Activation of this pathway is indepen-brane and SR, 3) InsP3-gated Ca2/-permeable channels, 4)

Ca2/ release-activatable Ca2/ channels (ICRAC), and 5) dent of InsP3 production (and thus distinct from the InsP3-activated Ca2/ channels), since various procedures thatATP-activated or G protein-coupled ROCC.

Although its existence has been postulated, so far deplete internal stores (e.g., use of thapsigargin and CPA)are able to stimulate Ca2/ entry across the plasma mem-no morphological or molecular evidence supportive of a

direct linkage between the SL and SR has been published brane without affecting the intracellular InsP3 level (1073).The currents that underlie capacitative Ca2/ entry (ICRAC)for VSM. However, an intriguing hypothesis was put for-

ward a few years ago, in an attempt to unify the many have been demonstrated using the patch-clamp technique(in mast cells, basophilic leukemia cells, hepatocytes,apparently irreconcilable observations made about ago-

nist-stimulated Ca2/ entry; this hypothesis came from a thyrocytes, and fibroblasts). In addition to its activationby Ca2/-releasing agents, ICRAC is activated by intracellularstudy of nonmuscle cells (491). By analogy with the close

geometric and functional coupling between RyR and DHP perfusion of the Ca2/ buffers, EGTA or BAPTA, due proba-bly to store depletion, but inhibited by elevated [Ca2/]ireceptors in the skeletal muscle triad, Irvine (491) formu-

lated a minimalist model in which there is physical inter- (431). The channels underlying ICRAC seem to have a lowunitary conductance (20 fS in 110 mM Ca2/) and an ex-action between the InsP3 receptor in the ER membrane

and the putative InsP4 receptor, which serves as an SL tremely high Ca2/ selectivity that is comparable to that ofVOCC, and they are inhibited by Cd2/ and La3/, suggestingCa2/-permeable channel. In this model, Ca2/ release

through the InsP3 receptor and Ca2/ entry through the some resemblance to VOCC. However, their lack of inhibi-tion by standard VOCC blockers and their insensitivity toInsP4 receptor are both blocked when the two receptors

are in close association (i.e., in unstimulated conditions). voltage change suggest that ICRAC is different from VOCC.The poor voltage dependence of ICRAC indicates that mem-Upon PLC activation, however, an increased cytosolic

InsP3 concentration with a decreased luminal Ca2/ con- brane hyperpolarization (e.g., by concomitant activationof K/ conductance during receptor activation) may resulttent or decreased InsP4, which is supposed to synergize

with InsP3, led to the dissociation of the two inositol phos- in an increased rate of Ca2/ entry through this channeland a more elevated [Ca2/]i; this has been confirmed byphate receptors, thereby activating Ca2/ mobilization and

influx mechanisms due to disinhibition. In fact, InsP3-acti- experiments in which the current and [Ca2/]i were re-corded simultaneously (892).vatable and InsP4-modulatable Ca2/-permeable channels

(or pathways) have been recorded from the plasma mem- Activation of capacitative Ca2/ entry may involve sec-ond messengers liberated from the emptied stores, ratherbrane of several different cell types. Furthermore, a recent

immunochemical labeling study has revealed that a 240- than the direct coupling of store and plasmalemmal Ca2/

channels that was originally envisaged. Thus a recent bio-kDa protein, which is reactive to the type I InsP3 receptormonoclonal antibody, is present in the cell surface mem- chemical investigation has demonstrated that whole cell

extracts made after store depletion, or partially purifiedbrane of several different tissues, including aortic endo-thelium, keratinocytes, and intestinal SM (306). Interest- small-molecular-weight (õ500) phosphate-containing



anions extracted from them, can activate Ca2/ influx that these mechanisms are voltage independent. 3) Specificblockers for VOCC (such as DHP or methoxyverapamil)across the plasma membrane quite effectively (the active

constituent has been named ‘‘Ca2/ influx factor,’’ CIF; are ineffective, but potent blockers of capacitative Ca2/

entry (e.g., Ni2/, Co2/, La3/) are effective in blocking theRefs. 878, 916). This factor 1) seems to be translocatedfrom the SR to the SL fraction upon store depletion (916); rises in tension or [Ca2/]i (360, 844, 1188). Moreover, re-

placement of Na/ with Li/, which would be expected to2) was abolished by alkaline phosphatase, but potentiatedby okadaic acid (a protein phosphatase inhibitor); and 3) inhibit Na//Ca2/ exchange, is without effect (844). 4) In

some SMC, Mn2/ entry is also observed upon store deple-was not detected in NG115–401 cells, which lack capaci-tative Ca2/ entry (1086). Randriamampita and Tsien (919), tion. 5) Tyrosine phosphorylation may play an essential

role in activating this Ca2/ entry (154, 360).using astrocytoma cells, reported that degradation of CIFwas inhibited by okadaic acid and cyclosporin A (an inhib-itor of calcineurin), and also that both agents potentiated 4. Investigation of Ca2/ mobilization in the cytosolthe Ca2/ elevation caused by low doses of CIF, thapsigar- from measurements of electrical activitiesgin, or carbachol. In lymphocytes, okadic acid potentiatedthe Ca2/ elevations due to low doses of phytohemaggluti- Two phenomena appear to be closely associated with

the cyclical release of stored Ca2/ in VSM, namely, STOCnin and increased the amount of extractable CIF. Further-more, CIF degradation could be observed in cell-free ho- (71; also named Ioo, Ref. 847) and spontaneous transient

inward currents (STIC; Ref. 1148). The first report ofmogenates of lymphocytes and was prevented by phos-phatase inhibitors, an effect that could at least partly STOC came from a patch-clamp study in the rabbit jeju-

num and ear artery, in which depolarized membrane po-explain their potentiation of Ca2/ influx. Degradation ofCIF is also prevented by lowering [Ca2/]i; this could act tentials (typically more positive than 040 mV) were asso-

ciated with periodic clustered openings of Ca2/-depen-as a feedback mechanism to enhance Ca2/ influx whencells are depleted of Ca2/. In addition to CIF, other partici- dent K/ channels (maxi-KCa; Ref. 71). The STOC activity

was abolished in Ca2/-free solution; ceased after applica-pants in capacitative Ca2/ entry may be 1) a small molecu-lar cytosolic G protein (basophilic leukemia cells and lac- tion of caffeine, ACh, ryanodine, or heparin; and was vari-

ably affected by VOCC blockers (ear artery, partial inhibi-rimal acinar cells), 2) a cGMP-generating system (rat por-tal vein SMC) or tyrosine phosphorylation (human tion; jejunum, almost ineffective), suggesting that Ca2/

entry via both voltage-dependent and -independent path-fibroblasts), and 3) the cytochrome P-450-related metabo-lite 5,6-epoxyeicosatrienoic acid (see below in relation to ways may be required to fill and discharge the internal

stores in a repetitive manner. Numerous reports followedendothelium-derived hypolarizing factor, Ref. 364).In SMC, the capacitative Ca2/-entry hypothesis has this work and confirmed that the properties of STOC are

basically similar across a variety of VSMC. It is now sup-been supported by evidence from work on vascular SMC(154, 360, 367, 418, 825, 1181), urinary bladder SMC (783), posed that the mechanism responsible for generating

STOC involves both IICR and CICR, but to a varying extentintestinal SMC (844, 870), and airway SMC (53, 240). Forexample, using CPA, Ohta et al. (844) found that in rat ileal (581, 655, 950, 1166).

In terms of functional importance, STOC were at firstSMC, the emptying of intracellular Ca2/ stores may activatea Ca2/ influx not associated with VOCC. In airway SMC, almost disregarded; indeed, they were treated as an artificial

phenomenon that can be observed only under special ex-Amrani et al. (26), using thapsigargin, concluded that thap-sigargin-sensitive Ca2/ stores contribute significantly to the perimental conditions. However, recent reports have re-

vealed an interesting new aspect to their story. In theiractivation of human tracheal SMC, which suggests a rolefor these stores in the subsequent induction of Ca2/ influx. original study, Benham and Bolton (71) postulated that the

site of Ca2/ release would be localized to a small local areaThese SR appear to be controlled by Ca2/-ATPase(SERCA2b isoform; determined by the use of antibodies; under the membrane (Ç3% of total membrane area), and

as such it would be insufficient to induce any detectablesee below) which could also participate in the regulationof Ca2/ influx through the SL. The following summarizes contractions. Indeed, the observed amplitude of individual

STOC can account for only a small number of KCa (nothe major conclusions that can be drawn from these stud-ies, taken collectively. 1) Thapsigargin or CPA (or caffeine) more than 100) being simultaneously open. Nelson and co-

workers (810, 811) substantiated this idea in the rat cerebralinduces, in proportion to their concentration and the lengthof exposure, maintained rises in muscle tension or [Ca2/]i artery by imaging spontaneous local [Ca2/]i increases (an

elementary ryanodine-sensitive Ca2/ release, ‘‘Ca2/ sparks,’’that are dependent on the external Ca2/ concentration (26,154, 360, 844, 1170). 2) These rises decline with depolariza- 808) under confocal microfluorometry. In this cell, the ma-

jority of Ca2/ sparks are restricted to the subsarcolemmaltion of the membrane (caused by excess K/ or electricaldepolarization) and conversely are enhanced by hyperpo- region (within 1 mM of the plasma membrane), and a close

relationship is found in terms of temporal and pharmacolog-larization (360). This suggests that the Ca2/ entry closelyfollows the electrochemical gradient for Ca2/, and thus ical profiles between STOC and Ca2/ sparks. These observa-



tions led them to propose that the frequency or magnitude C. Factors That Decrease [Ca2/]i in VSMC

of Ca2/ sparks, which probably result from the dischargingof ryanodine-sensitive stores near the SL membrane and An increase in [Ca2/]i to 600–800 nM caused by ago-the subsequent activation of KCa, may have great functional nist stimulation will normally be reduced to within 100–importance in controlling the overall cell K/ conductance, 150 nM of the resting level by two processes in VSMC.which in turn determines the resting membrane potential These are 1) reuptake of Ca2/ into the SR through theand thereby regulates the transmembrane Ca2/ influx activation of Ca2/-ATPase and 2) extrusion of Ca2/ fromthrough VOCC. Consistent with this idea, they observed, the cytosol, through the Ca2/-ATPase distributed in theusing pressurized cerebral arteries, that the specific blocker SL and also by Na//Ca2/ exchange diffusion throughof KCa (iberiotoxin) and store-depleting agents (such as the SL.thapsigargin and ryanodine) produce membrane depolariza-tion and concomitant vasoconstriction. Such a novel role

1. Ca2/-ATPase in the SL and SR (Ca2/ pump)for localized Ca2/ release from the stores, limited to mem-brane-localized activities rather than as a means of changing In VSMC, two different Ca2/-ATPases are distributed

in the SL and SR [plasma membrane Ca2/-ATPase (PMCA)the overall [Ca2/]i , would certainly be an important aspectof store function and will deserve further extensive explora- and sarco(endo)plasmic reticulum Ca2/-ATPase

(SERCA), respectively]. Furthermore, the Ca2/-ATPase intion (1059). Presences of STOC in the swine tracheal SMChave also been demonstrated (962, 963). the SL is encoded by at least four genes (PMCA1, -2, -3,

and -4; molecular masses estimated to be 127 to 137 kDa),In addition to its activation of STOC, increased cyto-solic Ca2/ can also evoke STIC. These currents were first and each PMCA can express four mRNA by alternative

splicing (at sites A, B, C, and D). Similarly, SERCA isreported by Large’s group (423, 424, 1148, 1151) in therabbit portal vein. These electrical currents were Ca2/ encoded by at least four genes (SERCA1, -2, -3, and -4;

molecular masses estimated to be 127–139 kDa). Bothdependent and evoked by depolarization or agonist stimu-lation, as also observed for STOC, and they are therefore PMCA and SERCA possess 10 membrane-spanning re-

gions (M1-M10), and both Ca2/-ATPases have long hydro-postulated to be due to activations of both the ryanodine-and InsP3-sensitive Ca2/ channels in the SR. Spontaneous philic regions between M2 and M3 and between M4 and

M5, although they differ in that the COOH-terminal regiontransient inward currents were found to be blocked orreduced by Cl0 channel antagonists such as anthracene- is longer in PMCA than in SERCA (701, 1040). SERCA1 is

associated with two mRNA (a and b); SERCA1a is exclu-9-carboxylic acid (AC-9) or SITS (1148). The time constantof decay of STIC is much longer than that of STOC, and sively expressed in the fast skeletal muscle of adult ani-

mals, whereas SERCA1b represents the neonatal isoform.Hogg et al. (424) postulated that the decay of STIC repre-sents voltage-dependent closure of the Cl0 channel rather Other SERCA genes expressed four mRNAs by alternative

splicing (classes 1–4 or a-d). SERCA2 class 2 and 3than a fall in [Ca2/]i , and thus the time constant of decayapproximates to the channel mean open time. mRNAs are expressed as cardiac, slow skeletal, SM, and

nonmuscle types, and SERCA2b class 4 mRNA in SM en-Distributions of Ca2/-dependent Cl0 channels havealso been reported in other VSMC (rat anococcygeus mus- codes the SM and nonmuscle isoform SERCA2b. Differ-

ences in function between the Ca2/-ATPase in fast skeletalcle, Ref. 152; rabbit ear artery, Ref. 23). These currentswere also blocked by Cl0 channel blockers such as AC-9, muscle (SERCA1a) and that in cardiac muscle are small,

although the latter was inhibited by phospholamban,SITS, and DIDS. More recently, in the human mesentericartery, Klockner (587) found that caffeine depolarized the whereas the former was not.

In the case of PMCA genes, the primary transcript maycell membrane and elicited an action potential. Underpatch-clamp conditions, caffeine produced an inward cur- be alternatively spliced at four sites (A, B, C, and D; Ref.

1039) so that multiple isoforms may be derived from eachrent with a mean amplitude of 0.22 pA and a single-chan-nel conductance of 2.8 pS. This current disappeared when of the four genes. Two of these potential splice sites, A

and C, have been more extensively investigated than theCl0 concentration was reduced. From this, Klockner (587)concluded that agonist-induced depolarization of the hu- others. Splicing at site C affects the CaM-binding domain

and has been observed for PMCA1 to -4. Splicing at siteman mesenteric artery may be mediated by activation ofthe Ca2/-dependent Cl0 channel through an increase in A affects the putative phospholipid-binding domain and

apparently occurs only in PMCA2. The phosphorylationthe cytosolic Ca2/. Akbarali and Giles (19) also observedthe Ca2/-dependent Cl0 channel in rabbit esophageal site for PKA would be spliced out at site D and would thus

be present in isoform PMCA1e, but lacking in PMCA1dSMC. They reported that the reversal potential of the tailcurrent was dependent on the Cl0 equilibrium potential (913). Kahn et al. (531) found in the rabbit stomach and

aorta that 83–86% of the PMCA message was accounted(approximately 020 mV) and that this current was inhib-ited by common Cl0 channel blockers, such as niflumic for by PMCA1b, the remainder coming from PMCA1a.

A number of differences in the features of SL and SRacid, AC-9, DIDS, and SITS.



Ca2/-ATPase (PMCA and SERCA) have been elucidated. was much lower than in either cardiac muscle (913) orbovine aorta (1162). Cornwell et al. (215), on the basis of1) Vanadate had a higher affinity for the SL Ca2/-ATPase

than for the SR Ca2/-ATPase (inhibition constant Å 1–2 immunocytochemical studies, suggested that PKG andphospholamban were localized in the same cellular re-mM in the SL but 50 mM in the SR). 2) In the SR, the Ca2/

pump was inhibited by vanadate with the following order gion and, in phospholamban isolated from the aorta,higher levels of phospholamban phosphorylation wereof potencies: SERCA3 ú SERCA2a ú SERCA2b (677).

However, this agent is not selective and also acts on other observed with cGMP than with cAMP. It has been re-ported that, in the guinea pig mesenteric artery, activa-ATPases (235, 721). 3) Differential effects on SERCA and

PMCA have been reported for a number of agents, namely, tion of PKA (via cAMP) may increase Ca2/ in the SR,whereas activation of PKG (via cGMP) may lower [Ca2/]ithapsigargin (an inhibitor of SERCA2a, SERCA2b, and the

SERCA3 family), 2,5-di(tert-butyl)-1,4-benzohydroquinone by activation of Ca2/ extrusion mechanisms (503). Ko-bayashi et al. (594) and Magliola and Jones (686) dem-(tBuBHQ, Refs. 233, 767; although other investigators

have reported that this agent also inhibits the InsP3-Ca2/ onstrated in cultured and rat aortic SMC, respectively,that nitroglycerin and sodium nitroprusside (thesereceptor, Refs. 233, 857), and CPA. All three selectively

blocked SERCA and had no effect on PMCA; thus thapsi- agents are inducers of cGMP through liberation of NO)both accelerate Ca2/ extrusion from the cells and thatgargin completely inhibited SERCA1, -2, and -3, tBuBHQ

inhibited Ç90% of SERCA1a and -2b, and CPA inhibited this acts as the main source of muscle relaxation. Atrio-peptine (an inducer of cGMP synthesis) also reduced90% of the activities of SERCA1a, -2a, and -2b (179, 241,

677, 678). 4) The ratio of SERCA to PMCA may differ [Ca2/]i , but it is thought that this agent increases theaffinity of the Ca2/ extrusion mechanism by an actionwidely between different types of SMC. Eggermont et al.

(264) compared the level of 100-kDa SR and 130-kDa SL on the pump. In cultured SMC, such results were con-firmed by Furukawa and Nakamura (321), Vrolix et al.phosphoprotein intermediates in isolated membrane frac-

tions from different SMC. They found that the amount of (1137), Furukawa and co-workers (321, 323), and Imaiet al. (463). However, it is now thought that activationthe 130-kDa form was much higher in the pig stomach

than in the bovine aorta and main pulmonary artery. of the Ca2/ pump in the SL by cGMP is not a directaction (463, 600, 913, 1138, 1163). It has been reportedThe SERCA found in VSM have different properties

from those found in skeletal and cardiac muscles. The that PKC also activates the Ca2/ pump in the SL (309,463). On the other hand, McGrogan et al. (718), whoSERCA-induced pump in the SR of cardiac muscle is regu-

lated by protein kinases via phosphorylation of the associ- investigated the mechanism underlying cyclic nucleo-tide-induced relaxation of canine tracheal SMC, con-ated protein phospholamban (1068). Wuytack et al. (1181)

reported that the highest levels of expression of phospho- cluded that Ca2/ uptake into the SR plays a major rolein the relaxation induced by cGMP, but not in the cAMP-lamban are found in cardiac and slow skeletal muscles

(SERCA2a), and the level is lower in fast skeletal muscles induced relaxation. They also concluded that L-typeCa2/ channels must be available for the relaxant action(SERCA1). Phospholamban in the SR is phosphorylated

by PKA and Ca2/-CaM protein kinase (912, 913), and this of Ca2/ pumping into the SR and that Isop-induced re-laxation may not be primarily dependent on elevationsubstance has been found in many SMC (bovine main

pulmonary artery, Refs. 264, 265; bovine aorta, Refs. 1126, of cAMP. It was also reported by de Alfonzo et al. (237)that Ca2//CaM-dependent kinase and a 17-kDa polypep-1162; rat cultured aortic cells, Ref. 969; canine ileum and

iliac artery, Ref. 290; pig stomach, Ref. 1126). Human and tide are involved in Ca2/ transport in SR vesicles pre-pared from tracheal SMC.rat platelets express two types of SERCA: a 100-kDa SER-

CA2b isoform and a 97-kDa SERCA isoform. Moreover, Mitochondria also contribute to Ca2/ removal inVSMC. Recently, Drummond and Fay (253) observed thethe rat 97-kDa isoform is identical to the SERCA3 protein,

and this isoform is upregulated in spontaneously hyper- effects of the inhibition of mitochondial Ca2/ uptake andof depolarization of the mitochondiral membrane poten-tensive rats. In addition, this SERCA3 mRNA is also ex-

pressed in endothelial and epithelial cells (33). Further- tial induced by tetramethylrhodamine ethyl ester. Theyconcluded that, in VSMC, mitochondria play a significantmore, during application of PDGF, a SERCA2-type Ca2/-

ATPase was activated (686). role in removing Ca2/ from the cytosol after stimulation.The uptake of Ca2/ into the mitochondria would, ac-It is now well attested that activations of PKA and

PKG induced by cAMP and cGMP, respectively, reduce cording to their hypothesis, stimulate mitochondrial ATPproduction, thereby providing a means for matching in-the [Ca2/]i . Recently, Vaandrager and de Jonge (1113)

reviewed the evidence that the principal targets of cGMP creased energy demand, i.e., the cell’s rise in [Ca2/]i ,would be followed by increased cellular ATP production.are 1) phosphodiesterases, resulting in interference with

the cAMP pathway; 2) cGMP-gated cation channels; and However, despite all this work, it is not yet clear how thedegree of Ca2/ removal from the cytosol correlates with3) PKG. Furthermore, in the bovine pulmonary artery,

the degree of stimulation of Ca2/ uptake by PKA or PKG relaxation in VSMC.



2. Na//Ca2/ exchange diffusion through the SL under physiological conditions (778). Furukawa et al.(322) found that in VSMC, cGMP stimulates Na//Ca2/ ex-

In general, in VSMC, the Na//Ca2/ exchanger has a change. Ganitkevich and Isenberg (338), using micro-stoichiometery of 3:1. In this respect, it thus differs from fluorospectrometry in the guinea pig coronary artery, con-that described in the rods of the retina (4Na/:1Ca2/ / cluded that the ryanodine-sensitive intracellular Ca2/

1K/). With regard to Na//Ca2/ exchange kinetics, two pos- stores can modulate the contribution made to [Ca2/]i bysible systems have been postulated; these are the consecu- Na//Ca2/ exchange either by amplifying it via Ca2/ releasetive (Ping-Pong) and simultaneous (concurrent) systems. or by reducing it by Ca2/ sequestration. However, the Na//The former involves a one-way transport of ions at any Ca2/ exchanger would seem to be of minor importancegiven moment, followed by transport in the opposite di- for the [Ca2/]i transient as long as intracellular Na/ is atrection (supported by Refs. 408, 566, 662, 814). In contrast, its physiological level of 10 mM.the latter supposes that both Na/ and Ca2/ bind to theexchange molecule and are translocated at the same time

D. Ca2/ Sensitization of Smooth Muscle(supported by Refs. 94, 420, 661). At the moment, theContractionresolution of this problem is elusive.

In VSMC, the nature of the Na//Ca2/ exchanger hasbeen investigated by many workers. It may be true that Although contraction is initiated by Ca2/, the amountthis exchange diffusion occurs in VSMC, and in vascular of Ca2/ and the amplitude of maintained contraction aretissues, and there is evidence that this exchange is more not always in the same proportion to each other. This isprominent in large elastic arteries than in small resistance because of modulation by many factors that regulate ei-vessels. However, most of the experiments were carried ther the myosin-linked [phosphorylation of 20-kDa myosinout under extreme conditions, such as in Na/-free media light chain (MLC20)] or actin-linked regulatory mecha-or in the presence of ouabain (a Na/-K/ pump inhibitor), nisms. Such modulation occurs in intact and skinned SMand with a variety of experimental procedures. In fact, tissues and is termed ‘‘Ca2/ sensitization’’ or ‘‘Ca2/ desen-the ionic current generated during Na//Ca2/ exchange dif- sitization.’’ For instance, in SMC from the rabbit mesen-fusion, an electrogenic process, has been recorded in SMC teric artery, phenylephrine, NE, histamine, and ET-1 all(995). Unfortunately, in SMC, it is not yet certain whether produce much higher levels of MLC20 phosphorylationorthodromic exchange (Na/ influx and Ca2/ efflux) or and contraction than those induced by high K/ at givenantidromic exchange (Na/ efflux and Ca2/ influx) plays concentrations of [Ca2/]i (501, 506, 1210). In fact, the ten-an essential role under physiological conditions. As yet, sion-to-[Ca2/]i ratio is higher during activation by agoniststhe minimum concentrations of extracellular Ca2/ or in- than on application of high K/ (to produce membranetracellular Na/ needed just beneath the SL to trigger or- depolarization) in various types of SM, indicating thatthodromic exchange diffusion have not been determined. myofilament Ca2/ sensitivity in SMC is higher in the pres-

Opinions as to the role played by this exchange diffu- ence of agonist than with high K/. This enhancement ofsion under physiological conditions remain controversial. contraction at a given concentration of [Ca2/]i is calledOn one side, Blaustein et al. (95) concluded that the Na// Ca2/ sensitization. Conversely, the tension can decreaseCa2/ exchanger is relatively prevalent, is voltage sensitive, even though the [Ca2/]i is maintained, indicating a de-and promotes Ca2/ entry into mesenteric SMC when the crease in the myofilament’s Ca2/ sensitivity. For instance,Na/ gradient across the the SL is reduced or the mem- when the high concentration of [Ca2/]i evoked by an appli-brane is depolarized. In addition, they felt that it not only cation of high K/ or caffeine produces a smaller contrac-plays an important role in the extrusion of Ca2/ after an tion than that expected from the contraction or MLC20elevation of [Ca2/]i , but it most likely contributes to the phosphorylation observed on application of agonist, thisprolonged elevation of [Ca2/]i during the maintenance of is called Ca2/ desensitization (3, 4, 501, 544, 817, 922, 923,a tonic tension. The predominant effect of a small reduc- 1069).tion in the Na/ gradient in quiescent SMC would be an At present, at least three possible mechanisms canincrease in the amount of Ca2/ sequestered into the SR. be considered for Ca2/ sensitization: 1) an inhibition ofThey concluded that Na//Ca2/ exchange diffusion in myosin light-chain phosphatase, 2) an inhibition of thinVSMC plays a key role in controlling the amount of Ca2/ filament-linked regulatory mechanisms (such as those in-stored in the SR and this, in turn, regulates muscle con- volving calponin and caldesmon), and 3) a direct actiontractivity. On the other side, Furukawa et al. (323) re- on contractile proteins involving the slowing of the rateported that, under physiological conditions, this Na//Ca2/ of cross-bridge detachment. It is believed that an increaseexchanger plays only a minor role in regulating [Ca2/]i , in [Ca2/]i in SMC first produces phosphorylation of MLC20compared with that played by Ca2/-pump mechanisms in (at serine-19) through an activation of a Ca2//CaM-depen-the SL and SR. However, when the Na/-K/ pump is inhib- dent enzyme, MLCK, and then activates actin-activated

myosin Mg-ATPase activity, thus initiating a contractionited, this exchanger may play a more dominant role than



(387, 504, 537). On the other hand, when [Ca2/]i is loweredin SMC, muscle relaxation occurs through dephosphoryla-tion of MLC20 by protein phosphatase (MLCP). Therefore,it is considered that MLC20 phosphorylation and dephos-phorylation primarily modulate the Ca2/ sensitivity of SMcontraction. Because the net extent of MLC20 phosphory-lation is determined by the relative activities of MLCKand MLCP, an inhibition of MLCP could produce Ca2/

sensitization (hypothesis 1). According to this hypothesis,an inhibition of MLCP would be expected to increase thelevel of MLC20 phosphorylation at any given [Ca2/]i , thuscausing an enhancement of the tension.

The basis for the second and third hypotheses toexplain Ca2/ sensitization is that the size of the contrac-tion is not always correlated with the level of phosphory-lation of MLC20. In addition to the activities of MLCKand/or MLCP (which confer a myosin-linked regulatorymechanism), a possible role for a thin filament-linked reg-ulatory mechanism has recently been suggested for theregulation of SM contraction, especially with respect toits tonic phase. Two actin-binding proteins, caldesmonand calponin, are known; both inhibit actin-activated myo-sin Mg2/-ATPase in vitro. A calponin peptide (T2) greatlyattenuated the inhibitory action of calponin on actomyo-sin Mg2/-ATPase activity both in vitro and in skinned vas-cular SM. This peptide enhanced the Ca2/-induced con-traction without an increase in the level of MLC20 phos-phorylation (Figs. 11 and 12). The calponin peptide itselfdid not produce contraction in Ca2/-free solution (underlow MLC2 -phosphorylation conditions), but it shifted therelationship between [Ca2/]i and tension to the left (Ca2/

sensitization). This peptide also shifted the relationshipbetween MLC20 phosphorylation and tension to the left

FIG. 11. Effects of T2 peptide and calmodulin on relationship be-(507). These results suggest that calponin may not play a tween Ca2/ and tension (A) and on that between Ca2/ and 20-kDa myosin

light chain (MLC20) phosphorylation (B) in b-escin-skinned smooth mus-role as a switch for the development of force, but rathercle of rabbit mesenteric artery. Slope of concentration-response relation-act as a modulator of myosin phosphorylation-dependentship (of Ca2/ against tension and against MLC20 phosphorylation) is

force through a thin filament-linked regulatory mecha- shown by Hill coefficient (N) and midpoint position (pK Å 0log K,where K is dissociation constant). These parameters were obtained bynism. Because it has been found that Ca2//CaM proteinfitting data points for each curve to the following equation by a nonlinearkinase II and PKC can phosphorylate calponin and thusleast-squares method: F/Fo Å (C/K)N/(1 / C/K)N, where C represents

attenuate its inhibitory action on actin-activated Mg2/- concentration of Ca2/. In A, F is amplitude of contraction at any givenconcentration of Ca2/ and Fo is maximum response evoked by 10 mMATPase activity, it is conceivable that calponin phosphor-Ca2/ expressed at a relative tension of 1.0. [From Itoh et al. (506).]ylation plays a role in agonist-induced Ca2/ sensitization.

Moreover, it has been found that mitogen-activated pro-tein kinase phosphorylates caldesmon, causing a relief of

concentrations may contribute to tension maintenance atthe latter’s inhibitory action on actomyosin Mg2/-ATPaselow levels of MLC20 phosphorylation and at low energyactivity. Unlike the calponin peptide T2, a caldesmon pep-cost (820, 821). Thus it has been suggested that Ca2/ sensi-tide induced a contraction in Ca2/-free solution (Ca2/-tization may be provoked in a myosin-phosphorylationindependent contraction) (552). However, it still remainsless-dependent fashion, when Mg-ADP is accumulated inunclear whether, during the stimulation of SM by variousSMC under some conditions.means, caldesmon and calponin act as switches for the

Let us now turn to Ca2/ desensitization. This coulddevelopment of force or as modulators of myosin phos-be produced 1) by an inhibition of MLCK activity at aphorylation-dependent force through phosphorylationgiven concentration of [Ca2/]i , thus causing a decrease inand dephosphorylation.the level of MLC20 phosphorylation; or 2) by a directIt was recently suggested that, in a-toxin-treated

skinned vascular SM, accumulation of ADP at millimolar inhibitory action on contractile proteins or an accelera-



by agonists than when they are depolarized (348, 545). Insaponin- or a-toxin-treated skinned vascular SM, it wasfound that GTPgS, given alone, enhances Ca2/-inducedcontraction with a parallel increase in the phosphoryla-tion of MLC20 (507, 582–584). Agonist with GTP or withGTPgS also enhanced the Ca2/-induced contraction withan associated increase in the level of MLC20 phosphoryla-tion, and the effects were inhibited by GDPbS (582, 818).In skinned tracheal SMC, GTPgS enhanced the Ca2/-in-duced force with a proportional increase in MLC20 phos-phorylation at serine-19 (613). Guanosine 5*-O-(3-thiotri-phosphate) also enhanced the contraction induced byCa2/-independent MLCK in Ca2/-free solution, but thisagent had no effect on Ca2/-induced contraction in the

FIG. 12. Effect of calmodulin and T2 peptide on relationship be-presence of okadaic acid (a protein phosphatase inhibi-tween tension and MLC phosphorylation in b-escin-skinned smooth mus-

cle of rabbit mesenteric artery. Curves were obtained by fitting data tor). Under these conditions, GTPgS had no effect on thepoints to the following equation by a nonlinear least-squares method: MLCK activation induced by Ca2//CaM, but the dephos-F/Fo Å (C/K)N/(1 / C/K)N, where N is the Hill coefficient and K is the

phorylation of heavy meromyosin by phosphatase wasdissociation constant. C and F/Fo represent phosphorylation of MLC20

(mol PO4/mol MLC20) and relative tension, respectively. Fitted values of significantly inhibited by GTPgS. On the basis of theseN and K were, respectively, 2.4 and 0.26 mol PO4/mol MLC20 in control results, Kubota et al. (614) suggested that GTPgS activates(g Å 0.996) and 1.6 and 0.13 mol PO4/mol MLC20 in presence of T2

G proteins and thus inhibits myosin phosphatase activity,(g Å 0.992). [From Itoh et al. (506).]causing an increase in the myofilament Ca2/ sensitivity.Other investigators have supported the above view that

tion of the cross-bridge cycling rate. A possible physiologi- agonist- and GTPgS-induced increases in Ca2/ sensitivitycal role for MLCK phosphorylation by Ca2//CaM protein are mediated by inhibition of MLC phosphatase (412, 584,kinase II has been described. In this scheme, the phos- 595, 947, 1015, 1043, 1078). Concerning the action ofphorylation of MLCK at site A by Ca2//CaM protein kinase GTPgS in relation to sensitization of contractile protein,II decreases the sensitivity of the enzyme to activation Wu et al. (1180), recently reported that 8-bromo-cGMPby Ca2//CaM, thus causing an effective desensitization to accelerated both the dephosphorylation of the regulatory[Ca2/]i of the phosphorylation of MLC20 (461, 1043). MLC20 and the relaxation of permeabilized rabbit ileum

Inorganic phosphate decreases the force induced by SMC. These effects were independent of MLC kinase ac-Ca2/-dependent and -independent means in skinned SM, tivity. Similarly, 8-bromo-cGMP relaxed GTPgS-inducedCa2/-independent contraction being observed in MLC thi- force and inhibited the GTPgS-induced increase in MLCophosphorylated muscle or on application of trypsin- phosphorylation at constant Ca2/ concentration. There-treated MLCK (502). Moreover, Pi inhibited the maximal fore, they concluded that cGMP-dependent kinase, acti-force and also shifted the relationship between Ca2/ and vated by 8-bromo-cGMP, increases smooth muscle MLCtension to the right, indicating that Pi lowers the myofila- phosphatase activity and counteracts the inhibition of thement’s Ca2/ sensitivity (502). It has been suggested that, latter enzyme by G protein.at low levels of activation, the cross bridge imposes a Protein phosphatases are generally divided into phos-mechanical resistance to shortening and that Pi dissoci- phoserine/threonine-specific and phosphotyrosine-spe-ates this (866), suggesting that Pi may tend to decrease cific enzymes (437). Because SM myosin is phosphory-the number of strong cross bridges. Thus, in addition to lated at serine-19 (and threonine-18), serine/threonine-the activity ratio of MLCK to MLCP (which decides the specific phosphatases could well be the responsiblelevel of MLC20 phosphorylation), many factors seem to enzymes. The serine/threonine-specific phosphatases arecontribute to the regulation of myofilament Ca2/ sensitiv- further subdivided into two types of enzymes accordingity through distinct cellular regulatory mechanisms. In the to their inhibition by one or both of two acid- and heat-following sections, we focus on the mechanisms underly- stable proteins (inhibitor-1 and -2) (459). Type 1 proteining the regulation of myofilament Ca2/ sensitivity, mainly phosphatase (PP1), which is sensitive to inhibitor-1 andthose that act through a modulation of myosin-linked inhibitor-2, dephosphorylates the b-subunit of phospho-mechanisms. rylase kinase (470) and is present in both cytosolic and

particulate fractions of the cell (434). On the other hand,1. Mechanisms underlying myofilament Ca2/

type 2 protein phosphatase (PP2) dephosphorylates thesensitization

a-subunit of phosphorylase kinase and is largely presentin the cytosolic fraction of the cell. Type 2 protein phos-In intact SMC, less [Ca2/]i is required for half-maxi-

mal MLC20 phosphorylation when tissues are stimulated phatase has been further subdivided into PP2A, PP2B, and



PP2C (204); of these, PP2A has significant phosphorylase phosphate (822). The InsP3 releases Ca2/ from the Ca2/

storage sites, and DAG activates PKC. Some agonists,phosphatase activity (204).Myosin phosphatases have been isolated from SMC such as ET-1, greatly enhance the production of DAG (370,

371). It has been suggested that PKC plays a primary rolein many laboratories and classified as either PP1 or PP2A(20, 277). The representatives of the PP1 class of phospha- in the agonist-induced Ca2/ sensitization of contractile

proteins in vascular SM (819, 838), although evidence totases are multimeric structures composed of a catalyticsubunit and one or more accessory subunits that target the contrary has also been reported (772, 982). It has also

been reported that, in intact and skinned SMC, phorbolthe catalytic subunit to a specific substrate (437). Theglycogen-associated PP1-G purified from skeletal muscle ester increased the myofilament Ca2/ sensitivity with or

without a corresponding increase in the phosphorylationis well known as a typical type of PP1, being composedof a 37-kDa catalytic subunit complexed to a 124-kDa of MLC20. These results suggest that PKC may contribute

to the agonist-induced increase in the myofilament’s Ca2/glycogen-targeting subunit (437). The PP2A type has atrimeric form: a 36-kDa catalytic subunit, a 65-kDa regula- sensitivity in SMC. However, Oishi et al. (853) reported

that in stomach SM tissues, the ACh-induced increase intory subunit, and a third variable subunit of between 54and 130 kDa (437). It has recently been found that the myofilament Ca2/ sensitivity was not prevented by cal-

phostin C (a relatively specific inhibitor of PKC) (982),myosin-bound phosphatase purified from avian SMC (mo-lecular mass 230 kDa) is also composed of three subunits: suggesting that PKC may not contribute to the increase

in Ca2/ sensitivity induced by ACh in this tissue. The role130, 37–38, and 20 kDa (20, 978, 979). The 37- to 38-kDacomponent is the d-isoform of the catalytic subunit of of PKC in the G protein-mediated increase in myofilament

Ca2/ sensitivity has also been studied using ET-1 plustype 1 protein phosphatase (PP1), and this binds only tophosphorylated myosin, referred to as the b-isoform (20). GTP, or GTPgS alone, in b-escin-treated skinned SMC

from the rabbit mesenteric artery (506, 1210). In theThe other two subunits are thought to be involved in thetargeting of the phosphatase to myosin, being putative skinned SMC, PDBu, ET-1 plus GTP, and GTPgS alone all

increased both the contraction and the MLC20 phosphory-regulatory subunits. These results suggest that smoothmuscle MLCP is a type 1 protein phosphatase. Pharmaco- lation induced by a given concentration of Ca2/ and

shifted to the left the relationship between tension andlogical evidence obtained using various types of phospha-tase inhibitors also supports this notion for okadaic acid Ca2/, or that between Ca2/ and MLC20 phosphorylation.

None of these agents changed the relationship betweenand for calyculin A.The pathway activated by agonists inhibiting MLCP tension and MLC20 phosphorylation. Under these condi-

tions, PKC-(19{36) (an inhibitor of PKC) completely in-is believed to rely on ligand occupancy of the receptor,coupling with and activation of heterotrimeric G proteins, hibited the PDBu-induced increase in the myofilament’s

Ca2/ sensitivity, only partly (byõ30%) attenuated the ET-and initiation of their coupling to intracellular targets forthe inhibition of MLCP. It has been found that the myofil- 1 response, but had almost no effect on the GTPgS-in-

duced response. Thus these results suggest that agonist,ament Ca2/ sensitization induced by GTPgS, but not thatinduced by agonist with GTP, is inhibited by neomycin, in causing an increase in myofilament Ca2/ sensitivity in

SMC, relies on both PKC-dependent and -independentwhich binds phosphatidylinositol 4,5-bisphosphate andprevents agonist-stimulated phosphoinositide hydrolysis mechanisms.

B) ARACHIDONIC ACID AND OTHERS. Calcium-mobilizing(506). These results suggest that, in addition to theinvolvement of heterotrimeric G proteins, some unidenti- agonists, such as phenylephrine, thromboxane analogs,

and carbachol in intact SM and GTPgS alone in skinnedfied G proteins, such as small-molecular-weight G pro-teins, may play a role in the GTPgS-induced Ca2/-sensiti- SM tissues, increase the levels of arachidonic acid and

DAG (358). Under these conditions, an increase in thezation mechanism (416, 961). However, the mechanismsunderlying the agonist-induced increase in myofilament concentrations of arachidonic acid and DAG precedes the

force development induced by GTPgS. In a-toxin-treatedCa2/ sensitivity have not yet been identified. To date, sev-eral possible underlying mechanisms have been consid- skinned SM, exogenously applied AA increased, at con-

stant Ca2/, the levels of both tension and MLC20 phos-ered to have a possible involvement (653). Thus 1) PKCactivated by DAG may be involved; 2) arachidonic acid phorylation and also slowed the rate of relaxation and

MLC20 dephosphorylation after the removal of Ca2/ (356).(AA), which is produced by activation of phospholipaseA2, may be the responsible second messenger; or 3) the These effects of arachidonic acid were not modified by

inhibitors of arachidonic acid metabolism (indomethacin,activation of small G proteins, such as rho- and ras-pro-teins, through the activation of unidentified pathways, nordihydroguaiaretic acid, or propyl gallate), by inhibitors

of PKC (a pseudosubstrate peptide), or by GDPbS. A non-may be involved.A) PKC. It is well established that Ca2/-mobilizing ago- metabolizable arachidonic acid analog, 5,8,11,14-eicosate-

traenoic acid, had effects similar to those of arachidonicnists bind to their receptor and synthesize InsP3 and DAGthrough the hydrolysis of phosphatidylinositol 4,5-bis- acid. A purified oligomeric MLCP isolated from gizzard



SMC was dissociated into subunits by arachidonic acid, Recently, three different species have been identifiedof Rho-interacting proteins with molecular masses ofand its activity toward heavy meromyosin was inhibited,

but that toward phosphorylase was not (356). Therefore, Ç128, 164, and 180 kDa (21, 1161). Of these, p128 wasidentified as protein kinase N (PKN) (21, 782). Proteinthese authors concluded that arachidonic acid may act as

a messenger promoting protein phosphorylation through kinase N is a serine/threonine kinase with an NH2-terminalregulatory domain and a COOH-terminal catalytic domaina direct inhibition of the form of protein phosphatase(s)

that dephosphorylate MLC20 in vivo. Recently, it has been (782). The catalytic domain of PKN is closely related tothat of PKC, and the substrate specificity of PKN is closelyfound that the Ca2/-sensitizing effect of 5-hydroxytrypta-

mine (5-HT) in permeabilized rabbit mesenteric artery related to that of PKC (781). RhoA kinase (p164) is alsoa serine/threonine kinase, and its kinase activity is spe-SMC can be abolished by a phospholipase A2 inhibitor,

quinacrine (885). Although the action of arachidonic acid cifically stimulated by GTPgSrRhoA, suggesting thatRhoA kinase is a putative target serine/threonine kinasein causing dissociation of oligomeric MLCP is the subject

of controversy (450, 451), these results seem to suggest for RhoA, and might serve as a mediator in the Rho-depen-dent signaling pathway. The substrate specificity of RhoAthat arachidonic acid released by Ca2/-mobilizing agonists

may contribute to the agonist-induced increase in myofil- kinase differs from that of PKN 708). In the rat, RhoAkinase phosphorylates the COOH terminus of the myosin-ament Ca2/ sensitivity through its direct (i.e., independent

of G proteins) inhibitory action on MLCP. binding subunit of MLCP (amino acids 740–1034), andGTPgSrRhoA stimulates the RhoA kinase activity towardLysophosphatidylcholine, which is released by acti-

vation of phospholipase A2, can also increase Ca2/ sensi- the myosin-binding subunit by Ç15-fold (708). Ichikawaet al. (450, 451) also described a kinase that phosphory-tivity through a G protein-independent mechanism, al-

though the level of lysophosphatidylcholine did not in- lates the myosin binding subunit (130 kDa) of MLCP. Themajor site of this phosphorylation was threonine-653 increase during stimulation with NE (521). In addition,

polyamines such as spermine, which inhibits SM PP1, in- the 130-kDa subunit, and the phosphorylation caused areduction in the activity of the phosphatase. However, thecreased Ca2/ sensitivity in skinned intestinal SM (1066).

However, as yet, the physiological role of lysophosphati- protein kinase that phosphorylated the 130-kDa subunitand resulted in an inhibition of MLCP has not been finallydylcholine and polyamines in agonist-induced Ca2/ sensi-

tization remains uncertain. identified. It was also reported that Rho kinase, whichis activated by GTPrRhoA, phosphorylated the myosinC) SMALL G PROTEINS. In saponin-treated skinned SMC

from the rabbit mesenteric artery, Hirata et al. (416) found binding subunit and consequently inactivated myosinphosphatase. Overexpression of RhoA or activated RhoAthat the increase in the Ca2/-induced contraction caused

by GTPgS was inhibited by application of either epidermal in NIH 3T3 cells increased the phosphorylation of themyosin binding subunit and MLC. Thus Rho appears todifferential inhibitor (EDIN), prepared from Staphylococcus

bacteria, or C3, prepared from Botulinus toxin, because of inhibit myosin phosphatase through the action of Rhokinase (432). It has been found that thiophosphorylationthe ribosylation of rho p21. The GTPgS-binding form of

rho p21, when introduced into the EDIN-treated skinned of the 130-kDa subunit of myosin-bound phosphatase afteran application of ATPgS in Ca2/-free solution caused anvascular SM, restored the Ca2/-induced contraction. Under

these conditions, EDIN and C3 themselves did not modify increase in myofilament Ca2/ sensitivity in a-toxin-perme-abilized SMC (1104). Under these conditions, there was athe contraction induced by Ca2/ alone. Thus it is postulated

that rho p21 may contribute to the GTPgS-induced increase slowing in the rate of decrease in MLC20, after the re-moval of Ca2/ in the presence of ML-9 (a MLCK inhibitor),in the myofilament’s Ca2/ sensitivity in SMC (357, 416). In

addition, in SMC from the guinea pig vas deferens, NE acti- suggesting that thiophosphorylation of the 130-kDa sub-unit of MLCP is associated with a decrease in the activityvates an a1-adrenoceptor-coupled heterotrimeric G protein

and thus increases the contraction induced by a low concen- of MLCP in situ. It remains unclear, however, whether themyosin-binding subunit serves as a physiological sub-tration of Ca2/. This action of NE was abolished in C3-

pretreated muscle, but it was not inhibited by PKC-(19{31) strate for RhoA kinase (708) and/or for MLCP-associatedkinase (452). Further studies are needed to clarify this(a PKC inhibitor). These results suggest that rho protein

may contribute to the NE-induced increase in the myofila- point.It has been demonstrated that RasrGTPgS, anotherment’s Ca2/ sensitivity through the activation of a PKC-inde-

pendent pathway. Furthermore, it was also found that, in type of small G protein, also mimics the Ca2/-sensitizingeffect of GTPgS. Satoh et al. (962) found that the Ca2/-permeabilized cultured SMC from the pig aorta, GTPgS not

only enhanced the Ca2/-induced MLC20 phosphorylation but sensitizing effect of the ras protein can be partially re-versed by a tyrosine kinase inhibitor, suggesting theattenuated the rate of dephosphorylation of MLC20 after the

removal of Ca2/. C3 abolished both these actions of GTPgS, involvement of an unidentified tyrosine kinase in the rasp21-mediated increase in the Ca2/ response. It has alsosuggesting that rho-protein may contribute the GTPgS-in-

duced inhibition of MLCP. been shown that a stimulation of ras p21 is involved in



the mitogen-activated protein kinase activation cascade channels can be classified in various ways: 1) from theanalysis of its cDNA, the a-subunit of some cloned K/(656, 1117). It was recently recognized that agonist-stimu-

lated coupling of G proteins stimulates the tyrosine phos- channels contains six transmembrane segments (Shaker,Shal, Shaw, or Shad), whereas that of other cloned K/phorylation of mitogen-activated protein kinase in vascu-

lar SMC (8, 248, 1108). Thus an activation of the mitogen- channels contains two transmembrane segments (IRK1,2,3; GIRK 1,2,3,4; KAB-1/ROMK1; uKATP-1; or RACTK1);activated protein kinase cascade by G protein-coupled

receptors after stimulation may play a role in the agonist- 2) from their voltage dependency, they are classified aseither delayed rectifier (KV), rapid delayed rectifier (KVR),induced increase in myofilament Ca2/ sensitivity. How-

ever, the mechanisms underlying the regulation of the slow decayed rectifier (KVS), inward rectifier (KIR) K/

channels, A channel (KA), or SR (KSR) K/ channels; 3)function of ras p21 during agonist-induced Ca2/ sensitiza-tion remain to be clarified. from their Ca2/ dependency, K/ channels are classified

as either maxi-K/ (big, high; BKCa), intermediate (IKCa),or small conductance (SKCa) K/ channels; 4) from their

VII. SUMMARY AND CONCLUSIONS receptor dependency, they are classified as muscarinic-inactivated (KM), atrial muscarinic-activated (KACh), or 5-HT-inactivated (K5-HT) K/ channels; and 5) as intracellu-Smooth muscle cells are mainly concerned with the

regulation of the motility of tissues through changes in lar ATP-sensitive (KATP), intracellular Na/-activated (KNa),or cell volume-sensitive (Kvol) K/ channels. However,their contraction-relaxation cycle, although they are also

involved in an excitation-secretion coupling. These ac- Ca2/-dependent K/ channels (3 subtypes) are also voltage-dependent K/ channels. Similarities and differences be-tions are mainly regulated by the level of Ca2/ in the

cytosol. This in turn is determined by 1) influx of Ca2/ tween of KATP and KIR in relation to SK/ have been dis-cussed. However, the features of these channels have yetthrough VOCC, ROCC, antidromic Na//Ca2/ exchangers,

and so on; 2) release of Ca2/ from the Ca2/ stores (mainly to be completely clarified. Furthermore, Ca2/-dependentoscillatory outward or inward currents (STOC and STIC)the SR); this release takes the form of CICR and IICR

(induced by activation of RyR and InsP3 receptors); 3) can be recorded when depolarizing pulses or agonist stim-ulation are applied. The above K/ channels are not alwaysreuptake of Ca2/ (into the SR by Ca2/-ATPase and by

nucleus and mitochondria); and 4) extrusion of Ca2/ (via recorded from VSMC, and in some tissues, a lack of somesubtypes has been reported. There has been considerableCa2/-ATPase and orthodromic Na//Ca2/ exchange).

Thanks to the considerable progress made in recent years debate as to the identity of the type of K/ channel respon-sible for the generation of the resting potential in VSMC.in developing and improving techniques to measure dy-

namic changes in the location of Ca2/, using various Ca2/ However, a satisfactory answer has not yet been provided.Furthermore, the contribution of K/ channels to the repo-dyes with the confocal microscope, it is clear that, during

the initial stage of cell activity, the cytosolic Ca2/ is not larization phase of the action potential and to plateauformation has been much discussed in relation to the pos-evenly distributed. Various terms have been used to de-

scribe the phenomena involved in creating this nonhomo- sible role of Ca2/-dependent, voltage-dependent K/, anddelayed rectifying K/ currents. Unfortunately, once again,geneous distribution. These include Ca2/ spark in the SL,

Ca2/ spike in the SR, quantal release in the SR, capacita- there is no clear answer as yet. In addition, the rolesplayed by intracellular ATP- and extracellular glyburide-tive Ca2/ entry, Ca2/ oscillations, and Ca2/ waves. These

take place in different parts of the cell. In addition, func- sensitive K/ channels in VSMC, especially in vascular andurinary tissues, have been the focus of considerable atten-tional coupling between the SR and SL has been involved

to explain Ca2/ mobilization, although we still need to tion in relation to inward rectifying K/ channels.Let us look at the influx of Ca2/. The VOCC in VSMCunderstand the functional role of the caveoles distributed

on the SL before we can fully explain Ca2/ mobilization are of two main subtypes (L type: 20–25 pS, high-voltageactivated and showing slow inactivation; and T type: 6–12in SMC. Moreover, the tissue and species differences be-

tween VSMC make the picture very complicated and, for pS, low voltage-activated and showing slow inactivation).However, in terms of increases in cytosolic Ca2/, the mainthat reason, it is difficult to give a detailed, yet brief,

description of the qualitative and quantitative differences role is played by the L-type Ca2/ channel. Some VSMCpossess both channels, whereas others possess only theamong VSMC in relation to Ca2/ mobilization. However,

this complexity may reflect a pattern of physiological dif- L type. The role of the T-type Ca2/ channel has yet to beclarified (although it may be involved in depolarization inferences between individual SMC that has a significant

relation to the variety of functions performed by the vari- pacemaker cells in the GI tract). It is plausible that asignificant influx of Ca2/ can occur through activation ofous tissues in the various species.

In VSM, K/ channels play essential roles in holding the L-type Ca2/ channel. However, to shift the amount ofCa2/ needed to trigger the full mechanical response re-the membrane potential at its resting level and in repolar-

izing the membrane after activation of VOCC. These K/ quires the further activation of the CICR mechanism. This



is because of the presence of Ca2/ buffering processes membrane potential and action potentials, and also aboutthe actions upon them of drugs such as oxytocin andjust beneath the cell membrane. Other phenomena may

also serve to increase the cytosolic Ca2/. These include relaxin. The story is not a simple one because, in thesecells, the receptors and ion channels are markedly af-1) repetitive spike generation (a burst discharge between

silent periods) as observed in the GI tract, myometrium, fected by changes in hormonal conditions, especially bythe levels of estrogen and progesterone. Such changesand portal vein; 2) plateau formation, as observed in the

ureter and myometrium; and 3) large slow potential occur during the estrus cycle, through gestation, duringparturition, and postpartum. Current thinking is that re-changes, as observed in the GI tract and myometrium,

and receptor-regulated various patterns of Ca2/-related ceptors for these steroid hormones are not only locatedintracellularly, with their activation leading to the produc-currents. Furthermore, the relative density of VOCC and

their activation and slow inactivation kinetics in VSMC tion of a specific form of mRNA, they are also distributedon the SL. As a result of this thinking, a whole new areamay also be important in determining their contribution

to Ca2/ influx through the so-called window current. of VSM research could open up, leading to the betterunderstanding of the hormonal modulation of myometrialEven when VSMC may possess VOCC, action poten-

tials may not be recorded by the microelectrode or cur- activities in different physiological states.In recent years, remarkable advances in variousrent-clamp procedures (e.g., in the aorta, iris dilator, stom-

ach fundus, and trachea; some are quiescent, whereas branches of biological research have resulted from theapplication of techniques developed in molecular biology;others generate graded responses). Presumably, a low

density of VOCC and/or fast activation of a voltage-depen- VSM research is no exception. Discoveries made by meansof such techniques are enabling us to build up a picturedent K/ channel (which induces a transient outward cur-

rent with a relatively low threshold) may mask the genera- of the distribution and function of the numerous subtypesand isoforms of ion channels and receptors in a wholetion of a spike potential. Furthermore, although a Na/

inward current can be recorded from various tissues using variety of tissues and species. However, as yet, the pictureis far from complete; we should probably feel more likethe voltage-clamp procedure, no Na/ spike is recorded

using the microelectrode method. At least two Na/ cur- a child anxiously contemplating a jigsaw puzzle with manypieces still not in place than an artist admiring his com-rents, namely, TTX-sensitive (nM order) and -insensitive

(mM order) currents, are presently recognized. However, pleted canvas.One point of view is that the list of subtypes andalthough the physiological role of such currents may lie,

as has been proposed, in the propagation of excitation, isoforms is growing too rapidly for us to have a realisticchance of attaching a function to each of the new discov-this is not yet certain.

Some VSMC generate spontaneous action potentials, eries. Do some of them actually have a function, we some-times wonder, or could they be evolutionary relics whosebut others do not. In the former type, there may be a

prepotential (generator potential; in pyloric region of the function has long since vanished? We can certainly saythat our knowledge is sorely inadequate to explain theureter or in the myometrium) or slow potential (in the GI

tract). The prepotential is thought to be related to the physiological function of the galaxy of subtypes and iso-forms that have been identified in VSMC in recent years.activation of a transient inwardly rectified current (funny

current); this current has been observed only in spontane- Because there is unlikely to be a moratorium on futurediscoveries, the reader will not be surprised to learn thatously active cells, and its kinetics are not yet completely

understood. On the other hand, in the GI tract, particular our final thought is as follows. Many workers will makemany detailed investigations on individual SMC and on acells, the ICC, serve to generate the pacemaker potential

through activation of T-type Ca2/ channels. variety of tissues and organs in different species beforethe picture shown on the jigsaw puzzle is revealed to usIn many VSM tissues, the passive membrane proper-

ties are expressed in terms of cable properties. This is in anything like a recognizable state.because individual SMC are connected by paths with elec-

We are grateful to Dr. R. J. Timms for correcting the Englishtrically low resistance. In VSM, gap junctions may play an and for giving comments. We also thank Yurie Taniguchi forimportant role rather than tight junctions, intermediate typing the manuscript.junctions, desmosomes, or nonjunctional membrane ad- Financial support was kindly provided by Chugai Pharma-hesions. However, neither the number nor the location of ceutical, Tokyo, Japan.gap junctions is stable, being altered by various factorssuch as Ca2/, Mg2/, and pH or by reproductive hormones

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