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Physiol. Rev. 78: 811-920, 1998;
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PHYSIOLOGICAL REVIEWS   Vol. 78 No. 3 July 1998, pp. 811-920
Copyright ©1998 by the American Physiological Society

Physiological Features of Visceral Smooth Muscle Cells, With Special 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
II. REGIONAL AND SPECIES DIFFERENCES IN THE RESTING MEMBRANE AND ACTION POTENTIALS IN VISCERAL SMOOTH MUSCLE CELLS
    A. Resting Membrane Potential and Passive Membrane Properties of VSMC
    B. Action Potentials Generated in VSMC
III. NEURAL CONTROL OF MEMBRANE ACTIVITIES IN VISCERAL SMOOTH MUSCLE CELLS
    A. Nerve Plexuses, Nerve Terminals, Varicosities, and Cotransmitters as Studied by Electrophysiological Methods
    B. Features of Varicosities Deduced From Electrophysiological Investigations
    C. Multitransmitter Release Inferred From the Generation of EJP and IJP and Features of Postjunctional Receptors
IV. CHARACTERISTICS OF ION CHANNELS IN VISCERAL SMOOTH MUSCLE CELL MEMBRANES
    A. K+ Channels
    B. Na+ Channels
    C. Ca2+ Channels
    D. Cl- Channels
    E. Nonselective Cation Channels
V. RECEPTOR-OPERATED ION CHANNELS IN VISCERAL SMOOTH MUSCLE CELLS
    A. Purinoceptor-Coupled Channels
    B. G Protein-Coupled Cation Channels
    C. Cytosolic Ca2+-Activated Nonselective Cation Channels
    D. Unclassified Cation Channels
    E. Muscarinic Receptor-Inactivated K+ Current: M Current
VI. MOBILIZATION OF CALCIUM AND MAINTENANCE OF CALCIUM ION HOMEOSTASIS IN VISCERAL SMOOTH MUSCLE CELLS
    A. Ca2+ Concentration in the Cytosol of VSMC
    B. Factors That Increase [Ca2+]i in VSMC
    C. Factors That Decrease [Ca2+]i in VSMC
    D. Ca2+ Sensitization of Smooth Muscle Contraction
VII. SUMMARY AND CONCLUSIONS
REFERENCES

    ABSTRACT
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Kuriyama, H., K. Kitamura, T. Itoh, and R. Inoue. Physiological Features of Visceral Smooth Muscle Cells, With Special 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 homeostasis in biological systems. The features of these cells differ markedly by tissue and by species; moreover, there are often regional differences within a given tissue. The biophysical features used to investigate ion channels in VSMC have progressed from the original extracellular recording methods (large electrode, single or double sucrose gap methods), to the intracellular (microelectrode) recording method, and then to methods for recording from membrane fractions (patch-clamp, including cell-attached patch-clamp, methods). Remarkable advances are now being made thanks to the application of these more modern biophysical procedures and to the development of techniques in molecular biology. Even so, we still have much to learn about the physiological features of these channels and about their contribution to the activity of both cell and tissue. In this review, we take a detailed look at ion channels in 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+ movements and Ca2+ homeostasis in the cell. In sections II and III, we discuss research findings mainly derived from the use of the microelectrode, although we also introduce work done using the patch-clamp procedure. These sections 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 Cl-; 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 of Ca2+ in VSMC using the patch-clamp procedure, biochemical procedures, measurements of Ca2+ transients, and Ca2+ sensitivity of contractile proteins of VSMC. We discuss the way in which Ca2+ mobilization occurs after membrane activation (Ca2+ influx and efflux through the surface membrane, Ca2+ release from and uptake into the sarcoplasmic reticulum, and dynamic changes in Ca2+ within the cytosol). In this article, we make only limited reference to vascular smooth muscle research, since we reviewed the features of ion channels in vascular tissues only recently.

    I. INTRODUCTION
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The "father" of the electrophysiology of visceral smooth muscle cells (VSMC) was, undoubtedly, Professor Emil Bozler. He founded visceral smooth muscle (VSM) physiology by his pioneering experimental procedures using extracellular recording, mainly from 1938 to 1948, and he recognized that VSMC could be classified as either single- or multiunit smooth muscle cells (SMC). In particular, he elucidated the myogenic propagation of excitation using the rat myometrium, and he also discovered the basis of automaticity (the prepotential) in VSMC of the intestine and ureter. In addition, differences in the shape of the action potential in different VSMC (from myometrium, ureter, intestine, and stomach) were first demonstrated by Bozler using extracellular recording procedures. He concluded that in VSMC, an all-or-none response to a single-threshold stimulus may show either a single spike, a series of spikes, a plateau, or a combination of these elements. His research interests were not limited to the study of smooth muscle (SM); he also studied skeletal muscle, cardiac muscle, and neurons. Bozler (121) summarized his brilliant work on SM cells in 1948. Thereafter, Bozler studied skinned muscle tissues and elucidated the role of Ca2+ in skeletal muscle cells using EDTA, ATP, and divalent cations (Ca2+ and Mg2+).

If Professor Emil Bozler was the father of SM physiology, the "mother" was definitely Professor Edith Bülbring. In 1954, she first applied microelectrode techniques to the measurement of the electrical properties of VSMC (135). Her scientific family is distributed the world over, and her "grandchildren" are playing major roles in advancing SM research. Professor C. Ladd Prosser was also one of the pioneers of SM physiology, and he and his students (who are mainly distributed in the United States) have made important contributions to the progress of research, mainly in gastrointestinal (GI) SM physiology.

A powerful new technique for the study of ion channels and receptors, the patch-clamp method, was introduced by Neher and Sakmann (807) and Hamill et al. (381), the first report using the method in VSMC being published by Benham and Bolton (71). Since then, many articles on ion channel and receptor activities have been written. A new technique for making measurements of intracellular free Ca2+ ([Ca2+]i) using aequorin (see review by Blinks et al., Ref. 96) was first applied to VSMC by Fay et al. (287) and Morgan and Morgan (775). Since then, similar techniques have been employed by others, using various dyes (see sects. IV and VI). At present, at least three different procedures for measuring [Ca2+]i are in use, namely, Ca2+ transient measurements using various indicators and methods using Ca2+-sensitive microelectrodes (1188) or nuclear magnetic resonance (NMR). Combined techniques for measurements of 1) the ionic current and Ca2+ transient, 2) the ionic current and mechanical response, or 3) the Ca2+ transient and mechanical response have been successfully applied to the investigation of VSMC activity. Furthermore, the introduction of the confocal microscope has enabled the determination of the regional distribution and dynamic changes in the location of Ca2+ in the cytosol. Recent remarkable progress in molecular biological techniques has enabled us to identify the primary amino acid sequence and three-dimensional structure of the proteins that make up ion channels and receptors, as well as giving clues to their functional expression. Such procedures have also been successfully applied to the investigation of the membrane characteristics of VSMC.

Visceral smooth muscle cells develop from the mesoderm, although the developmental steps involved in the formation of individual VSM tissues differ according to the type of tissue. Moreover, the membrane potential and the shape of the action potential differ by region and by species. When studying the cellular and subcellular characteristics of VSMC, it is necessary to know something about the basic cellular functions of VSMC (as determined using the microelectrode method: resting and action potential of the membrane) before attempting to understand the features of the ionic mechanisms at the subcellular level. In addition, an understanding of the neuronal modifications of SMC membrane activities is essential. For this reason, section II introduces the fundamental membrane properties of VSMC measured by the microelectrode and examines the ionic currents responsible for forming the membrane potential and action potential. In section III, neuromuscular transmission in VSMC measured mainly by the microelectrode method and some features of the prejunctional regulation of transmitter release are discussed. Sections IV and V review the general properties of the voltage-dependent and -independent ion channels and of the receptor-operated ion channels found in VSMC, together with their underlying mechanisms (including the role of GTP-binding proteins, G proteins). This area of study involves measurements made mainly by patch-clamp methods (whole cell, cell-attached, and cell-free patch-clamp recordings). Section VI deals with Ca2+ homeostasis in resting and active cells, as studied mainly by making measurements of Ca2+ transients and by electrophysiological techniques. The ultimate functions of VSMC are control of motility and of processes allied to secretion. These actions are induced or modulated by contractile proteins and their regulatory proteins and are activated by changes in [Ca2+]i and by non-Ca2+ processes. The level of [Ca2+]i is regulated by the influx and efflux of Ca2+ across the cell membrane, and also by the uptake and release of Ca2+ from the SR. Furthermore, Ca2+ sensitization and desensitization are discussed in relation to the actions of agonists. Because the specific characteristics of vascular SMC, together with their pharmacology, have been reviewed recently by the present authors (626), we do not intend to incorporate information specific to vascular SMC in this article. However, features of ionic channels and receptor properties relevant to vascular SMC are included wherever they relate to, or exemplify, the general functions of VSMC.

    II. REGIONAL AND SPECIES DIFFERENCES IN THE RESTING MEMBRANE AND ACTION POTENTIALS IN VISCERAL SMOOTH MUSCLE CELLS
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A. Resting Membrane Potential and Passive Membrane Properties of VSMC

1. Resting membrane potentials in VSMC

The resting membrane potential, as measured in various VSMC, varies from -35 to -70 mV. Such variations in the value of the membrane potential may, partly, be from the different experimental conditions used. Because the resting membrane potential and action potential in both longitudinal and circular muscle layers in the myometrium are greatly modified by hormonal conditions, such as occur during the estrus cycle, during the course of pregnancy, and also post partum, we will use the myometrium as an example.

The cells of the myometrium are spindle shaped, ranging in size from 2 to 10 µm in diameter and from 200 to 600 µm in length. As gestation progresses, hormonal conditions change from nonpregnant (5-10 µm in diameter; resting membrane potential -35 to -45 mV) through pregnancy (15-17 days of gestation: 10-15 µm in diameter, -55 to -65 mV). During gestation, cell diameter and resting membrane potential increase more or less in line with each other through to the last stage of gestation (21-22 days of gestation: 15-30 µm, membrane was slightly depolarized compared with midpregnancy). The above data, derived by the following authors from various species, appear to be reliable, but some experimental error may be present in the measurements in the nonpregnant uterus due to the small diameter of the myocytes. In general, the resting membrane potential in rat and mouse myometriums is larger in circular muscle cells than in longitudinal muscle cells, both before and during the early stages of gestation. Moreover, when pregnancy is well under way, the membrane is hyperpolarized in both layers ("progesterone blockade"; Refs. 621, 624). However, at term, the membrane of SMC in both layers is depolarized to a certain extent. In the rat myometrium, at midterm, the membrane potential was -60 mV, whereas at term, this value had fallen to -45 mV (695). The highest membrane potential in the rat myometrium during pregnancy was seen on day 16 (27, 632). In midpregnancy, the membrane of longitudinal SMC in the placental region showed a consistently higher membrane potential than those in the nonplacental region (540). However, it has been reported that there is no change in the membrane potential of SMC in the circular muscle layer during pregnancy or 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, whereas that of progesterone decreases. Progesterone, given intracutaneously to estrogen-primed ovariectomized rats, produced a consistent hyperpolarization of the longitudinal SMC membrane (632). Estrogen itself given to ovariectomized rats in the nonpregnant condition also hyperpolarized the membrane. During pregnancy, however, estrogen did not modify the membrane potential, although a decrease in the concentration of estrogen at near-term occurred at the same time as a depolarization of the membrane potential. On the other hand, in vitro treatment with progesterone or estradiol produced different actions on the membrane potential and contraction in circular and longitudinal muscle layers of the rat myometrium (831). Thus, on muscle membranes, progesterone enhanced electrical activity in the longitudinal muscle layer and inhibited it in the circular muscle layer. In contrast, estrogen produced the reverse of these actions in the two muscle layers. However, at term and postpartum, estrogen produced excitatory actions on the circular muscle layer.

Steroid hormones, such as progesterone and estrogen, may act on the cell nucleus and increase the production of mRNA. Erulkar et al. (278) concluded that gonadal steroids exert an influence on the expression of different populations of ionic channels in isolated cells of the immature rat uterus. On the other hand, it is conceivable that these steroid hormones also act directly on the surface of cell membranes through unknown mechanisms. Erulkar et al. (278) observed the effects of gonadal steroids (17beta -estradiol and progesterone) on K+ currents in cells isolated from the myometrium of immature rats. In their study, predominantly outward K+ currents with an early transient component were recorded in response to depolarization pulses (from a holding potential of -90 mV). This current was rarely observed in adult myometrial cells, was inactivated at -40 mV, and was blocked by 4-aminopyridine (4-AP). Depolarization also generated a second sustained outward current. 17beta -Estradiol reduced the probability of occurrence of the transient outward current, and progesterone had only a slight effect on the currents. Treatment with 17beta -estradiol (applied before isolation) led to a shorter time constant of decay for the transient outward current, whereas progesterone caused the time constant to increase.

2. Passive membrane properties

A) MORPHOLOGICAL FEATURES OF CELL-TO-CELL CONNECTIONS. To understand the passive membrane properties of VSMC, it is essential first to know something about the morphological and physiological features of gap junctions between cells. In studies of cell-to-cell connections, several morphological structures have been described: tight junctions (nexuses), intermediate junctions, desmosomes, gap junctions, and nonjunctional membrane adhesions. Many VSMC possess cell-to-cell connections through gap junctions. These gap junctions play two major roles: propagation of excitation (including ion and small molecule transport) and mechanical coupling. Moore and Burt (768) studied the features of gap junctions and reported that each junctional channel is composed of two hexamers of proteins terminating in connexins. One hexamer originates from each cell, and they join in the extracellular space to form a patent channel. Gabella (329) reviewed the features of gap junctions in VSMC in detail and described them as follows. In the gap junction, the intercellular space is narrower than 3 nm. In contrast, if the space is larger than 60 nm, this structure is called an adherens-type junction. The gap junction contains two dense bands, one from each cell, linked to bundles of actin filaments. The intramembrane particle, the connexon, is 8-10 nm in diameter, and its main component is connexin (Cx). The presence of Cx43 has been detected in myometriums and also in colonic muscle (82, 932). These particles lie always strictly parallel to each other, and there is no encroachment of the dense material onto the cytoplasmic side (329, 331). The spatial density of connexons in gap junctions in VSM is ~7,000/µm2, this value being much smaller than that observed in cardiac muscles. The largest junctions may contain some 1,400 connexons from each of the two membranes, whereas the smallest ones are made of only 3-6 connexons. One of the densest distributions of gap junctions has been found in the circular muscle of the small intestine. In fact, in the circular muscle of the guinea pig duodenum and ileum, 0.5 and 0.2%, respectively, of the cell surface is occupied by gap junctions; these values correspond to 25 and 11 µm2/cell or 175,000 and 77,000 connexons/cell, respectively (329). In contrast, in the adjacent longitudinal muscle, although the dye Lucifer yellow will penetrate between the cells, gap junctions are either absent or few in number (1224). Much the same finding has been reported in the longitudinal muscle of the guinea pig taenia coli, although, again, a dense distribution was observed in the adjacent circular muscle (329). Another tissue with a very high density of gap junctions is the sphincter pupillae of rodents (actually, the highest yet found). However, other SMC are virtually, or completely, devoid of gap junctions; these include the detrusor muscle of the rat bladder (329) and the corpus cavernosum of the penis (195, 196).

In electrophysiological terms (space constant and other characteristics), the role of the gap junction is not completely settled. In the myometrium, gap junctions are formed rapidly and in large numbers within a few hours before parturition, and they come to occupy 0.2-0.4% of the cell surface (344). Furthermore, progesterone suppresses the formation of myometrial gap junctions, whereas estrogen promotes it (226, 744). Sakai et al. (951) observed the effects of the antiprogesterone compounds RU-486 and ZK-299 on cell-to-cell coupling in the guinea pig myometrium during pregnancy. They found that the myometrial SMC of guinea pigs are moderately well coupled before the onset of labor and that the coupling increases further, just before spontaneous delivery. It also increases on treatment with antiprogesterones in nonpregnant animals. They reported an input resistance of 44.6 MOmega on days 44-45 of gestation, falling to 22.9 MOmega on days 59-69, and then to 13.1 MOmega , and finally, at term, to 17.7 MOmega . Application of antiprogesterones reduced the input resistance to the same levels at three different stages of pregnancy. They postulated that these events may be required for the synchronization and coordination of the electrical, metabolic, and contractile events of labor. Ramondt et al. (917) reported that estradiol administration during continuous infusion of naproxen (an inhibitor of PG synthesis) increased the area occupied by gap junctions and improved the coordination of myometrial activity. Thus the formation of myometrial gap junctions that is induced by estradiol is not mediated via PG.

As mentioned above, the gap junction is composed of connexins. The connexins are a gene family of at least 12 members, but in vascular SMC, only two members have been found, namely, Cx43 and Cx40 (297, 768, 1173). Connexin-43, but not Cx40, has been localized to the SM layer of large vessels, whereas Cx40 and Cx43 are both clearly present in the SM layer of some resistance vessels and in endothelial cells (665-667, 1103). Moore and Burt (768) reported that the connexin mRNA expressed in the rat mesenteric artery was that related to Cx43. They reported a unitary conductance of 75 pS and a junctional conductance of 11.8 nS. The connexin in the pig coronary artery was also related to Cx43, although the unitary conductance was somewhat smaller (59 pS) and the junctional conductance larger (20.5 nS). In contrast, in the human coronary artery, the mRNA expressed Cx40, and two unitary conductances were obtained (51 and 107 pS), the junctional conductance being 14 nS. Moreover, cultured cells from the aorta of the rat (A7r5) expressed both Cx43 and Cx40 and showed three different unitary conductances (70, 108, and 141 pS) and a junctional conductance of 11 nS. In arterioles, cell-to-cell coupling differed between SMC and endothelial cells (769), and connexin-specific antisense oligodeoxynucleotides blocked the expression of gap junction channels (769). Phosphorylation of connexins by protein kinase A (PKA) or by protein kinase C (PKC), which is activated by 5-hydroxytryptamine (5-HT, serotonin) and by dopamine and other agonists, can apparently modify the unitary conductance and voltage dependency of the gap junction (773, 926).

B) PASSIVE MEMBRANE CHARACTERISTICS. To aid the determination of the characteristic constants of each membrane, various electrical models have been proposed, e.g., the leaky condenser model and cable models (1-dimensional, 2-dimensional, and cable model for limited length). In VSM tissues, the one-dimensional model has been successfully applied using the insulated partition stimulating method of Abe and Tomita (5) and Tomita (1099). Thus, if longitudinal muscles of the portal vein possess a cable property, as suggested by the presence of many gap junctions, the relationship between the time needed to reach the half-amplitude of the electrotonic potential and the distance from the stimulating partition should be linear and could be expressed by tau /2lambda , where tau  is the time constant of the membrane at erf-1 (measured value 420 ms) and lambda  is the space constant of the membrane (421, 553).

Toro (1101) measured the passive membrane properties of isolated cultured SMC from the caudal artery and vein using the microelectrode method. In their study, the characteristics of cells prepared from artery and vein, respectively, were as follows: resting membrane potential, -56 and -66 mV; input resistance (Rin), 590 and 450 MOmega ; membrane time constant (tau m), 19 and 19 ms; membrane capacity (Cm), 1.3 and 1.0 µF/cm2; and length (space) constant, lambda  (measured using limited-length cable theory), 900 and 1,300 µM. It is plausible that, because of the lack of a syncytial structure, the isolated cell may show a much higher membrane resistance and internal resistance than would a multicellular tissue.

1) GI tract. Using multicellular VSM tissues, many investigators have measured passive membrane properties from VSMC using the partition stimulating procedure of Abe and Tomita (5). In these calculations, internal resistance (Ri) is assumed to be 125-300 Omega ·cm (based on impedance measurements in the longitudinal direction in guinea pig taenia coli, where the Ri was estimated to be 100 Omega ·cm by Tomita (1096). In the GI tract, the lambda  and tau m values obtained for longitudinal and circular muscle layers of the guinea pig stomach (antral region) were as follows: 2.2 and 1.4 mm, and 130 and 180 ms, at membrane potentials of -58 and -54 mV, respectively. The calculated conduction velocities were 1.2 and 1.6 cm/s, respectively (636, 863). In longitudinal (taenia coli) and circular muscles of the guinea pig caecum, the corresponding values were as follows: 1.5 and 1.7 mm and 100 and 250 ms, at membrane potentials of -55 and -52 mV, with conduction velocities of 6 and 5.2 cm/s, respectively (495, 1095). In the longitudinal muscle layer of the guinea pig rectum, the values of lambda , tau m , and conduction velocity were 0.81 mm, 84 ms, and 4.4 mm/s, respectively (1279). In contrast, in the common bile duct of the guinea pig (longitudinal direction), values of 0.8 mm, 100 ms, -51 mV, and 0.5 cm/s were reported (220). Crist et al. (225) compared the passive and active membrane properties of the circular muscle from the proximal and distal parts of the esophagus of the opossum and found no differences between the proximal and distal sites in terms of any of these parameters.

The passive membrane properties of the longitudinal and circular muscle layers were compared in both the human and dog colon by Huizinga and Chow (441). The lambda  value for the circular muscle layer was longer (2.14 mm; short axis; 0.43 mm) and tau m value was shorter (160 ms) than the corresponding values for the longitudinal muscle (1.63 mm and 500-800 ms, respectively) in the human colon, but no difference between the two muscle layers was observed in the dog colon. It is clear that, in the two muscle layers, electrotonic coupling occurs, and it seems to be related to the features of the gap junctions in the human colon, but not in the dog colon in spite of the fact that gap junctions were scarce or absent in the longitudinal muscle layer. It is interesting that, in the longitudinal muscle layer of the guinea pig intestine, the lambda  and tau m values could be calculated from electrotonic potentials recorded at any given distance from the stimulating electrode, yet the density of gap junctions was sparse if assessed using electron microscopy (329, 331).

2) Myometrium. Myometrial cells possess a syncytial structure, and the passive membrane properties of this tissue have been determined under various conditions. The values obtained for the passive membrane characteristics have been as follows: tau m , 100-300 ms in rat, guinea pig, sheep, and human myometrium in midpregnancy; lambda , 1-3 mm in the same species. Kuriyama and Suzuki (632) reported that in the nonpregnant rat longitudinal myometrium, the values of the membrane potential, lambda , and tau m were -56 mV, 1.5 mm, and 128 ms, respectively. At the middle stage of gestation (11-15 days), the corresponding values, again in the longitudinal muscle layer, were -68 mV, 2.6 mm, and 180 ms, respectively. At 18-19 days of gestation, the values had changed to -64 mV, 2.6 mm, and 198 ms, respectively. On the last day and during parturition, they were -54 mV, 2.9 mm, and 228 ms, respectively, and finally, postpartum (6-10 h) they were -51 mV, 3.2 mm, and 241 ms, respectively. In midpregnancy in the circular muscle layer, lambda  was 1.0 mm (555). However, Parkington (882) reported that, in circular muscle cells of the guinea pig myometrium, the tau m value remained unchanged throughout pregnancy (days 30 and 60 of pregnancy and in labor) but that the values were higher in estrus (330 ms) than in diestrus (210 ms). In contrast, the lambda  value increased during the progress of gestation, but no change in this value was seen at term in comparison with the last stage of gestation. In the circular muscle layer in sheep, despite no change in the membrane potential during gestation or at term, the lambda  value increased progressively (~1 mm at 50 days of pregnancy, 2 mm at 140 days of pregnancy and 4 mm in labor, at 145 days). In contrast, the tau m value did not change during gestation (remained at 130 ms) but was markedly increased in labor (510 ms; Ref. 882). Parkington (882) therefore postulated that the onset of labor in the ewe is associated with rapid and drastic changes in both the passive and active properties of the circular muscle of the uterus. Presumably, a reduction in the Ri may contribute to these changes through modifications of cell-to-cell connections. In the guinea pig, although the lambda value increased during gestation, with no further change in labor, the values for tau m and the membrane potential remained unchanged throughout gestation and labor (881, 883). In the upper margin of the human myometrium (monolayer culture), Pressman et al. (904) measured passive membrane properties and showed that the resting membrane potential was -49 mV, the specific membrane resistance 6 kOmega ·cm2, and the specific membrane capacitance 1.6 µF/cm2. In the pregnant rat myometrium (18-19 days), Mollard et al. (766) found that in the longitudinal muscle layer the membrane potential was -54.5 mV, the action potential had an overshoot (+7.8 mV), the specific membrane resistance was 14.8 kOmega ·cm2, and the specific membrane capacitance was 2.3 µF/cm2. In the pregnant human myometrium, Inoue et al. (487) reported a lambda  value of ~1.0 mm and a tau m of 396 ms.

In the pregnant mouse myometrium, Osa (862) measured a conduction velocity of excitation that was 20 cm/s in the longitudinal direction and 0.7 cm/s in circular muscle cells. In the rat myometrium in late pregnancy, Miller et al. (744) reported that the conduction velocity of excitation was 9 cm/s before delivery, increasing to 11 cm/s during delivery.

3) Vascular SMC. If we look at vascular SMC, we find that the reported lambda  and tau m values for elastic and resistance vascular tissues vary from 1.0 to 2.0 mm and from 200 to 300 ms, respectively (e.g., guinea pig portal vein, Refs. 496, 631; rabbit abdominal aorta, Refs. 727, 728; guinea pig aorta, Ref. 621; rabbit pulmonary artery, Refs. 169, 170; rabbit carotid artery, Refs. 726, 730; canine coronary artery, Refs. 728, 731; monkey coronary artery, Ref. 731; rabbit aorta, Refs. 378, 729; for more details on vascular SM tissues, see Ref. 626). Because elastic and capacitive vessels do not produce action potentials under physiological conditions, gap junctions in these tissues may be more important for the transport of low-molecular-weight substances (including ions) between cells than for a propagation of excitation or for tightening the structure of the SM (for review, see Ref. 626).

4) Vas deferens. Most electrophysiological investigations on the vas deferens have been made because of an interest in the innervation or in the neurotransmitters involved, rather than in the function of the SMC of the vas deferens themselves. In the vas deferens of rodents, such as guinea pig, rat, and mouse, the muscle is arranged in three layers: outer and inner longitudinal layers with circular muscle in between. The morphological arrangement of these muscle layers is loose everywhere but looser in the epididymal regions than in the amplulla region (144, 146, 586, 1032). The passive membrane properties of the longitudinal muscle layer of the vasa deferentia of guinea pig and mouse have been investigated (77, 417, 732). For instance, in guinea pig vas deferens, Holman et al. (427) identified two cell populations, active and inactive cells, on the basis of spike generation induced by intracellular stimulation. The former had an membrane resistance of 10 kOmega ·cm2 and a Cm of 2.5 µF/cm2, whereas in the latter, the corresponding values were 1 kOmega ·cm2 and 3 µF/cm2. When the extracellular, rather than intracellular, stimulating technique was used, the corresponding values were 10 kOmega ·cm2 and 1 µF/cm2, respectively, and a tau m of 100 ms was obtained (1097). Thus the passive electrical characteristics of the guinea pig vas deferens were much the same as those observed in the guinea pig taenia coli. However, they were not the same as in mice; lambda  of the tissue was much shorter (nearly zero) in the mouse vas deferens, and no longitudinal propagation of excitation was recorded. Moreover, on membrane depolarization evoked by electrical stimulation, the maximum frequency of action potentials was different in the vasa deferentia of the two species; it was 1-2 Hz in the guinea pig but 30 Hz in the mouse (and rat) (316, 427). It is of interest that Holman et al. (427) reported that electrotonic potentials elicited by extracellular stimulation spread in a longitudinal direction in the guinea pig vas deferens but that there was no such longitudinal spread in the mouse vas deferens. Interestingly, in the guinea pig, rat, and mouse, the cells were electrically quiescent, whereas in rabbit and human, the cells were spontaneously active, the cells of the former group being more sparsely innervated than those of to the latter. Friel (304) measured the passive electrical properties of the rat vas deferens using the whole cell voltage-clamp technique. In freshly cultured cells, the membrane potential was -37 mV (a much lower value than that measured by the microelectrode method from the intact tissue, -58 to -65 mV, Ref. 625; -67 mV, Ref. 1005). Their Rin was 2.9 GOmega , their C was 37 pF, and their tau m was 0.1 s. After denervation, spontaneous activity was seen in the vasa deferentia of the guinea pig and the rat, and it has also been reported that denervation, by sectioning the hypogastric nerves, led to an accelerated propagation of excitation, presumably because of an increase in the number of tight junctions (994). Not only female reproductive organs, but also male reproductive structures, such as the vas deferens, are modulated by hormones. For example, automaticity and conductivity were increased after castration in male guinea pig, and such increases were suppressed by treatment with testosterone (993).

B. Action Potentials Generated in VSMC

1. Spike potentials

Some VSMC generate spontaneous spike potentials (e.g., longitudinal SMC of the pyloric region of the stomach, ileum, and taenia coli, pyloric region of the ureter, myometrium, and portal vein in all experimental animals tested). However, some VSMC are electrically quiescent (longitudinal SMC in the stomach fundus, SMC of conduit and capacitive vessels, and the trachea). Quiescent SMC generate electrical activity on field stimulation of peripheral nerves; these include SMC in the mesenteric arteries and vasa deferentia (for review, see Refs. 106, 426, 625). In the trachea of many species, such as guinea pig, dog, and cow, peripheral nerve stimulation generates a slow potential change without spike generation. However, after treatment with tetraethylammonium (TEA), 4-AP, or procaine, oscillations or spike potentials can be recorded, after direct or indirect (nerve) stimulation, from canine and bovine tracheas (467, 497, 571). It is known that, in spontaneously active VSMC, the amplitude of the spike potentials recorded is irregular (except in a few examples, such as the estrogen-dominated, or at-term, rat myometrium; Ref. 625), and an overshoot potential is not consistently observed. However, after treatment with TEA or 4-AP, spikes of regular amplitude with an overshoot can be recorded from intestinal and vascular SMC (625). In quiescent VSMC, such as those in the canine trachea, the Ca2+ antagonist-sensitive L-type Ca2+ current could be recorded after treatment with TEA or 4-AP. This implies that, under normal conditions, a large outward K+ current (Ca2+ sensitive and delayed rectifying) may mask the inward Ca2+ current and prevent the generation of spike potentials (625, 785). However, in bovine and human iris dilator SMC, high concentrations of K+ solution produced a contraction that could be blocked by atropine. Presumably, the high K+ depolarized nerve terminals so that they released ACh, which then produced a contraction. Alternatively, in this case as well as in guinea pig and porcine coronary arteries and canine and guinea pig pulmonary arteries, in the presence of TEA or 4-AP, the SMC generated only graded action potentials; Refs. 169, 464, 625). Therefore, it is probable that the density of channel distribution and its ability to carry charge via voltage-operated (-dependent) Ca2+ channels (VOCC) is low in these tissues.

Figure 1 shows various types of action potentials recorded from longitudinal and circular myometrial cells of rats under different physiological conditions (pregnancy, at delivery, and post partum) and after treatments with estradiol and progesterone. In the nonpregnant rat, mouse, and guinea pig myometriums, circular muscle produced slow potential changes with or without initial spike. In the nonpregnant conditions, longitudinal muscle produced bursts of spikes with variable quiescent intervals. In the rat myometrium, at the middle stage of gestation (10th-15th day of pregnancy), circular muscle generated only a slow plateau potential with or without spike potentials, as gestation progressed, the amplitude of the plateau became smaller and either bursts of spikes of irregular amplitude or slow oscillations occurred on the plateau potential; at term, bursts of spikes of uniformal amplitude masked the generation of the plateau (882). In contrast, in the rat longitudinal muscle layer, in the middle stage of gestation, bursts of spikes with irregular amplitude occurred on a small plateau potential. However, as gestation progressed to term, the amplitude of the plateau potential became much smaller, and bursts of spikes of regular amplitude were superimposed on it at regular intervals. Much the same pattern of electrical activity as that observed in the last stage of gestation can be induced during the middle stage of gestation by treatment with estradiol (3-4 days, 0.1 mg/day) without any marked change in the membrane potential (Fig. 1). On the other hand, when progesterone (2-3 days, 5 mg/day) was given on the 22nd day of gestation in the rat, SM membranes were hyperpolarized in both longitudinal and circular muscle cells. Moreover, cells of both layers showed a plateau potential of reduced amplitude, with spike discharges of irregular amplitudes superimposed on it. Furthermore, in the middle stage of gestation, the rate of rise in the spike recorded from rat and mice myometriums was greater than that observed in the last stage of gestion and, after treatment with progesterone, the rate of rise was increased more than after treatment with estrogen (with progesterone from ~10 to ~20 V/s; Ref. 621). In the mouse myometrium, much the same responses could be observed as in the rat myometrium both during gestation and also after separate treatment with estrogen or progesterone (834, 861, 864). When studying the effect of estrogens on the Ca2+ current in cultured SMC of the late pregnant rat myometrium, Yamamoto (1200) found that beta -estradiol inhibited the Ca2+ current, in a voltage-dependent manner, and shifted the steady-state inactivation curve toward more negative potential levels. A synthetic estrogen, diethylstilbestrol, also had actions on the Ca2+ current similar to those of beta -estradiol, at a lower concentration, may directly act on the surface membrane, and inhibited VOCC more potently than delayed rectifying K+ current.


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  		FIG. 1.   Spontaneous or induced electrical activities recorded from longitudinal (A) and circular (B) muscle cells during gestation, at delivery, postpartum (18 h after delivery), 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 from the 20th day of gestation. [From Kishikawa (577).]

In the pregnant rat myometrium, Miyoshi et al. (764) reported that the inward currents consisted of Ca2+ and Na+ currents, whereas the evoked outward current consisted of a fast TEA-insensitive current and a large TEA-sensitive delayed rectifying outward current. Mershon et al. (733) examined the hypothesis that parturition is associated with significant changes in the expression of the alpha 1-subunit of the VOCC at the mRNA or protein level. They concluded that the number of L-type Ca2+ channels, although large in the rat myometrium, does not change before, or at the onset of, myometrial contraction. Intriguingly, mRNA was markedly decreased at parturition. How changes in isoform expression occur during labor is not yet known, but multiple unknown factors probably contribute to the initiation of parturition. Figure 2 shows the effects of 17beta -estradiol on ionic currents in excised pregnant rat myometrium.


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  		FIG. 2.   Effects of 17beta -estradiol (10 µM) on Ca2+ and K+ currents in excised pregnant rat myometrium (longitudinal muscle at 19 days of gestation). A: control. B: 17beta -estradiol. Depolarization pulses to -30, -10, and 30 mV from a holding potential of -50 mV were applied (500 ms). (From K. Okabe, unpublished observations.)

Parturition is a complex process involving the interplay of several endocrine factors. Kishikawa (577) reported that in the rat, progesterone content rapidly decreased and estrogen content increased 1 day before delivery. Therefore, the ratio of progesterone to estrogen decreased rapidly on that day. The events occurring in the last stage of gestation and during delivery differed from those seen in the middle stage of gestation. At the end of gestation, a plateau potential was not apparent in the longitudinal muscle layer, and the amplitude of the plateau potential was reduced in the circular muscle, despite the increase in the estrogen content of the sera. In addition, the amplitude of the plateau potentials seen in both muscle cell types did not increase with estrogen treatment but did increase with progesterone treatment in the last stage of gestation.

In the pregnant rat myometrium, the placental region shows different electrical properties from the nonplacental region. Thus the membrane potential measured from the placental region showed a consistently higher value than that from the nonplacental region up to the last stage of gestation. Moreover, the conduction velocity of excitation was consistently low in the placental region (about one-tenth of that in the nonplacental region; days 7-22). Furthermore, propagation of excitation seems to be blocked from the cells of the nonplacental region to the placental region, but not vice versa. The shape of the spike in the nonplacental region changed markedly during the progress of gestation, whereas the electrical activity of muscle cells in the placental region was not consistent. However, the electrical activity of muscle cells recorded at full term was much the same in both regions (550).

2. Pacemaker potentials and slow potential changes

A pacemaker potential (i.e., a gradual depolarization which, when it reaches a threshold level, triggers a spike potential) occurs in some VSMC. The first description of the typical pacemaker potential was published by Bozler (121), who used an extracellular electrode on the dog ureter. In pacemaker cells (sinoatrial node) in the heart, the current responsible for generating the pacemaker potential has been called a "funny" current (If) or hyperpolarization-activated current (Ih; Refs. 828, 1204).

A) SLOW WAVE. In many VSMC, the membrane potential exhibits slow spontaneous changes in activity in the absence of any influence from nerve activity or endogenous substances [mostly examined in the presence of tetrodotoxin (TTX)]. Slow potential changes are defined here as those changes that occur very slowly up to -20 to -30 mV and exhibit repolarization also with a very slow time course. The total time course of such slow potentials exceeds 1 s, and they occur in the GI tract, urinary bladder, myometrium, and oviduct. In some cells, these slow potential changes evoke a spike or spikes when the depolarization exceeds threshold, but in other cells, spike generation does not occur on slow potential changes (e.g., oviduct and stomach fundus). In other words, generation of a spike potential is not necessarily a function of the generation of slow potential changes.

The spontaneous slow potential change (slow wave) that occurs in the GI tract is also called a pacemaker potential, because this potential occurs without the generation of a prepotential. The features of this slow wave recorded from the GI tract have been reviewed extensively (954, 1066, 1100). Confusingly, the slow waves generated in the GI tract are resistant to Ca2+ channel blockers, but their amplitude and rate of rise are dependent on the Ca2+ concentration. To try to resolve this discrepancy, a role for the T-type VOCC in the generation of the slow wave might seem worthy of investigation. Unfortunately, however, the presence of the T-type Ca2+ channel in the GI tract has not yet been confirmed (L type alone found in newt stomach, Ref. 156; dog stomach, Ref. 985; rabbit ileum, Ref. 490; dog proximal colon, Ref. 1160). In this context, an analysis has been made of the features of the anomalous L-type Ca2+ channel and of the voltage dependency of the L-type Ca2+ channel. Huizinga et al. (442, 443) reported in canine colon that the generation of the slow potential change involves a non-L-type Ca2+ conductance. In freshly dispersed human uterine SMC, Young and Herndon-Smith (1217) suggested that the T-type current may be primarily involved with action potential transmission and the L-type current primarily with increasing [Ca2+]i by bulk Ca2+ transport. However, as yet, there is insufficient experimental data for us to determine the validity of this idea. In cat colon circular muscle, Venkova and Krier (1124) studied the feature together with the effects of norepinephrine on slow wave in the presence of TTX with atropine and concluded that the circular SMC near the submucosal border had a resting membrane potential of -76 mV and exhibited electrical slow waves (frequency, 4-6 Hz; upstroke potential, -40.7 mV; plateau potential, -44 mV, and duration, 4.9 s), and the circular SMC near the myenteric border had a resting membrane potential of -51 mV and did not exhibit electrical slow waves.

In fact, the mechanisms underlying the generation of slow waves in the GI tract have been investigated by many researchers over the past 30 years. Prosser's group (213) postulated that the periodic appearance of slow waves in intestinal SMC may correspond to oscillations in the activity of the electrogenic Na+-K+ pump. In contrast Tomita's group (835, 836) postulated that the slow waves in stomach circular muscle consist of two components: pacemaker activity, triggered by a non-voltage-dependent process, and a later, ionic conductance. Experiments using a multicellular double sucrose gap method with the partition stimulating procedure of Abe and Tomita (5) revealed that changes in the membrane potential modify the amplitude of the slow wave but not its frequency of generation (in circular muscle of guinea pig stomach, Ref. 629). Also in guinea pig stomach, Tsugeno et al. (1108) observed the effects of phosphodiesterase (PDE) inhibitors, such as caffeine, theophylline, IBMX (a nonselective PDE inhibitor, Ref. 996), and rolipram, as well as the effects of cAMP synthesis accelerators, such as isoproterenol, forskolin, dibutyryl cAMP (DBcAMP), and 8-bromo (8-BrcAMP). They reported that an increase in cAMP reduces slow wave frequency without changing either the membrane potential or the slow wave configuration. They concluded that an increase in intracellular cAMP inhibits the pacemaker activity of the slow waves. In their view, 1) an increase in K+ conductance is unlikely to be a major factor in this inhibition, and 2) slow waves appear to represent compound electrical activity in a groupof muscle cells, which is likely to be disintegrated by xanthine derivatives.

B) INTERSTITIAL CELLS OF CAJAL. Some elegant work has been carried out by Sanders' group (644, 645, 652, 906, 952, 953) and also by other investigators to clarify the origin of pacemaker activity in the GI tract in relation to the interstitial cells of Cajal (ICC). Their conclusion was that it actually lies in the activity of ICC. For example, Langton and co-workers (644, 645) reported that ICC isolated from the pacemaker region of the canine colon showed spontaneous electrical activity of an amplitude and with characteristics similar to those of the electrical slow waves recorded from muscle strips. They suggestted that the ion channels responsible for the electrical events are Ca2+ channels and Ca2+-dependent K+ channels. Publicover et al. (906), using freshly dispersed and cultured ICC prepared from the canine colon, reported that the spontaneous electrical activities they recorded were due to oscillations in [Ca2+]i that appeared to be secondary to the periodic influx of Ca2+. Lee and Sanders (652) further observed that when currents recorded from the ICC were compared with those recorded from SMC from the same region of circular muscle in the canine colon, depolarization of both types of cells initiated a transient inward current followed by a slowly inactivating outward current. When the inward current and Ca2+-dependent outward current were both blocked, the ICC then generated a more 4-AP-resistant outward current (rapidly activated and inactivated current; A-like current) than did circular SMC. Furthermore, the outward currents recorded from the ICC were deactivated at a more negative potential (half-inactivation at -53 mV) than were those recorded from circular SMC (-20 mV). Thus the features of the outward currents in the two types of cell are not exactly the same. Furthermore, when the inward currents were compared in terms of their current-voltage relationship (-100 mV to +20 mV), the ICC showed the presence of both L- and T-type VOCC (generation of a hump at potentials within the range -70 mV to about -40 mV), whereas SMC showed the L-type current alone. The function of ICC is generally agreed to be difficult to investigate in intact GI muscles because of the structural complexity of the tissues. Attempts to remove ICC surgically or chemically may fail to remove the cells in sufficient quantities or may damage adjacent SMC or neurons, or introduce nonspecific pharmacological effects (952). Sanders and Ozaki (956) thus postulated that this voltage-dependent T-type Ca2+ channel and a negative potential-activated outward current may combine to generate the slow potential changes seen in the GI tract. Recently, Sanders (953) reviewed recent views concerning roles of ICC as a pacemaker cell: 1) electrophysiological experiments on dissected muscle strips show that slow waves originate from specific sites. These pacemaker areas are populated by networks of ICC that make gap junction with SMC. Removal of pacemaker regions interferes with slow wave generation and propagation. 2) Chemicals that label ICC histochemically can damage ICC and abolish rhythmicity. 3) Isolated ICC are spontaneously active, and several voltage-dependent ion channel are expressed. 4) Interstitial cells of Cajal are innervated by enteric neurons, and they respond to neurotransmitters. Interstitial cells of Cajal may produce nitric oxide (NO) and amplify inhibitory neurotransmission. 5) Some classes of ICC fail to develop in animals with mutations in c-kit or stem cell factor, the ligand for c-kit receptors. Without ICC, electrical slow waves are absent.

Xue et al. (1189) studied the expression of NO synthase (NOS) in ICC of the canine proximal colon. They concluded that the submucosal component of the circular muscle layer, which lies along the surface of the septum in the myenteric region between the longitudinal and circular muscle layers, expresses a constitutive form of NOS. They therefore suggested that the synthesis of NO by ICC may influence electrical rhythmicity and may serve to amplify and even propagate enteric inhibitory neurotransmission.

C) C-KIT IN RELATION TO THE PACEMAKER ACTIVITY. Recently, the involvement of ICC in pacemaker activity was elegantly demonstrated (682, 960, 1094, 1101, 1155). They made use of c-kit, which is a proto-oncogene encoding a tyrosine kinase receptor, c-kit, a member of a platelet-derived growth factor (PDGF)/mast cell colony stimulating factor (CSF) receptor family, namely, the PDGF/CSF-1 receptor family (1212). Maeda et al. (682) reported that some of the cells in the SM layers of the developing intestine express c-kit, and blockade of its function for a few days postnatally by an antagonistic anti-c-kit monoclonal antibody (ACK2) results in a severe anomaly of gut movement, which in BALB/c mice produces a lethal paralysis. On this basis, they postulated that c-kit plays a crucial role in the development of a component of the pacemaker system that is required for the generation of automatic gut motility. Torihashi et al. (1100) confirmed the above view using cells that express c-kit-like immunoreactivity in the mouse small intestine and colon. Furthermore, in the colon they found cells that were labeled with ACK2 in the region of the myenteric plexus as well as deep muscular plexus of the small intestine and in the subserosa (in the myenteric plexus region), within the circular and longitudinal muscle layers, and along the submucosal surface of the circular SMC. The distribution of cells that expressed c-kit was the same as that of interstitial cells. A decrease in ICC was accompanied by a loss of electrical rhythmicity in the small intestine and reduced neural responses in the small bowel and colon. Ward et al. (1155) and Huizinga et al. (444) suggested that the c-kit function may be important in the development of the network of ICC, because W/WV and W/+ mutant mice (179, 346) had few ICC in the myenteric plexus region, and this was associated with a loss of slow electrical activity. Furthermore, when the excitability of c-kit-expressing cells in primary culture was examined using the nystatin-perforated patch-clamp procedure, the majority of c-kit-expressing cells showed rhythmic current waves with an amplitude and frequency of 263 nA and 2.3 cycles/min, respectively, when the membrane was depolarized to -40 mV. The reversal potential level of the rhythmic current was close to the Cl- equilibrium potential. These electrical rhythms were blocked by SITS, and such responses were not observed in tissue samples containing SMC alone. Therefore, they concluded that the intestinal c-kit expressing cells, but not SMC, exhibit a rhythmic Cl- current oscillation, which suggests that they participate in the pacemaker activity responsible for peristaltic gut movements. Slow wave was D600 (gallopamil) resistant, and thus differentiation between action potential and slow wave can be differentiated (Fig. 3). Moreover, Sato et al. (962) found that the effects of ACh, bradykinin, and PGF2alpha on the ACK2-treated murine intestinal tract was to increase both the contractile responses and receptor sensitivity of its longitudinal SMC and that ACh produced a larger depolarization in the SMC of the ileum in the ACK2-treated mice than in controls. Circular SM responses to these substances, as measured by changes in intraluminal pressure, were not altered by ACK2 treatment. They concluded that ICC play an important role not only in the development of the pacemaker system of the small intestine but also in the functional development of the contractile properties of intestinal SMC. Liu et al. (667) studied the relationship between the SR Ca2+ and periodicity of the biochemical clock to determine the pacemaker frequency using canine colon and reported that since cyclopiazonic acid (CPA) inhibited the SR Ca2+ pump and reduced the pacemaker frequency, the Ca2+ refilling cycle of the inositol trisphosphate (InsP3)-sensitive Ca2+ stores associated with the myoplasma membrane may determine the frequency of the pacemaker activity generated by the submuscular ICC-SM network.


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  		FIG. 3.   Action potentials recorded from small intestine of normal (c-kit positive) and W/Wv mutant (c-kit negative) mice. +/+, Preparation from wild-type mice having normal interstitial cell of Cajal network; W/Wv, preparation from W mutant mice. In normal small intestine, slow wave generation 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 Huizinga et al. (444).]

On pacemaker cells of proximal colon, Kobayashi's group (597, 795) described that, in the canine proximal colon, tissue near the submucosal surface of the circular muscle layer (innermost pacemaker muscle cells; P layer) produces spontaneous mechanical contractions, synchronized with electrical slow waves. The P-layer SMC have slow distinguishable features from the bulk circular SMC: 1) flattened and shorter shapes of the cell and nucleus, 2) numerous caveola on the cell surface and abundant mitochondria, and 3) frequent gap junction formation. Neither slow waves nor spontaneous mechanical rhythmicity was recorded from the submucosal or connective tissue or from the bulk circular muscle. They concluded that the inner sublayer characterized by special SMC with a delicate nerve plexus is essential for producng spontaneous activities of circular muscle coat in the canine proximal colon. Furthermore, they (795) reported that these specified SMC in the slow wave (pacemaker) was inhibitory innervated by nitrinergic nerves, and NG-nitro-L-arginine methyl ester (L-NAME) enhanced the spontaneous mechanical rhythms, whereas L-arginine suppressed the L-NAME-induced enhancement. In fact, the ICC and P-layer cells are the same cells.

To conclude, the pacemaker activity that generates the slow wave (slow potential change) may indeed prove to be originating from the ICC. Although the ICC was originally thought to be neural origin (914), they are now known to be specialized SMC and derived from the neural crest and to develop independently from the enteric nervous system (650). However, it is possible that the origin may differ in different tissues in the GI tract (on the basis of their biophysical features). More detailed information can be expected in the near future on pacemaker activity in the GI tract in relation to activities of individual ICC. At present, unsolved problems remain; for example, how is the activity of individual ICC cells coordinated (the role of the ICC network), how do the actions of ICC correlate with those of the myenteric plexus (not only by nitric oxide), how does the heterogeneous distribution of ICC in ileum and cecum relate to functional differences in the initiation of pacemaker activity, and so on. We also need more detailed data to clarify the features of pacemaker activity in spontaneously active SMC.

3. Plateau potentials

With the use of the microelectrode method, a plateau phase of high amplitude that is generated after a spike or spikes can be recorded in guinea pig and rabbit ureters (592, 593, 630), whereas a plateau phase of low amplitude can be recorded in the uterus (estrogen-treated longitudinal and circular muscle layers; Refs. 693, 834). Zhang et al. (1226) classified the cells of the guinea pig renal pelvis into three types on the basis of their electrical activity: 1) pacemaker cells (10% of the population), with a simple action potential comprising relatively slow rising and repolarizing phases triggered on top of a slowly developing prepotential; 2) driven cells (75%), with a complex action potential comprising a rapid initial spike, followed by a period of membrane oscillation and a plateau of 0.2-2 s duration; and 3) intermediate cells (15%) that fired action potentials with an initial rapid phase and then a long plateau phase. The generation of a spike potential is a prerequisite for plateau potential generation in the ureter, but not in the colon, where a plateau-potential-like sustained depolarization occurs with or without an initial spike. Furthermore, in the case of the myometrium, early pregnant circular muscle cells generated only slow potential changes, but later in the progress of gestation, there was a typical sustained depolarization after spike generation. At term, a burst of spikes of regular amplitude was superimposed on a low-amplitude plateau potential. In the myometrium, the duration of the plateau phase was reduced in high extracellular Ca2+ concentration. In guinea pig and cat ureters, in experiments using the microelectrode procedure, the generation of a plateau potential was inhibited when Na+ was removed from the extracellular medium (593, 630), and when extracellular Ca2+ was increased, the amplitude of the spike was enhanced, whereas the duration of the plateau potential was reduced (620). However, such responses induced by changes in Na+ or Ca2+ have yet to be confirmed using the patch-clamp procedure. Incidentally, the spike potential generated in the ureter is due to activation of VOCC.

In guinea pig ureter, the contribution made by the Ca2+-dependent K+ current to action potential formation appears to be relatively small, and a delayed rectifying K+ current is lacking; as a consequence, spike potentials appear repetitively at depolarized potential levels and produce a plateau phase by virtue of the slow inactivation of the Ca2+ current (466). These authors also postulated, with regard to the plateau potential and from the action of norepinephrine on the ureter, that Ca2+ channel activity is regulated by dual mechanisms: a Ca2+-dependent inhibition and a G protein-mediated potentiation. Under physiological conditions, Ca2+-inward and Ca2+-dependent K+ currents were both reduced by norepinephrine. However, the reduction in the Ca2+-dependent K+ outward current was much larger than that in the Ca2+ current, thus resulting in an increase in net inward current during the action potential plateau and a prolongation of the action potential's duration (788). In the ureter, Aickin et al. (17) reported the presence of Na+/Ca2+ exchange diffusion, and Kazarian et al. (558) suggested that Na+/Ca2+ exchange diffusion may contribute to the formation of a plateau potential. It is plausible that plateau formation in VSMC may not be due to activation of a specific ion channel but may be due to the concerted activation and inactivation of several ion channels.

    III. NEURAL CONTROL OF MEMBRANE ACTIVITIES IN VISCERAL SMOOTH MUSCLE CELLS
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Neurotransmitters (endogenous agonists) activate individual receptors distributed on VSMC, and this induces two main actions. Transmitters released from nerves 1) modify the ionic permeability directly, with or without activation of G proteins, and induce either depolarization or hyperpolarization of the membrane (ionotropic action) or 2) activate receptors leading to the synthesis of second messengers through the action of a G protein and enzymes such as phospholipase C (PLC) or adenylate and guanylate cyclases (metabotropic action). These synthesized second messengers phosphorylate various proteins in individual cells and also indirectly regulate ion-permeable channels.

A. Nerve Plexuses, Nerve Terminals, Varicosities, and Cotransmitters as Studied by Electrophysiological Methods

1. GI tract

A) MYENTERIC PLEXUS. In the myenteric plexus of the proximal colon of the guinea pig, Messenger et al. (735) classified plexus neurons electrophysiologically as either afterhyperpolarization-forming (AH) neurons (morphologically these cells typically had a large, oval soma and several long tapering processes that sent branches into many adjacent ganglia) or S neurons (these neurons were uniaxonal and many of them had axons ending in an expansion bulb in the myenteric plexus; they typically had broad, lamellar processes or short, spiny processes). The AH neurons were characterized electrophysiologically by the presence of a slow afterhyperpolarization after their action potential. Internodal strand stimulation in the intestine evoked the slow excitatory synaptic potential, but not a fast excitatory synaptic potential. The S neurons lacked a slow afterhyperpolarization, and internodal strand stimulation usually evoked fast excitatory synaptic potentials (although some neurons did show slow excitatory synaptic potentials). A subpopulation of AH neurons displayed a rhythmic oscillation in their membrane potential (which could trigger an action potential). The S neurons could be subdivided into tonic and phasic firing types. About 80% of the AH neurons examined by Messenger et al. (735) were immunoreactive for calbindin, as were 10% of S neurons. A further 17% of S neurons, but no AH neurons, were calretinin immunoreactive. Roughly equal proportions of S neurons had orally or anally directed projections. However, almost all the S neurons that were immunoreactive for calbindin or calretinin projected orally.

Sang and Young (959) studied the presence and colocalization of a range of putative transmitter in myenteric plexus of the small and large intestine of mouse. They classified the two major classes of circular muscle motoneurons in the small intestine: one class was characterized by the presence of NOS, vasoactive intestinal polypeptide (VIP), plus neuropeptide Y (NPY), and the second class contained calretinin plus substance P. There were seven classes of neurons that innervated myenteric ganglia; these contained NOS, VIP, NOS plus VIP, NPY, calretinin plus calbindin, substance P, or 5-HT. In the large intestine, there were five major classes of neurons that contained NOS, NOS plus VIP, GABA, substance P, or calretinine plus substance P, and seven major classes of neurons that innervated myenteric ganglia and contained NOS, VIP, calretinin plus calbindin, calretinin, substance P, GABA, or 5-HT. Distributions of galanin-containing neurons were also detected in the canine GI tract, i.e., galanin-like immunoreactive (GAL-Li) nerves and cell bodies were distributed in esophagus, stomach, and intestine. Thus it was estimated that GAL-Li nerves are distributed more widely in the canin enteric nervous system than previously recognized.

In the canine intestine, according to Furness et al. (317), VIP fibers run anally in the myenteric plexus of both small and large intestine, whereas substance P fibers run orally in the large intestine and both orally and anally in the small intestine. The innervation of the muscularis mucosa and mucosa by substance P- and VIP-containing fibers was not affected by myectomy or extrinsic denervation, and these structures are, therefore, likely to be innervated from nerve cells in the submucous ganglia. Furness et al. (315) reported that the myenteric plexus contains both excitatory and inhibitory nerves, of which the former contain ACh and tachykinins such as substance P and neurokinin A (NKA), whereas the latter contains VIP, peptide histidine methionine, and peptide histidine isoleucine (PHI). The myenteric reflex response to distension of the guinea pig small intestine has been extensively investigated, by means of electrophysiological methods, by Smith and co-workers (1000, 1001). Distension of the tissue generated excitatory junction potentials (EJP) in the oral region but inhibitory junction potentials (IJP) in the anal region. Crist et al. (221) studied circular SMC of the guinea pig distal ileum and found that intramural nerve stimulation produced a fast EJP and a late EJP at oral sites as well as an initial IJP and EJP and a prolonged hyperpolarization at anal sites; there was a also a late IJP, which was observed only at sites anal to the stimulus point, and a late EJP, which was observed only at sites oral to the stimulus point. The neural inhibition of circular and longitudinal colonic SMC (in relation to the submucosal ICC) have also been investigated, using electrophysiological procedures by Huizinga et al. (439). Endogenous alkaline phosphatase activity is located in enteric neurons (Dogiel type 1, but not type II) of the guinea pig small intestine, and this activity is associated with NOS-containing neurons. The latter includes inhibitory motoneurons to the circular muscles and anally directed interneurons to other myenteric and submucous neurons. Paterson and Indrakrishnan (891) investigated in opossum esophageal SMC whether or not the distension-induced esophageal peristaltic reflex involves a polysynaptic pathway. They concluded that this reflex is not polysynaptic, but rather involves long descending neurons that depend on NO as a final mediator.

B) SUBMUCOSAL PLEXUS. In the submucous plexus of the guinea pig small intestine, Bornstein et al. (114) have classified neurons according to their synaptic inputs. They studied 130 neurons and found that 82 cells were VIP reactive, of which 23 cells were NPY sensitive. Electrical stimulation of internodal strands evoked EJP (lasting 20-30 ms; fast response) in all 130 neurons. Most VIP-reactive neurons and some VIP-negative neurons, but no NPY-sensitive neurons, exhibited inhibitory synaptic potentials (ISP; amplitude in the range 2-30 mV, and lasting 150-1,500 ms). Most VIP-reactive neurons exhibited a slow excitatory synaptic potential (ESP) that could be evoked by a single stimulus, lasted 5-20 s, and was associated with an increase in input resistance. In contrast, in some NPY-reactive neurons, a single stimulus evoked an ESP lasting 500-1,500 ms with an associated fall in the input resistance. Therefore, Bornstein et al. (114) concluded that neurochemically distinct populations of submucous neurons can be distinguished physiologically on the basis of the particular combination of synaptic inputs they receive.

Evans et al. (281) also studied the guinea pig submucous plexus and reported that five distinct groups of cells could be distinguished. These were as follows. 1) S cells (neurons) with an inhibitory input (73 specimens = 61%) were immunoreactive to VIP and showed a Dogiel type III morphology; their varicosities and tufts of varicosities were observed surrounding other cell bodies as well as blood vessels. 2) S cells without an inhibitory input (19%), of which most were immunoreactive to NPY, also showed Dogiel type III morphology but possessed a shorter axonal projection. 3) AH cells (8%), which most likely contained substance P, lacked a synaptic input and exhibited Dogiel type II morphology; they branched more extensively than the S cells and also formed varicose tufts within other ganglia. 4) S-AH cells (5%), which combined the electrophysiological properties of the type of S cells with an inhibitory input (i.e., type 1 above) with those of AH cells, did not show consistent morphological characteristics. 5) Glial networks were typical and unusual networks. On this basis, they drew three conclusions: that VIP-containing S cells may act as interneurons, mediating a slow ESP; that NPY-containing S cells, which are known to be cholinergic, may play a role as cholinergic interneurons mediating the nicotinic fast ESP; and that AH neurons, too, may provide cholinergic innervation to other submucosal neurons in addition to their dual projection into the mucosa and the myenteric plexus.

From these observations, it is clear that both plexuses contain at least five different types of cells, on the basis of their electrophysiological properties, but that there are a greater number of morphological and chemical categories. After studying neurons and their projections in the proximal colon of the guinea pig, Messenger (734) reported that the myenteric and submucous plexuses were not uniform around the enteric circumference. At the mesenteric aspect of the colon, there was almost no longitudinal muscle, and the circular muscle was unusually thick and cordlike. In this region, there was no tertiary plexus of fibers, and the ganglia of the myenteric and submucous plexuses were elongated in the direction of the circular muscle. Neurons containing VIP, gastrin-releasing peptide, galanin, calbindin, or NADPH-diaphorase all lay in anally projecting pathways within the myenteric plexus, whereas enkephalin and somatostatin appeared in orally projecting nerve pathways. Few NPY neurons were present within the myenteric plexus of the proximal colon. The longitudinal muscle was innervated by fibers containing VIP, substance P, enkephalin, or NADPH diaphorase. The circular muscle was innervated by axons each containing one of the substances investigated, with the whole list, except NPY, being represented. Galanin, NPY, somatostatin, and VIP fibers, all dense in the mucosa, largely arose from nerve cell bodies in the submucous plexus. Messenger et al. (735) concluded that the chemically specified neural population in the proximal colon is more similar to that of the distal colon than that of the ileum, but that neurochemical and anatomic differences exist between the populations of the proximal and distal colon. Furthermore, it has been reported that, in the myenteric and submucous ganglia, the GABA fibers contained NOS activity (823).

2. Esophagus

In the canine and human esophageal sphincter, Tsumori et al. (1108a) found that VIP and NPY are synthesized in the same neuron, stored in the same axon terminal, and released together to act on sphincter SMC. Singaram et al. (993) reported that, in the human esophagus, most myenteric neurons (55%) were nitrergic. Most (96%) received terminals containing VIP, calcitonin gene-related peptide (CGRP) (80%), and galanin (56%). Furthermore, of neuronal somata, 14% contained VIP, whereas 10% contained galanin. They postulated that NO is an inhibitory (I) nonadrenergic noncholinergic (NANC) mediator and has a possible interactive role with the peptidergic system. In the feline extrahepatic biliary tree, the distributions of substance P, VIP, and cholecystokinin (CCK) have been investigated by Dahlstrand (227) using immunohistochemical procedures. Nerve terminals containing substance P were distributed to the SM layer and to acetylcholinesterase (AChE)-positive ganglion cells in the intrinsic plexuses. Substance P was also located in cell bodies of the intrinsic plexuses as well as in vagal axons, and VIP had a similar distribution. Using opossum esophagus, Christensen et a