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PHYSIOLOGICAL REVIEWS Vol. 78 No. 3 July 1998, pp. 811-920
Copyright ©1998 by the American Physiological Society
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. ClChannels
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
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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.
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. A. Resting Membrane Potential and Passive Membrane Properties of VSMC
1. Resting membrane potentials in VSMC
I. INTRODUCTION
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II. REGIONAL AND SPECIES DIFFERENCES IN THE RESTING MEMBRANE AND ACTION POTENTIALS IN VISCERAL SMOOTH MUSCLE CELLS
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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.
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 (17
-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. 17-Estradiol reduced the probability of occurrence of the transient outward current, and progesterone had only a slight effect on the currents. Treatment with 17-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 M
on days 44-45 of gestation, falling to 22.9 M on days 59-69, and then to 13.1 M, and finally, at term, to 17.7 M. 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 
/2
, where is the time constant of the membrane at erf-1 (measured value 420 ms) and 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 M; membrane time constant (m), 19 and 19 ms; membrane capacity (Cm), 1.3 and 1.0 µF/cm2; and length (space) constant, (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 ·cm (based on impedance measurements in the longitudinal direction in guinea pig taenia coli, where the Ri was estimated to be 100 ·cm by Tomita (1096). In the GI tract, the and 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 , 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 value for the circular muscle layer was longer (2.14 mm; short axis; 0.43 mm) and 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 and 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: m , 100-300 ms in rat, guinea pig, sheep, and human myometrium in midpregnancy; , 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, , and 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, was 1.0 mm (555). However, Parkington (882) reported that, in circular muscle cells of the guinea pig myometrium, the 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 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 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 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 value increased during gestation, with no further change in labor, the values for 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 k·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 k·cm2, and the specific membrane capacitance was 2.3 µF/cm2. In the pregnant human myometrium, Inoue et al. (487) reported a value of ~1.0 mm and a 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 and 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 k·cm2 and a Cm of 2.5 µF/cm2, whereas in the latter, the corresponding values were 1 k·cm2 and 3 µF/cm2. When the extracellular, rather than intracellular, stimulating technique was used, the corresponding values were 10 k·cm2 and 1 µF/cm2, respectively, and a 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; 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 G, their C was 37 pF, and their 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-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 -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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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
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 17-estradiol on ionic currents in excised pregnant rat myometrium.
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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 to20 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 PGF2
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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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 al. (197) investigated distributions of NADPH-diaphorase and concluded that, in the circular muscle layer, NADPH-diaphorase-positive fibers were most abundant at the cephalic end of esophageal body with a significant decline toward and through the esophagogastric sphincter. In the longitudinal muscle layer and the longitudinally oriented muscularis mucosae, these nerve fibers were most abundant at the esophagogastric sphincter, with a significant decline toward and through the striated-smooth muscle junction. Constitutional nitric oxide synthase (cNOS) immunoreactivity colocalized with NADPH-diaphrase activity, and CGRP-Li were distributed like the NADPH-diaphorase-positive fibers. They further described that fibers stained for immunoreactivity to the VIP, galanin, and substance P showed no clear differences in distribution along the esophagus in any of the muscle layer.3. Airway
In trachea of sheep, Cocoran (192) investigated distributions of nerve fibers containing CGRP, VIP, NPY, substance P, and the catecholamine (CA) / enzyme maker dopamine-hydroxylase (DBH) and concluded that moderate to large numbers of CGRP-Li nerve fibers were present in all parts of the trachea. Substance P nerve fibers had a similar distribution to CGRP, but they were absent from epithelial cells and only small numbers of fibers were present in other areas. Moderate numbers of VIP-Li and NPY-Li were present in SMC. Large numbers of DBH-Li nerve fibers were present in the SMC, and they had a similar distribution to NPY. The presence of both NPY and DBH in most DBH-Li nerve fibers was established in the SMC and dense interconnecting network. In plexus of the ferret trachea, Dey et al. (245) studied neuroanatomic features and concluded that 1) most cholinergic nerve do not contain VIP, NOS, or substance P; 2) cholinergic neurons are predominantly located in the longitudinal trunk ganglion; 3) VIP, NOS, and substance P are predominantly located in the superficial muscular plexus ganglia; and 4) nerve terminals containing exclusively substance P, suggesting possible sensory origin, are closely associated with some neurons in the plexus. In human airway, Fischer and Hoffman (296) investigated NOS-containing fibers and concluded that the innervation density of airway SMC by NOS-containing nerve fibers decreased significantly from trachea to large-diameter bronchi to small-diameter bronchi, whereas NOS-containing nerve fibers were completely absent from bronchioli. Colocalization of NOS with VIP but not with substance P was frequent in these nerve fibers.
B. Features of Varicosities Deduced From Electrophysiological Investigations
1. Features of varicosities
The nerves innervating all VSMC carry many varicosities on their peripheral portions; however, a distribution of one varicosity per cell is not necessary because of the syncytial structure of SMC. These varicosities in arteries and veins are ~2 µm long, 1 µm in diameter, and are spaced at 5- to 10-µm intervals. The terminal branches of each individual fiber bear several hundred varicosities, and the number of varicosities varies with the size of the vessel. Varicosities contain two types of dense-cored vesicles: small dense-cored vesicles (600-1,000 Å) may contain norepinephrine (NE) and ATP, and large dense-cored vesicles (1,200-1,500 Å) may contain NE, ATP, and neuropeptides, such as CGRP (241), NPY, or VIP (32, 260). Luff and McLachlan (674) studied the sympathetic neuromuscular innervation of arterioles (diameter 50 µm) in the guinea pig ileum and found that 13% of the varicosities made contact with SMC and that most of these (92 or 82% in 2 preparations) formed junctions. Iontophoretically applied NE and nerve stimulation produce much the same depolarization, in terms of shape. Stjärne (1036), who studied the release of neurotransmitters in the rat tail artery, estimated that the average varicosity contained 25 large dense-cored vesicles and 500 small dense-cored vesicles whose contents are released by exocytosis. In the former case, release was from "random sites," and in the latter from a "preferred release site" on the varicosity membrane (1037). Stjärne et al. (1037) noted that the nerve impulse is propagated along to the terminal at 0.5 m/s, invades all the varicosites, and activates the voltage-dependent N-type Ca2+ channel. Within the terminal, it travels only at ~0.5 mm/s, and release of transmitter may require Ca2+ with a permissive factor, "x." The ATP released in this way acts locally and probably only on postjunctional SMC, whereas the released NE acts both on postjunctional SMC and on the prejunctional nerve terminal (uptake of NE and activations of2-adrenoceptors occurs at nerve terminals). Recently, Stjärne and Stjärne (1038) reviewed the geometry, kinetics, and plasticity of release and clearance of ATP and NE, as well as their roles in neurogenic contraction in the vas deferens of the guinea pig and mouse and in the rat tail artery. They again emphasized their hypothesis ("the string model") about the quantal release of ATP and NE: 1) most sites ignore the nerve impulse and only a few (<1%) release a single quantum of ATP and NE, 2) the probability of monoquantal release is extremely nonuniform, but 3) there is a high probability from sites on "active strings," and 4) an impulse train causes repeated quantal release from these sites. Concerning exocytosis, they proposed that the coincident presence of at least two factors, Ca2+ and specific cytosolic proteins, may be required to move a "fusion clamp," form a "fusion complex," and trigger exocytosis of a sympathetic transmitter quantum and that the availability of these proteins may regulate the probability of release.
The morphology and electrophysiology of the varicosities distributed in mouse vas deferens have been elegantly investigated by Bennett's group (77, 546, 646-648). Using the fluorescent dye 3,3-diethyloxardicarbocyanine iodide [DiOC2(5)] or Faglu fluorescence (catecholamine staining), they visualized varicosities and catecholamine distribution while simultaneously recording excitatory junctional currents (EJC). For this they used a small microelectrode (4-6 µm; placed over 1-3 varicosities) and a large-diameter microelectrode (20-50 µm; placed over 3-7 varicosities), each placed on the same nerve fiber. The size of the varicosities and the intervaricosity distance obtained using DiOC2(5) were 1.09 and 5.53 µm, respectively. With the use of Faglu fluorescence, corresponding values were 1.05 and 5.12 µm, respectively. They further found that the mean and variance of the evoked EJC was similar to that of the spontaneous EJC, suggesting that a given varicosity secreted at most one quantum of transmitter(s) on arrival of a nerve impulse. Furthermore, they found that the mean quantal content of the EJC declined by over threefold along the length of a single sympathetic nerve terminal between adjacent sets of varicosities. Moreover, they described close-contact varicosities (~50 nm from the muscle) and loose-contact varicosities (at a greater distance). They interpreted these findings in relation to the generation and shape of EJP; thus 1) an EJP may result from transmitter release from close-contact (formed intermittent fast component) or loose-contact (formed nonintermittent slow component) varicosities, and 2) each of the varicosities could secrete a quantum of transmitter with a particular probability, after a delay that is characteristic for that varicosity (77).
Klemm (585) reported on the ultrastructure of the projections to the longitudinal SMC of the guinea pig ileum from nerves of the teriary plexus. It was found that there were two different types of neuromuscular junction; two-thirds of the junctions had many vesicles aggregated toward the area of junctional contact. Some 20% of these junctions had prejunctional membrane specializations. The remaining junctions were smaller than others usually found and covered a small area of membrane and contained only a few small vesicles; prejunctional membrane specializations were not found on these junction. Thus it was estimated from these observations and other physiological experiments that nearly released transmitters activate a different subset of receptors to externally applied transmitter substances.
Gabella (330) studied on the structural relations between nerve fibers and SMC in the urinary bladder of the rat, i.e., upon penetrating into the musculature, the nerve bundles branch repeatedly, and almost all turn into single fibers; their axons become varicose, and the separation between axonal membrane and SMC membrane is reduced to tens of nanometers. Varocosities contain mostly of the agranular type of vesicles. Terminal varicosities are often devoid of Schwann cell sheath and lie closes to a muscle cell (gap is often reduced to ~10 nm). Intervaricose segments vary in length and diameter, with the narrowest ones accompanying the more clear-cut varicosities. Some intervaricose segments are as small as 50 nm in diameter. Intracellular recordings were made from intramural neurons in the urinary bladder of guinea pig (383). From these procedures, the firing patterns were classified into two groups: prolonged firing of action potentials (tonic type; 86%) and one to three action potentials (phasic type) by depolarization of nerves. Single action potentials were followed by fast hyperpolarization, and the repetitive firing of action potentials was followed by delayed, slow hyperpolarization, which were diminished by 4-AP and Ca2+-free and high-Mg2+ solution. Electrical stimulation of nerve fiber tract evoked fast EJP. Presumably, two types of excitatory neurons may contribute in this tissue.
Because the features of synaptic vesicles incorporated in nerve terminals have already been extensively reviewed (52, 157, 561, 920, 965), here we do not intend to describe the features of synaptic vesicle biogenesis, docking, fusion, and exocytosis in relation to biochemical processes.
2. Presence of cotransmitters in nerves distributed on VSMC
Until about 30 years ago, it was thought that most VSMC are innervated by ACh-containing (cholinergic) and/or NE-containing (adrenergic) nerves. After pioneering investigations carried out by Burnstock (142, 148, 149), it became apparent that cotransmitters were also released from these nerves. The physiological roles of cotransmitters in adrenergic, cholinergic, I-NANC, and excitatory (E)-NANC nerves have been extensively investigated by many workers. For example, in vascular tissues and vasa deferentia, NE and ATP are released together. However, for the generation of an EJP after peripheral adrenergic nerve stimulation, ATP, rather than NE, seems to be the main transmitter in these nerves and, thus "NANC purinergic nerve" would seem a more suitable name for them. On the other hand, for the mechanical response, NE seems to play the major role, and not ATP. Thus, on that basis, they should be termed "adrenergic nerves." In contrast, in the GI tract, ATP seems to be the I-NANC transmitter in the guinea pig taenia coli (137) and jejunum (50, 51, 385), and this substance may or may not be involved as a cotransmitter with NE. In the GI tract, a single field stimulus evoked an apamin-sensitive inhibitory junction potential (IJP), whereas NE overflowing from the myenteric plexus on repetitive field stimulation may generate a slow hyperpolarization of the SMC membranes through-adrenoceptor stimulation (50, 51, 155). In SMC of the guinea pig ileum, Bauer and Kuriyama (50, 51) concluded, on the basis of electrophysiological and pharmacological procedures, that E-NANC fibers are more densely distributed in the terminal than in the proximal region, whereas in the case of I-NANC fibers, the situation is the reverse. Circular muscle cells are, however, homogeneously innervated by I-NANC nerves.
In SMC of the guinea pig urinary bladder, Hashitani and Suzuki (389) reported that nerve stimulation releases ACh and VIP, and the former produces contraction without changes in the membrane potential, whereas the latter generates the EJP that triggers an action potential and thus elicits contraction.
In the airway SMC of many species, a dual adrenergic and cholinergic innervation in many species has been reported, but the distribution of adrenergic nerves is not consistent. For example, adrenergic nerves are found in all parts of the canine airway system (886) but not in human bronchi (893). Recently, the densest form of innervation, cholinergic excitatory fibers, has been found to contain not only ACh but also I-NANC substances such as VIP and NO. The presence of I-NANC nerves had been postulated in airway SMC from the pharmacological effects on either electrical or mechanical responses of adrenergic and cholinergic receptor blockers (guinea pig, Refs. 203, 930, 931; cat, Ref. 498; pig, Ref. 760; baboon, Ref. 740). Recently, the heterogeneous inhibitory actions of NO and VIP in cat central and peripheral airways were described by Takahashi et al. (1069). Ward et al. (1154) observed the distribution of human I-NANC bronchodilator and NO-immunoreactive nerves and concluded that I-NANC neural relaxation appears inhomogeneously in airway system because of a decrease in the density of nitrergic innervation in the peripheral region. These observations may indicate that neither the amount nor the sites of transmitter release in the airway SM system exhibits a homogeneous distribution.
C. Multitransmitter Release Inferred From the Generation of EJP and IJP and Features of Postjunctional Receptors
1. EJP and IJP recorded from VSMC
Since 1959, pioneering electrophysiological investigations involving the recording of EJP and IJP from vasa deferentia, GI tract, and vascular tissues have been carried out by Australian groups such as those of Burnstock and Holman (150) and now by Hirst's group and Bennett's group. Field or transmural peripheral nerve stimulation evokes EJP (EJC) or IJP (IJC) in some VSMC, but not in all. The clearest excitatory potential changes (depolarization), especially EJP, were recorded from resistance vascular tissues, vas deferens, urinary bladder, or iris sphincter, as were the clearest inhibitory potential changes. The EJP was first recorded by Burnstock and Holman (150) from guinea pig vas deferens, and the IJP was first recorded from the GI tract (for review, see Refs. 142, 143, 145, 416, 615). For example, from guinea pig, rat, and rabbit mesenteric arteries, and from rabbit ear and rat tail arteries, EJP and spontaneously generated miniature EJP (sometimes depolarization exceeded 15 mV) were recorded. A mutual relationship between spontaneously generated EJP and evoked EJP has been inferred from studies of the properties of varicosities. Compared with that seen in the rabbit mesenteric artery, the EJP evoked by perivascular nerve stimulaton in the rabbit mesenteric vein exhibited a slower rising phase; however, in the guinea pig mesenteric vein, repetitive stimulation evoked only a slow fused depolarization (1057). The slow nature of the responses in the latter two cases may be explained by a lack of, or a low-density distribution of, P2x receptors or by the presence of larger numbers of loose-contact varicosities than of close-contact varicosities at peripheral nerve terminals (77). In the canine basilar artery, perivascular nerve stimulation evoked EJP with a prolonged smooth hyperpolarization. Repetitive stimulation fused the hyperpolarizations, but within several seconds, the fused hyperpolarization depolarized went back to the resting level because of a depression of the response. Both types of potential changes were blocked by TTX, but the hyperpolarization was not blocked by apamin or by-adrenoceptor blockers (308). In the spontaneously active guinea pig portal vein, perivascular nerve stimulation failed to evoke any depolarization of the membrane. In the rabbit ear artery (535), histograms plotting the generation of spontaneous EJP (recorded by the microelectrode method) show a skew distribution pattern in most cases, but in a few penetrations, a bell-shaped distribution pattern was observed (628). In mesenteric and tail arteries, and in the vas deferens, EJP show facilitation when the stimulus frequency exceeds 0.1 Hz, and when the depolarization reaches threshold, a spike potential is superimposed on it. The m of the falling phase of the EJP recorded from the guinea pig mesenteric artery was ~200 ms (at 35°C), whereas that of the resting membrane was ~130 ms (and was 0.8 mm in a longitudinal direction). Therefore, the decay of the EJP may not solely reflect a passive decay of the EJC. However, the decay of a spontaneous (miniature) EJP may mostly depend on the passive properties of the membane (m).
In the urinary bladder of most experimental animals, and humans, Brading and Inoue (122) concluded that there is a dual purinergic (ATP) and cholinergic (ACh) excitatory innervation. The former depolarized SMC membranes and increased the spike frequency (EJP-forming), whereas in most species, ACh did not modify the membrane potential, yet still produced contraction. However, in some species, a small depolarization was seen after the purinergic EJP (218). This response may reflect ATP-NE cotransmission in vascular SMC.
In the GI tract, Komori and Suzuki (602), who studied the circular muscle of the guinea pig stomach, reported that in the presence of guanethidine, transmural nerve stimulation evoked an atropine-sensitive EJP (reversal potential of 18 mV) in the fundus region and an apamin-sensitive IJP in the antrum region (reversal potential of 89 mV). After additional application of atropine to the bath, an IJP was evoked by transmural stimulation in both regions. After application of AChE inhibitors, such as physostigmine or neostigmine, cholinergic stimulation evoked an enhanced EJP and a subsequently produced depolarization of the membrane in the fundus region. In contrast, in the antrum region, the amplitude of the slow potential changes was further enhanced. Experiments with exogenously applied ACh showed that the threshold concentration for the depolarization of the membrane was 1,000 times higher in the antrum region (1 µM) than in the fundus region (1 nM). Thus whether an EJP or IJP is generated in a given region could depend on its ACh sensitivity. In SMC of the guinea pig stomach fundus, transmural nerve stimulation would normally evoke a cholinergic EJP, but in the presence of atropine, it would produce a NANC-IJP. Suramin enhanced EJP amplitude and inhibited the IJP with no significant effect on the membrane potential. Ohno and co-workers (839, 840) postulated a possible involvement of ATP in the generation of the NANC-IJP.
A) CATECHOLAMINE RECEPTORS AND RELATED SUBSTANCES. The 1-receptor is subdivided into 1A , 1B , 1C , and 1D (although 1C = 1D), and the presence has been claimed of 1L and 1N . The 2-receptor is subdivided into 2A , 2B , and 2C , and the -adrenoceptor is further subclassified into 1-, 2-, and 3-adrenoceptors (273).
1) -Adrenoceptors. When studying the actions of NE via 1A- and 2A-adrenoceptors in rat portal vein myocytes, Macrez-Lepretre et al. (680) tried to identify the type of PLC involved. They found that an inhibitor of PL-PLC, U-73122, inhibited the release of Ca2+ from the store sites induced by activation of the 1A-adrenoceptor related InsP3 production, whereas an inactive analog, U-73343, had no effect on the NE-induced release of Ca2+ from its stores. In contrast, both U-73122 and U-73343 inhibited the L-type Ca2+ channel. An inhibitor of phosphatidylcholine (PC)-PLC, called D-609, had no direct inhibitory effect on the L-type Ca2+ channel, but it inhibited the 2A-induced stimulation of Ca2+ channels, which had already been shown to be independent of phosphatidylinositol hydrolysis. They therefore postulated that the 2A-adrenoceptor activates a PC-PLC in vascular myocytes. However, it must be said that D-609 had other sites of action as it blocked both NE- and caffeine-induced Ca2+ release from the stores.
The presence of various 1-adrenoceptor subtypes has been described in vascular SMC. However, in the rabbit thoracic aorta, only 1B-adrenoceptors (antagonist, AH 11110A) and 1L-adrenoceptors (antagonist, JTH-601) have been identified, whereas in the rat thoracic aorta, the 1A- (antagonist, SNAP-5089), 1B-, and 1D-adrenoceptors (antagonist, BMY-7378) have been found. In human vas deferens, it was estimated that 1A-adrenoceptors contribute for mechanical responses induced by adrenoceptor agonists (320). It is not yet clear, however, what role is played by 1A- and 1D-adrenoceptors (132, 187, 788, 849).
Concerning actions of NE on VOCC in vascular SMC, Benham and Tsien (76) reported that NE increase the L-type Ca2+ channel of the rat mesenteric artery, and this action was enhanced by GTP. It was also reported using the same tissue by Nelson et al. (810) that NE shifted the Ca2+ activating potential level (threshold) to more negative potential level and enhanced the L-type Ca2+ current. Loirand et al. (671) observed the effects of NE on the rat portal vein that this agent enhanced the fast component of VOCC, and this action was further enhanced by guanosine 5'-O-(3-thiotriphosphate) (GTP
S). In SMC prepared from cultured rat aorta, Pacaud et al. (874) observed that NE enhanced the dihydropyridine (DHP)-insensitive (presumably T-type) Ca2+ channel, and NE inhibited the DHP-sensitive Ca2+ channel via an increase in the cytosolic Ca2+. However, Droogmans et al. (252) reported that NE inhibited L-type Ca2+ current. Further investigations are required to clarify this controversial observation.
When discussing vascular SMC, Stjärne et al. (1038) concluded that released ATP generates an EJP, whereas NE generates a slow and small depolarization via the 2-adrenoceptor. In contrast, the 1-adrenoceptor does not appreciably change the membrane potential (627), as also concluded by Nally and Muir (802), working on the rabbit saphenous artery. Moreover, Cheung and Fujioka (193) found that in the rat saphenous vein, perivascular nerve stimulation produced an EJP and sustained depolarization, and the latter, slow depolarization was due to activation of the 2-adrenoceptor, not the 1-adrenoceptor. In the rat tail artery, exogenously applied NE markedly depolarized the membrane, and this depolarization was blocked by the 2-adrenoceptor antagonist yohimbine, but not by the 1-adrenoceptor blocker prazosin.
In the dilator muscle of the rat iris, Hill et al. (409) reported that there are three different muscle cell types. In two of these, which were assumed to be myoepithelial cells, sympathetic nerve stimulation evoked EJP (with a long latency of several seconds and duration of several seconds). These EJP were blocked by prazosin and were insensitive to yohimbine; furthermore, they were blocked by chloroethylcholine. Thus the receptor involved was characterized as the 1B-adrenoceptor. Furthermore, EJP evoked by sympathetic nerve stimulation were blocked by reducing the external concentration of Cl. Consequently, they concluded that EJP are generated in this tissue by activation of Ca2+-dependent Cl channels through activation of the 1B-receptor and that Ca2+ released from the SR and subsequently synthesized InsP3 are involved in the generation of EJP and of contractions.
A mutual relationship between the hypogastric and pelvic nerves has been demonstrated, in as much as sympathetic nerves may end on ganglion cells in the pelvic plexus and act inhibitory nerves (238). In response to hypogastric nerve stimulation, the urinary bladder shows slight relaxation through the actions of mainly the 2-adrenoceptor, and in many species, the urethra shows biphasic response (contraction and subsequent relaxation) evoked via 2- and 2-receptors, respectively. However, in the rabbit urethra, the 2-adrenoceptor was more dominant (29). In the urinary bladder of dog, rabbit, and guinea pig, and after treatment with 1- and 2-adrenoceptor blockers, pelvic nerve stimulation produced a large EJP, presumably because of the release of ATP.
2) -Adrenoceptors. The presence of the -adrenoceptor in VSMC was described many years ago; activation of this receptor was found to hyperpolarize SMC membranes, probably via the synthesis of cAMP in the guinea pig vas deferens (138, 622). Moreover, Kuriyama and Makita (627) reported that isoproterenol (Isop) increased the amplitude of EJP through an action at a prejunctional receptor. Thus the -adrenoceptor may have both a pre- and postjunctional distribution. In urogenital organs, Isop and terbutamine each blocked the response evoked by nerve field stimulation, and these inhibitory responses were reserved by the application of sotalol. In the GI tract, -adrenoceptors have been identified in the guinea pig ileum (365), and this receptor is thought to be the 3-adrenoceptor (1118). In airway SMC, there is a sparse innervation by sympathetic (adrenergic) nerves, and -adrenoceptors are more sparsely distributed than -adrenoceptors (44, 45). Consequently, the contraction induced by -agonists was weak, being only 10-20% of the maximum response elicited by ACh or histamine (590). Both 1- and 2-adrenoceptors appear to be distributed in airway SMC, but with different densities (422). The canine trachealis, however, seems to be unusual in that the 2-receptor is apparently more densely distributed than the 1-adrenoceptor (44). The distributions of -adrenoceptors in airway SMC have been elucidated in several species, and the receptor most responsible for airway dilation is the 2-adrenoceptor. However, the terminal airways and alveoli contain a mixture of 2- and 1-receptors (355). Carstairs et al. (167) reported that the density of 2-adrenoceptors in airway SMC increases progressively from the trachea to the small bronchioles. It was reported in airway SMC that PKC markedly enhanced the relaxation induced by -adrenoceptor activation. On the other hand, vagal (cholinergic) nerves densely innervate airway SMC (39-44, 930). In tracheal SMC, Ca2+-dependent maxi K+ activity was induced by cAMP synthesized in response to epinephrine or forskolin (616-618). Kume et al. (617) further investigated the action of cAMP on the maxi-K+ channel and concluded that cAMP and the -subunit of the related G protein act independently, and also that channel openings they induced differed in terms of their kinetics. Therefore, they suggested that -adrenoceptor stimulation of maxi-K+ channel activity and relaxation of tone in this tissue both occur, in part, independently of cAMP formation. Recently, Kotlikoff and Kamm (609) reviewed recent data of -adrenergic relaxation of airway SMC; -adrenoceptor stimulation results in the opening of Ca2+-dependent maxi-K+ channels. Coupling between receptor and channel occurs by phosphorylation-dependent and -independent mechanisms. Furthermore, -adrenoceptor agonists can decrease the Ca2+ sensitivity of contractile proteins. This desensitization does not result from the phosphorylation of myosin light-chain kinase (MLCK) but may be associated with the activation of a MLCK phosphatase.
B) MUSCARINIC RECEPTORS AND RELATED SUBSTANCES. Muscarinic receptors sensitive to ACh are subdivided into five subtypes (M1-M5) (43). For example, the guinea pig airway contains M2 and M3 receptors, whereas, in humans, the receptor subtype is M3 and the presumed subtype is M3 in the bovine airway (445, 664, 688). However, in both human and porcine airway SMC, use of a cDNA probe has revealed the presence of the mRNAs for both M2 and M3 receptors (688, 689). Activation of the M receptors expressed by m1 , m3 , and m5 genes stimulated phosphatidylinositol turnover, whereas activation of those expressed by m2 and m4 gene decreased cAMP synthesis. In cardiac muscle, a K+ current was modified by a receptor expressed by the m2 gene and acting via by protein kinase A (89). Thus m1 and m3 genes were associated with activation of Gq/11 (synthesis of InsP3), and m2 and m4 genes corresponded with activations of Gi/o (inhibition of synthesis of cAMP, direct activations of K+ channels, and other actions) (111, 131, 250, 446). In addition, the presence has been deduced of the M1 receptor in parasympathetic ganglia and of the M2 receptor on parasympathetic nerve endings (688).
Selective antagonists for muscarinic receptors have been introduced. These include the following: for the M1 receptor, pirenzepine and telenzepine; for the M2 receptor, methoctramine and himbacine; for M3 , hexahydrosiladifenidol and p-hurohexahydrosiladifenidol; and for M4 , tropicanide. A selective antagonist has not yet been introduced for the M5 receptor.
Acetylcholine, in many VSMC, is known to act as an excitatory transmitter. However, in the lingual artery, Bevan and Brayden (81) reported that peripheral nerve stimulation induced relaxation and hyperpolarization of the membrane and that these inhibitory responses were blocked by atropine. This phenomenon indicates that cholinergic inhibitory nerves may be distributed in some vascular tissues.
1) Urogenital organs. Cholinergic innervations have been identified in the vasa deferentia of various species. Fukushi and Wakui (311-313) and Wakui and Inomata (1143, 1144), who studied the guinea pig vas deferens, reported that during muscarinic blockade, high concentrations of nicotinic agonists produced a depolarization. However, the interpretation of this result is not simple because it is known that many small ganglionic cells in peripheral hypogastric nerves are distributed in this tissue. To establish the presence of the nicotinic receptor in SMC in vasa deferentia would require more detailed experiments. It is clear that ACh in vasa deferentia activates the postjunctional M2 receptor and produces excitatory effects, but this action on SMC is weak by comparison with those of NE and ATP released from the adrenergic innervation. On the other hand, ACh also activates the M1 receptor distributed on adrenergic prejunctional nerve terminals and negatively controls the release of NE (301).
Brading and Mostwin (123) recorded the electrical and mechanical responses of guinea pig urinary bladder SMC to nerve stimulation and observed that the mechanical response to excitatory nerves is biphasic. The early response to the transmitter, which takes the form of EJP and an evoked spike (E-NANC), is attenuated by the desensitization of purinoceptors. The late response is mediated through muscarinic receptors, involves little membrane depolarization, and is unaffected by desensitization of purinoceptors.
2) Airway. In the airways, by means of an autoradiographic procedure (using [3H]QNB), the presence has been demonstrated of M2 and M3 receptors in the guinea pig, and of the M3 receptor in humans (687). However, using a molecular biological pocedure, Maeda et al. (681) and Mak and co-workers (688, 689) have reported that the mRNA for both M2 and M3 receptors are present in porcine and human airway SMC. Furthermore, with the use of pharmacological procedures, involving antagonists of M2 (AF-DX 116, methoctramine) and M3 (hexahydrosiladifenoidol) receptors, it was concluded that the M3 receptor possesses a more potent action than the M2 receptor on human, guinea pig, and bovine airway SMC (936, 937, 1087). It is thought in the rabbit airway SMC that M3 receptors have been associated with SM contraction and M2 receptors have been implicated in Gi-coupled inhibition of adenylate cyclase. Schramm et al. (970) reported that there exist inherent age-dependent differences in both the airway relaxant responsiveness to -adrenoceptor stimulation and muscarinic functional antagonism of -adrenergic relaxation, and the latter is attributed to mechanisms other than ontogenetic alteration in M2 receptor function or Gi protein expression in maturing rabbit tracheal SMC.
3) GI tract. The role of muscarinic receptors in the parasympathetic control of colonic activity in the rabbit was investigated in vitro by Blanquet et al. (92), who found that EJP evoked by parasympathetic (vagal) nerves were blocked by 4-diphenyl-acetoxy-N-methylpiperidine methobromide (4-DAMP) but facilitated by methoctramine. In contrast, IJP were blocked by methoctamine but unaffected by 4-DAMP. These results indicate the involvement of two different receptors. The former action may be mediated by activation of the M3 receptor and the latter by the M2 receptor. In guinea pig ileum and trachea, interaction between M2 (inhibition of adenylate cyclase) and -adrenoceptor (stimulation of adenylate cyclase) has been estimated. In guinea pig esophageal SMC, lack of interaction between M2 receptors and -adrenoceptors but an interaction between M3 receptor and -adrenoceptor has been suggested (1164).
4) Iris. Dual innervation by adrenergic and cholinergic nerves occurs in SMC and is examplified by the case of the iris sphincter and dilator muscles. The mammalian iris dilator seems to be innervated by excitatory adrenergic nerves, but investigators have revealed that this tissue also receives a cholinergic inhibitory innervation in cats (266), rats (813), and cattle (1063). In human iris dilator, Yoshitomi et al. (1215), confirming that this tissue is innervated by adrenergic excitatory and cholinergic inhibitory nerves, further suggested that the cholinergic inhibitory innervation may support cholinergic meiosis, as reported in cattle and rats (803, 1063). Apparently, in the monkey, iris dilator muscle receives 75% adrenergic and 25% cholinergic nerve terminals (829). Furthermore, in the bovine iris dilator muscle, excess extracellular K+ produced a biphasic response that was blocked by simultaneous treatment with phentolamine and atropine (when phentolamine was applied alone, it produced a slight relaxation). Thus the contraction induced by extracellular K+ is due to joint activations of adrenergic and cholinergic nerves and is not due to a direct action on SMC through an activation of VOCC (1162, 1163). Furthermore, Yoshitomi and Ito (1216) reported a double reciprocal innervation in dog iris sphincter and dilator muscles. Thus, in both sphincter and dilator muscles, nerve stimulation evoked an initial phasic contraction followed by relaxation. Atropine selectively suppressed the phasic contraction of the sphincter and the relaxation of the dilator muscle, whereas guanethidine selectively blocked the relaxation of the sphincter and the contraction of the dilator. In the rat iris sphincter, after treatment with phentolamine, an initial contraction followed by a relaxation was evoked by transmural nerve stimulation or ACh (1197). Furthermore, Watanabe's group (703) concluded that only relaxation of the dilator muscle is related to activation of the pertussis toxin (PTX)-sensitive G protein (Gi) and that contraction in the dilator muscle may be induced via M3 or M3-like receptor activation, as also obtained by Yamahara et al. (1197). The contraction induced by ACh may be mediated by PTX-insensitive G protein.
In iris sphincter and dilator muscles, at least four different M receptors seem to be distributed (M1-M4). The M2 receptor plays a role, on prejunctional cholinergic nerves, in the autoregulation of transmitter release (103, 104), whereas the M3 receptor predominantly mediates contraction through activation of PLC, leading to the synthesis of InsP3 and diacylglycerol (DAG). In the bovine iris sphincter, the mRNA encoding the M3 receptor is the predominant form found (429). Direct evidence in support of the idea that the M5 receptor subtype mRNA and the corresponding M receptor subtype has not been found in the iris sphincter, but Bognar et al. (103) reported that a receptor other than the M1-M4 receptors contributes to the mediation of the contraction in the rabbit iris sphincter.
Abdel-Latif (2) recently reviewed cross talk between cAMP and the polyphosphoinositide on signaling cascade in iris sphincter. Namely, cholinergically synthesized InsP3 and adrenergically synthesized cAMP showed a mutual interaction through metabolic path; cAMP inhibits PLC activation and stimulates InsP3 3-kinase activity, both of which can result in 1) reduction in InsP3 concentration and 2) reduction in InsP3-dependent Ca2+ mobilization, which may lead to muscle relaxation. In addition to InsP3-induced Ca2+ mobilization, changes in [Ca2+]i are the result of the interplay of many processes that may also serve as potential sites for cAMP inhibition.
C) PURINOCEPTORS AND THEIR RELATED SUBSTANCES. In VSMC, receptors sensitive to purine derivatives, purinoceptors, have been classified as either PI (P1) or PII (P2) receptors. The former, P1 , has been subdivided into A1 , A2A , and A2B , and the latter, P2 , has been subdivided into P2t , P2x , P2z (these 3 types are ionotropic receptors), P2u , and P2y (these 2 types are G protein-coupled types). Brake et al. (125) claimed that the sequences of their cloned P2x receptor predicted a subunit structure (P2x1-P2x3) resembling that of voltage-insensitive cation channels.
1) Urogenital organs. The innervation of the vas deferens in many species (guinea pig, rat, mouse, rabbit, dog, and human) has been investigated in detail using histochemical, electron microscopic, and pharmacological procedures, leading to prediction about the distribution of adrenergic and cholinergic terminals and receptors. There is a relatively dense innervation by adrenergic nerves in vasa deferentia, with the 1-adrenoceptor being located postjunctionally on SMC and the 2-adrenoceptor being predominantly prejunctional on nerve terminals (212). The former produces excitatory responses, and the latter produces inhibitory responses through inhibition of NE release. However, EJP and mechanical responses evoked by hypogastric nerves are not completely blocked by -adrenoceptor blockers, and the presence of a cotransmitter, ATP, was deduced. Burnstock (146, 147) hypothesized that ATP is stored with NE in the small-cored vesicles seen in adrenergic nerves, and also together with ACh in the small dense-agranulated vesicles seen in cholinergic nerves. This means that no purinergic fibers distributed to the vasa deferentia and that the role played by this substance is a cotransmitter (1037, 1038). However, Wagner and Sjöstrand (1139) reported that excitatory responses in human and dog vasa deferentia (in which the innervations are rather sparse in comparison with that of rodents) are blocked by -adrenergic blocking agents. Moreover, after denervation, the response induced by hypogastric nerve stimulation in the guinea pig vas deferens was blocked by phentolamine. Cheung (191) studied the guinea pig vas deferens to try to elucidate the role of the nerve action potential in synaptic transmission; they concluded that transmitter release is intimately related to presynaptic nerve activity.
During excitatory transmission in sympathetic nerves, the cotransmitters ATP and NE may act cooperatively under physiological conditions. In the isolated vas deferens of rat, rabbit, and guinea pig, Sneddon (1005) and Sneddon and Machaly (1007) reported regional variation with respect to purinergic and adrenergic responses: 1) NE was more potent in producing contraction in epididymal segments than in prostatic segments, whereas 2) ATP and ,-methylene ATP were more potent in producing contraction of prostatic segments than epididymal segments. Furthermore, they noted that in guinea pig vas deferens, the resting membrane potential was greater in SMC in the prostatic than in the epididymal region. Excitatory junction potentials in both regions were of similar amplitude and were almost abolished by the P2x purinoceptor antagonist suramin, but phentolamine had no effect. Furthermore, distributions of the SR (i.e., Ca2+ homeostasis) differed by regions in vas deferens (1128). McLaren et al. (720) measured the membrane potential and the contraction elicited in guinea pig vas deferens in the presence of the P2x purinergic antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). This agent produced a small potentiation of the phasic contraction induced by field stimulation (at 0.1 µM; suggesting a predominantly purinergic excitatory component), but high concentrations (3-10 µM) produced inhibition. Furthermore, PPADS inhibited contractions evoked by ,-methylene ATP. On the other hand, this agent had no effect on the tonic component of contraction (predominantly noradrenergic component). At 10 µM, PPADS reduced the amplitude of EJP and depolarized the membrane. However, the PPADS-induced depolarization was not modified by suramin. This implies that ATP and NE released from nerve terminals may play different roles in excitatory transmission. With regard to the action of PPADS, Piper and Hollingsworth (899) reported that this agent antagonized the action of ATP in the isolated guinea pig vas deferens but had no effect on responses to ATP in the guinea pig taenia coli, indicating that this antagonist induced selective for P2x over P2y purinoceptors.
It has been reported in rabbit prostate SMC (972) that this muscle tissue contains two muscle bundles: one forms the capsule and the other runs longitudinally in the outermost layer of the prostate. The former was contracted by exogenously applied NE and the latter by ACh. In fact, the NE-sensitive muscle receives an adrenergic excitatory and a NANC inhibitory innervation, whereas the ACh-sensitive muscle receives a cholinergic excitatory and a NANC inhibitory innervation. In the rat vas deferens, Kurz et al. (634) reported that electrical- and agonist-evoked ATP overflow correlated well with the contractile responses.
In the urinary bladder, the idea that ATP is an excitatory cotransmitter (142) has been supported electrophysiologically (158, 209, 305). It is possibly released with ACh from the parasympathetic (pelvic) nerves (1084). Furthermore, pelvic nerves, which innervate the lower urinary tract, contain many neuropeptides, such as NPY, VIP, somatostatin, substance P, and CGRP. However, none may be a primary neurotransmitter in the detrusor; rather, they may act as potent modulators of sympathetic and parasympathetic transmission (432).
2) Vascular tissues. There is considerable variation between tissues in terms of the receptors they carry. For example, the rabbit saphenous artery has been found to have approximately equal contributions from P2x purinoceptors and 1-adrenoceptors, whereas the ileocolic artery has mainly P2x purinoceptors with a smaller contribution from 1-adrenoceptors, the plantaris vein has mainly 2-adrenoceptors with a small contribution from P2x purinoceptors and 1-adrenoceptors, and the saphenous vein has only 1-adrenoceptors (626, 679). In the human saphenous vein, Rump and Kugelgen (945) reported that 1- and 2-adrenoceptors and ATP all contribute to the generation of vasoconstriction. In contrast, Evans and Suprenant (283), studying guinea pig submucosal arterioles, thought that postjunctional responses to sympathetic nerve stimulation were mediated solely through the action on the P2x receptor of ATP. In fact, Starke et al. (1028), reviewing the contribution of ATP to neurogenic vasoconstriction, concluded that it varies from 0 (in the rabbit pulmonary artery) to 100% (in the rabbit small jejunal artery) (915). The picture is further complicated by the assertion by Maynard et al. (712) that chronic electrical stimulation of the greater auricular nerve supplying the rabbit central ear artery may lead to a selective alteration in the postjunctional P2x purinoceptor, whereas the effects mediated by postjunctional 1-adrenoceptors remain unchanged. These accumulated results from work on vascular SMC indicate that the relative contribution made by the receptors for ATP (P2x receptor) and NE (1- and 2-adrenoceptors), with respect to the production of excitation and contraction, varies by tissue and also by species (as reported by Starke et al., Ref. 1028). Recent work in the rat portal vein by Pacaud et al. (876) has shown that ATP, in the presence of gallopamil and at a holding potential of 60 mV, induces an inward current and increase [Ca2+]i (as measured using indo 1). On the basis of results obtained in various ionic environments and with various drugs, they concluded that ATP releases Ca2+ from the intracellular stores without involving InsP3 , but via a Ca2+-release mechanism activated by Ca2+ influx through an ATP-gated channel (see sect. V).
In SMC of the rabbit facial vein, perivascular nerve stimulation produced a biphasic response: a depolarization followed by a hyperpolarization. This hyperpolarization was blocked by a -adrenoceptor blocker, implying that NE activates a -adrenoceptor distributed postjunctionally. In the rabbit ear artery, ATP activated the P2x receptor and evoked a transient inward current (69, 72, 75), whereas in the portal vein, ATP activated P2x and P2y receptors (1184) and evoked a biphasic response. With the use of the whole cell voltage-clamp procedure, it has been found that activation of the P2x receptor by ATP produces a transient phasic inward current mediated via cation channels (mainly Na+ and Ca2+), whereas activation of the P2y receptor evokes a sustained tonic inward current that is mediated via nonselective cation channel.
3) GI tract. Crist et al. (221-223) studied guinea pig ileum and opossum esophageal circular muscles and reported that the electrical events elicited by nerve stimulation could be classified as one of four types: early EJP or IJP and late EJP or IJP. The late IJP is recorded only at sites anal to the stimulus, and the late EJP is recorded only at sites oral to the stimulus. A slow NANC-IJP was thought to be generated by a decrease in the Cl conductance (in the esophagus). Furthermore, in guinea pig ileum circular muscle, Crist et al. (224) reported that both ATP and VIP are inhibitory transmitters and that they may be the transmitters responsible for the fast IJP and the slow IJP, respectively. Bridgewater et al. (128) reported in the guinea pig taenia coli that reactive blue 2, a purinoceptor antagonist, attenuated the fast IJP, suggesting that the NANC inhibitory transmitter responsible for the fast IJP is an ATP. Using circular SMC of the guinea pig ileum, He and Goyal (391) found that field stimulation induced a fast IJP that was blocked by TTX, apamin, and ,-methylene ATP and, after application of apamin and substance P, a slow IJP that was blocked by TTX and NG-nitro-L-arginine (L-NNA). Consequently, they concluded that NO is not involved in the fast IJP (which was mediated by ATP). However, NO is involved in the slow IJP, which is actually mediated by VIP and NO acting in series. The hyperpolarizing effects of VIP and the slow IJP are both normally masked by an overlapping depolarization due to the concomitant release of substance P induced by the peptide VIP. In rat anococcygeus muscles, Byrne and Large (151) reported that field stimulation evoked three types of TTX-sensitive electrical responses, a fast EJP (latency <100 ms and time to peak of 300 ms), a slow EJP (latency several hundred milliseconds and time to peak 1-2 s), and an IJP. Iontophoretically applied ATP produced a fast EJP-type response (latency <30 ms and time to peak 150-300 ms), and ionotophoretically applied NE produced a slow EJP-type response (latency 470 ms and time to peak 860 ms). These responses are much the same as those observed in vascular tissues (626).
2. Heterogeneous distributions of receptors, especially on nitrergic receptors, estimated from IJP and hyperpolarization
In recent years, NO has come to be a popular candidate for the role of inhibitory substance in VSMC such as those of the GI tract, including the anococcygeus muscle, and those in vascular beds. It is also thought to act in the same way in airway and in other VSMC. A) GI TRACT. After studying the NANC-IJP in the GI tract, Sanders's and Boeckxstaens's groups (98-102) extensively reviewed its features in relation to NO and concluded that many of the criteria necessary for NO to be considered a neurotransmitter had been satisfied (956). In the canine proximal colon, Thornbury et al. (1091) demonstrated that NO and the NO carrier S-nitrosocysteine can mimic the hyperpolarization induced by nerve stimulation. Daziel et al. (236), using the same preparation, suggested that the NO synthetic processes is involved in I-NANC neurotransmission and that the NANC-IJP is generated by released NO. Ward and Sanders (1158, 1159) found that S-nitrosocysteine breaks down fast enough to cause an IJP-like hyperpolarization and that oxyhemoglobin (oxy-Hb) blocks the hyperpolarization response to NO, S-nitrosocysteine, and the NANC-IJP. They further noted that NO enhanced the open probability of Ca2+-activated K+ channels, which produce hyperpolarization in response to NANC neurotransmission in colonic muscle. In this tissue, their group also reported that the effects of NANC nerve stimulation and NO may be mediated by cGMP. In the canine proximal colon (between the pyloric sphincter and the sphincter of Oddi, and using preparations of circular muscles from near the myenteric and submucosal surfaces), Bayguinov et al. (55) reported that cells near the submucosal surface showed predominantly EJP, whereas those near the myenteric border showed predominantly IJP or biphasic responses (small EJP followed by IJP). The EJP were blocked by atropine, and the IJP were apamin sensitive. Furthermore, in the canine circular pyloric sphincter, Bayguinov and Sanders (53) reported that the apamin-sensitive IJP they recorded were attenuated by L-arginine derivatives (analogs) and that such inhibitions were reversed by L-arginine. Oxyhemoglobin partly blocked and the combined application of oxy-Hb and NG-monomethyl-L-arginine (L-NMMA) completely blocked IJP. Exogenously applied NO hyperpolarized the membrane, and this action was blocked by apamin. Osthaus and Galligan (87), who studied nerve-mediated relaxation in the guinea pig ileum, concluded from the actions of antagonists of NOS that inhibitory NANC-induced relaxation is due to the synthesis of NO. In the canine jejunum, Stark and co-workers (1025, 1026) also concluded that NO mediates NANC neural inhibition and may act as a I-NANC transmitter. However, Stark et al. (1025) found, in the human and canine jejunum (circular muscle), that nerve stimulation evoked a rapidly developed IJP followed by a late sustained hyperpolarization. Exogenously applied NO mimicked only the late response in the case of the human jejunum, whereupon nerve stimulation evoked an IJP that consisted of a fast hyperpolarization, alone. When the effect of NOS inhibitors (L-arginine derivatives) on these hyperpolarizations was tested, they were found to reduce the amplitude of the initial IJP in the canine jejunum but to reduce only the late sustained hyperpolarization in the human jejunum. Thus these authors concluded that NO mediates neural inhibition in the circular muscle of both the human and canine jejunums, but through different mechanisms. Serio et al. (975) thought that, in circular muscle of the rat proximal colon, while NO plays an important role in NANC-IJP generation, another mechanism, peptidergic in nature, is also involved. Presences of nitrergic inhibitory nerves in dog sphincter of Oddi have been elucidated (1077). In the GI tract, NO may contribute as a relaxing substance, but distributions of nitrergic nerves and NO vary markedly by species, strains, and regions, and also aging. For example, NO hyperpolarized the membrane in the dog proximal colon (236, 980, 1025, 1092) but not in the rat proximal colon (1055) and guinea pig gastric fundus (685). Nitric oxide increased the synthesis of cGMP in many region of GI tract and hyperpolarized and relaxed tissues, but in some cases, increased cGMP did not correlate with relaxation (1075). Using JCL Wistar (Wistar), SLC Wistar-ST (Wistar-ST), and Sprague-Dawley (SD) lines of rats (8 wk old), Hata and Takeuchi (390) compared differences in participation of NO in relaxant responses of the intestine among intestinal regions and strains, and also the effects of aging (4, 8, and 50 wk old) on NO-mediated relaxant responses of intestine of Wistar rat. They concluded that 1) in longitudinal SMC of jejunum, NO-mediated and L-arginine reversed relaxing component were 30% of the total relaxing response in Wistar-ST, 0% in Wistar, and 60% in SD rats; those in longitudinal SMC in rectum were 0% in Wistar-ST, 0% in Wistar, and 65% in SD rats, and in longitudinal SMC in proximal colon were 90% in Wistar-ST, 90% in Wistar, and 70% in SD rats. Thus differences in contribution of NO on the relaxing responses observed in these strains indicate that NO seems to play a dominant role in the proximal colon, but it becomes less in more oral region or more anal region of GI tract. 2) When the effects of aging were compared in longitudinal SMC in jejunum of Wistar rats, NO-mediated relaxing components were changed from 40% of the total relaxing response in 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 rectum were 0, 0, and 0%, respectively, and those changes in longitudinal SMC of proximal colon were 80, 90, and 30%, respectively. Thus age-dependent change in NO distribution are also apparent (390). Much the same age-dependent reduction in relaxing response has been reported in the rat stomach fundus (1002) and ileum (1003). In anococcygeus muscle, the involvement of NO is well documented (351, 354, 363). However, Gibson et al. (350) questioned whether nitrergic transmission simply involves release of NO. They detailed several possibilities: 1) NO is released attached to a carrier molecule, perhaps in the form of a nitrosothiol; 2) NO is released in a modified redox form; 3) NO is released as a free radical, but is protected within the neuroeffector junction by other substances that preferentially interact with scavenger molecules; and 4) NO is released as a free radical which, because of a rapid and unhindered rate of diffusion over a short distance, is less susceptible than exogenous NO to scavenger molecules. As yet, they concluded, there is insufficient experimental evidence to decide which, if any, of these explanations is correct. Bridgewater et al. (128), in the guinea pig taenia coli, recorded two distinct IJP (fast and slow) under treatment with hexamethonium, atropine,
-conotoxin GVIA, or apamin. That the ability of -conotoxin GVIA to selectively abolish the fast IJP, leaving the slow IJP intact, suggests that separate nerves are involved in mediating these responses.
B) ESOPHAGUS. Murray and co-workers (789, 790) reported that, in opossum esophageal SMC, NO activates the maxi-K+ (Ca2+-dependent) channel via cGMP-dependent G protein pathways. Moreover, using the same tissue, Cayabyab and Daniel (178) observed that the IJP evoked by NO was dependent on cGMP elevation and the activation of quinine- and apamin-sensitive K+ channels. Robertson et al. (936) have studied in vascular SMC that the PKG-mediated activation of the Ca2+-dependent K+ channel may be involved in relation with the action of NO and other nitrovasodilators. The presence of NADPH-diaphorase in the intramural plexuses of the esophagus of the cat and opossum has been detected by Fang and Christensen (285). Du et al. (254) and Conklin and co-workers (210, 211) also concluded that NO, or a NO-like compound, probably mediates nerve-induced hyperpolarization and also that this hyperpolarization is induced via cGMP. In opossum esophagus-body SMC and in canine intestinal circular SMC, Christinck et al. (199) postulated that the generation of NANC-IJP is due to release of NO as final mediator. Christinck et al. (198) further reported that the response in the former tissue was apamin insensitive, whereas in the latter it was partially sensitive to apamin. The L-arginine analog L-NAME abolished the IJP in both tissues. In opossum esophagus circular muscle, Cayabyab and Daniel (178) reported that the IJP evoked by electrical field stimulation and the hyperpolarization induced by NO donors, such as nitroprusside and 3-morpholinosydnimine hydrochloride, were both more strongly inhibited by quinine than by apamin, but that TEA, charybdotoxin, 4-AP, and glibenclamide had no effect on these hyperpolarizations. Thus the IJP evoked in the esophagus depends on cGMP elevation and is secondary to activation of quinine- and apamin-sensitive K+ channels. Hydroquinoline inhibited only the hyperpolarization induced by S-nitrosocysteine. Apamin partly inhibited IJP and the hyperpolarization induced by NO, but not the nitroprusside-induced hyperpolarization. In addition, 8-bromo-cGMP also produced hyperpolarization, and this hyperpolarization was not inhibited by apamin, TEA, or glyburide. Furthermore, Jury et al. (530) reported that NO release activates K+ outward currents in the opossum esophagus circular muscle, which may depend on Ca2+ release from the SR stores.
C) GENITAL ORGANS. In male genital organs, J. Gillespie's group (243, 246, 732, 733) detected and studied a relaxing substance released from genital and anococcygeus muscles. This substance was named "inhibitory factor" (IF). Inhibitory factor is soluble in water, is not an adenine nucleotide or a peptide, is inhibited by hemoglobin, and increases the synthesis of cGMP but not that of cAMP. It is now clear that these properties of IF are the same as those of endothelium-derived growth factor. The presence of cNOS has been demonstrated in male reproductive tissues, using immunohistochemical methods or NADPH-diaphorase staining [the activity of cNOS is dependent on Ca2+, calmodulin (CaM), and NADPH]. Thus cNOS has been found in nerves of the bovine and rat retractor penis (228, 977), in nerves innervating penile arteries and cavernosal SMC, and also endotheliums of arteries and cavernosal sinusoids (7, 141). Participation of NO in excitatory neurotransmission in rat vas deferens was also evident (1131). In a bovine retractor penis preparation, L-NMMA has been reported to have a partially agonistic action (392, 393; although it inhibited the actions of L-NNA and L-NAME, Refs. 699, 700). In the bovine retractor penis and in the penile artery, L-NNA blocked the relaxation induced by activation of NANC nerves, as it did in the human and rabbit corpus cavenosum (reviews in Refs. 31, 586). It seems likely, but not certain, that there is a relationship between penile erection and the action of I-NANC nerves. In the deep penile artery of the horse, Simonsen et al. (985) found that relaxation induced by NANC nerve stimulation involves NO or a NO-like substance released from nitrergic nerves. Papka et al. (877) looked at the source of NOS-containing nerves supplying the rat uterus and which other peptides might coexist with NO in these nerves. They concluded that NOS-1/NADPH-diaphorase reactivity coexisted with VIP and substance P in parasympathetic nerves, but not in tyrosine hydroxylase-1 neurons of pelvic parasympathetic ganglia. Nitric oxide synthase-containing nerves in the uterus are autonomic and sensory and could play significant roles.
It would be interesting to know whether the NO involved in nitrergic neurotransmission is continously released under resting conditions, or is released only during nerve excitation in a constitutive manner when the cytosolic Ca2+ is increased. In the rabbit anococcygeus muscle, Kasakov et al. (547) found that NO is continously released from nitrergic nerve terminals so as to maintain a tonic relaxation. Moreover, they found that NO is also released during short-term (1-60 s) and long-term (10-120 min) electrical field stimulation. This NO further relaxed the preparation and modulated sympathetic transmission. It was further reported by Shuttleworth et al. (982) that sustained nitrergic neurotransmission in enteric neurons is due to the synthesis of NO through functional recycling of citrulline, a coproduct of NO, to L-arginine (the precursor of NO).
D) IRIS. In the isolated rabbit iris sphincter muscle, Chuman et al. (200) observed the role of NO in the postjunctional regulation and concluded that cholinergic contraction is NO sensitive, whereas tachykinergic contraction is NO insensitive. Therefore, they estimated that, in rabbit, the cholinergic and tachychinergic responses have different features for the fine adjustment of the iris sphincter muscle tone.
E) URINARY TRACT. Ehren et al. (267) investigated the localization of cNOS activity in the human lower urinary tract and its correlation with neuroeffector responses; they concluded that in areas where a marked relaxation was elicited by nerve stimulation, there was a high NO activity. Thus NO seems to be the mediator for neurogenic dilation of the bladder neck and urethra during the micturition reflex. Almost the same conclusion in the same tissue have been reached by others (30).
F) AIRWAY. Recently Zhou et al. (1234) reported, in Chinese hamster tracheal SMC, using the cell-attached patch, that 8-(4-chlorophenylthio)-cGMP (an activator of PKG) enhanced the open-state probability of the Ca2+-dependent maxi-K+ channels. These actions induced by PKG were inhibited by the PKG inhibitor KT-5823 and protein phosphatase inhibitors (microcystin and okadic acid). The catalytic subunit of protein phosphatase 2A mimicked the effects of the PKG on the open probability in excised inside-out patches. Therefore, Zhou et al. (1234) concluded that activations of the maxi-K+ channels by PKG, but not by PKA, required the activity of protein phosphatase 2A. In cat airway SMC, Takahashi et al. (1069) reported that free radical NO and NO-containing compounds are involved in the L-NAME-sensitive NANC relaxation, but that only free radical NO has a prejunctional action.
Although NO is normally considered to have inhibitory actions, it has been reported that this substance may also act as an excitatory substance. Thus Barthó and Lefebvre (49) reported that, in the longitudinal layer of the guinea pig ileum, NO produced a moderate relaxation followed by a TTX-sensitive (partly atropine-sensitive, the remaining part being sensitive to substance P) aftercontraction ("off-contraction"; Refs. 47, 48). Much the same nitrergic off-contraction in response to electrical stimlation of I-NANC nerves has been reported in the opposum esophageal body and in the cat distal colon (1124). Furthermore, not only a rebound contraction, but a directly NO-induced contraction apparently occurs in the longitudinal muscle of the rat ileum and in the longitudinal muscle of the opossum esophagus (47, 48). In fact, Barthó and Lefebvre (49), working on the rat ileum, found that contraction occurred with a lower concentration of NO than did relaxation. This NO-induced contraction in the opossum is related to the synthesis of cGMP (as is the NO-induced relaxation), but this is apparently not the case in the rat. The NO-induced contraction is not related to the ryanodine-sensitive Ca2+ channel but is sensitive to blockers of the L-type Ca2+ channel. According to the same authors, primary contractions due to NO can also be observed in the rat whole ileum and in rat cecal longitudinal muscle, whereas a rebound contraction induced by NO can be seen in the rat descending, transverse, and sigmoid colon, as well as in cat ileal longitudinal muscle. Furthermore, in the muscle at the neck of the urinary bladder in the sheep, Thornbury and Peake (1090) and Thornbury et al. (1089) found that NO plays a role in poststimulation contraction (rebound contraction) and that this phenomenon depended more on release of Ca2+ from stores than on Ca2+ influx through L-type Ca2+ channels.
3. Relationship between NO-induced hyperpolarization and apamin
In the GI tract, most of the IJP generated in the GI tract are sensitive to the bee venom apamin; those evoked in the opposum esophagus (199) were resistant to apamin. It was suggested that the generation of IJP in the GI tract is due to activation of K+ channels (1098), and the target K+ channel for apamin was reported to be a small-conductance K+ channel (140, 517, 765, 1042). However, more recently, Thornbury et al. (1090) demonstrated that application of NO to canine colonic SMC enhanced the open probability of Ca2+-activated maxi-K+ channels. The release of NO has been demonstrated from inhibitory enteric neurons (127, 1156, 1157), and a TTX-sensitive release of NO can be induced by field stimulation in the GI tract: gastric fundus (240, 737), intestine (102), internal anal sphincter (181). In the canine proximal intestine and the pyloric sphincter (53-55), apamin blocked evoked IJP. This finding was supported by experiments carried out on the "knockout" mouse by Huang et al. (435), who bred mice that lacked the-NOS gene (knockout). They reported that -NOS expression and NADPH-diaphorase staining were absent in the mutant mice but that the presence of a very low level of residual catalytic activity could be detected. Thus other enzymes may also generate NO to a minor extent. However, these mutant mice did not show any histopathological abnormalities in the central nervous system, and the most evident effect of disrupting the neural NOS gene was the development of a grossly enlarged stomach, with hypertrophy of the pyloric sphincter and circular muscle layer. They considered that this phenotype resembles the human disorder known as infantile pyloristenosis.
In general, NO released from nerve terminals induces a slow relaxation in precontracted VSM tissues. The main inhibitory actions of NO on VSMC are thought to be due to the inhibitory action of cGMP on contractile proteins and to activation of K+ channels distributed on postjunctional cells, and also to inhibition of excitatory transmitter release from nerve terminals. In some tissues, such as the GI tract, this agent is thought to generate an IJP or a slow hyperpolarization. However, the evidence is not straightforward, in that the actions of NO antagonists and apamin on these two inhibitory electrical processes are not consistent. For example, in canine proximal colon and jejunal muscles, L-arginine antagonists (L-NNA or L-NMMA) partly, but not completely, blocked IJP, with this inhibition being reversed by L-arginine (1026, 1092, 1156, 1157). In SMC, it is thought that L-arginine antagonists block cGMP-activated Ca2+-dependent maxi-K+ channels (90, 1092), as also observed in studies of the actions of cAMP on the maxi-K+ channel (612). Furthermore, it was reported by Gerland and McPherson (347) that NO does not induce hyperpolarization of the membrane in the rat mesenteric artery. However, Kitamura et al. (580) found in the rat gastric fundus that NO-cysteine and sodium nitroprusside hyperpolarized the membrane and that these responses were sensitive to apamin (but not in cat airway, Ref. 526). Kitamura et al. (580) further postulated that NO may activate both apamin-sensitive and -insensitive K+ channels. Unfortunately, however, neither the apamin-sensitive K+ channel nor the apamin-insensitive, L-arginine antagonist-sensitive K+ channel has yet been identified (Fig. 4).
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4. Mutual relationship between NO and VIP
A mutual relationship between NO and VIP in the GI tract has been hypothesized. According to this hypothesis, during GI tract electrical stimulation with EJP suppressed, a fast large-amplitude IJP may be generated by ATP and a sustained low-amplitude hyperpolarization by VIP and NO (391, 1025, 1027, 1071). Thus VIP and NO would be functionally linked cotransmitters as well as postjunctional regulators of SM activity. The NO released from nerve terminals by Ca2+ after nerve activation would regulate VIP release and, in turn, VIP would regenerate NO by activating a NOS present in the target SMC, according to Murthy et al. (792). They postulated that NO, whose synthesis is triggered by influxes of Ca2+ through activated cNOS, activates the release of VIP at nerve terminals and that the released VIP activates two different receptors (VIP-specific receptor and VIP-preferring receptors). The former recognizes VIP and blocked pituitary adenylyl (adenylate) cyclase activating peptide (PACAP), but not PHI, whereas, the latter recognizes VIP, PHI, and PACAP. The former receptor is thought to synthesize cGMP through activation of NO via activation of a NOS that is tightly bound to CaM in the plasma membrane. The latter receptor is thought to synthesize cAMP through activation of a G protein. To test the truth of this hypothesis in the GI tract, we need the answer to many unsolved problems. For instance, Desai et al. (244) reported that NO, but not VIP, is a vagal inhibitory neurotransmitter of the guinea pig stomach. In airway SMC, VIP and NO are both thought to be inhibitory transmitters, but recently a general consensus has emerged that NO probably plays the more dominant role as a relaxant (66, 246, 642). The release of NO occurs independently of the presence of epithelial cells in airway SMC (1165). In equine airways, Yu et al. (1219) found that adrenergic innervation is limited to the cranial part of the trachea, whereas NO-containing I-NANC nerves supply both the trachea and the central bronchi. Furthermore, Ward et al. (1154) and Belvisi et al. (65), who studied the distribution of human I-NANC bronchodilator and NO-immunoreactive nerves, and also the effects of various NO-related agents in the main, proximal, and distal airways of isolated human airways, concluded that I-NANC nerves innervate most densely the distal region and less so the proximal region. It is thought that the synthesis of NO in airway SM tissue occurs in sensory nerves, endothelial cells, vascular and airway SMC, inflammatory cells, and the airway epithelium (805). Kannan and Johnson (542) investigated whether or not NO released from I-NANC nerves is involved in producing SMC relaxation through the action of synthesized cGMP in airway SMC. They concluded that NO-induced relaxation is due to activation of both charybdotoxin- and iberiotoxin-sensitive Ca2+-dependent K+ channels. In the human trachea, Belvisi et al. (65) and Ellis and Undem (271) demonstrated that NO completely relaxed the contraction evoked by electical field stimulation. On the other hand, Ito's group postulated that VIP and NO are distributed differently from each other in cat airway SMC and exert coordinated inhibitory actions on cholinergic excitatory transmission. In fact, in the guinea pig, administration of-chymotrypsin or incubation with VIP antiserum reduced the mechanical response evoked by electrical stimulation (by 20-70%; Refs. 270, 650), the remaining relaxation being abolished by NOS inhibitors (660, 1110). In the dog trachea, but less so in the cat, the amplitude of fused EJP was evoked by repetitive stimulation (20 Hz) increased in a stimulus frequency-dependent manner (379, 497). After treatment with VIP antiserum or with VIP antagonists, the EJP recorded from the cat trachea showed marked summation in response to repetitive field stimulation (1182). Moreover, in the same tissue, S-nitrosocysteine and VIP did not modify either the membrane potential or input resistance of SMC, but did relax the tissue (498, 526). From these accumulated results, Ito's group (497, 498) postulated that VIP and ACh probably coexist in the same nerves and act as cotransmitters with each other (39, 40, 247, 640), just as VIP and NO are thought to coexist in the same nerve terminals (216, 246). In airway SMC, excitatory cholinergic transmission may be regulated by two inhibitory substances (VIP and NO) released from the same excitatory vagal nerve fibers (526). In isolated stomach of the guinea pig, Desai et al. (244) investigated a possible link between NO and VIP in mediating relaxation induced by vagal stimulation. They concluded that NO has a neural origin and support NO, but not VIP, as a major neurotransmitter of vagally induced gastric relaxation. Ideas about the role of NO in bronchial hyperresponsiveness have been reviewed by Nijkamp and Folkert (815).
On the basis of electrophysiological experiments, it has been postulated by Hakoda and Ito (379) that in the dog penile artery and vein, VIP acts as an I-NANC substance; the EJP was inhibited by VIP, but not by ,-methylene ATP. Hakoda et al. (378), also using electrophysiological procedures, investigated the possible role of VIP as a cotransmitter with ACh in the cat trachea after it had been immunized against a conjugate of VIP-BSA. In in vitro experiments, EJP were markedly enhanced, so VIP was postulated to be an I-NANC substance (these observations were also supported in the rabbit lower esophagus, Ref. 86). In the cat gastric fundus, NO is a main transmitter, but during sustained relaxation, VIP may act as a NANC neurotransmitter. In opossum internal anal sphincter, the increase in NO production in response to VIP occurs mainly from the myenteric neurons, with some contribution from the SMC (182). Furthermore, Daniel's group (182, 231, 529), working on the canine and opossum lower esophageal sphincters, reported that VIP did not contribute to the generation of IJP, whereas NO did (these conclusion was also supported in the rat proximal colon, Ref. 1054). Furthermore, evidence for coexistence of ATP and NO in NANC inhibitory neurons in the rat ileum, colon, and anococcygeus muscles was presented (63, 1004).
It has also been postulated on VIP receptors that the IJP is a VIP-induced response in esophageal circular SMC (224), and VIP has been nominated as an inhibitory I-NANC transmitter in the GI tract (230, 231, 362, 369). However, this idea was not supported by experiments on dog ileum, opossum esophagus, or guinea pig taenia coli (144, 199, 231, 529, 690). In airway SMC, although there is evidence in favor of NO as an I-NANC transmitter (64, 65, 542), VIP immunoreactivity and VIP receptors have also been identified. Moreover, after studying the circular muscle of the canine proximal colon, Keef et al. (559) concluded that the enteric plexus releases two I-NANC transmitters, NO and VIP, as cotransmitters released in parallel from the enteric inhibitory nerves. Their evidence was that 1) the electrical and mechanical effects of VIP did not depend on NO synthesis, 2) the VIP-induced changes in [Ca2+]i did not depend on NO synthesis, and 3) VIP did not cause the release of NO. Cotransmission via VIP and NO has also been suggested in the rat gastric fundus (240), whereas distributions of ATP and NO, as I-NANC substances, in guinea pig colon have been documented (1221).
With the use of immunohistochemical techniques, distributions of PHI and peptide histidine-methionine (which possess structural similarities with VIP) have been identified in airway SMC (247, 380, 709, 933). Furthermore, VIP receptors are found predominantly in large rather than small airways (166). In airway SMC of the cat, VIP antagonists dose dependently enhanced the amplitude of EJP without changing either the membrane potential or input resistance, although in those of the dog, such antagonists had no effect (1182). Thus this effect in the cat seems to be an example of VIP acting on prejunctional nerve terminals.
5. Role of CGRP
As well as VIP, other peptides also act on VSMC as cotransmitters released from cholinergic and adrenergic nerves (675, 1037). For instance, the immunohistochemical localization of CGRP and other cotransmitters was described in a subpopulation of postganglionic neurons in the porcine inferior mesenteric ganglion by Majewski and Heym (687). They subdivided these neurons according to size and cotransmitter content. Thus CGRP-immunoreactive (CGRP-IR) neurons were demonstrated to belong to the population of nonadrenergic neurons (tyrosine hydroxylase and DBH negative). Virtually all of the CGRP-IR neurons exhibited colocalization of CGRP with somatostatin, and some of them with NPY. Majewski and Heym (687) further reported that NPY-, somatostatin-, and NPY/somatostatin-IR subpopulations of adrenergic and NANC neurons were present. In the rat vas deferens, exogenously applied CGRP (human) did not modify the EJP but did inhibit contraction through an inhibitory actions on postjunctional SMC (361). However, the same group (948) reported that, in the large cerebral artery of the cat, 1) immunoreactive CGRP-like material was contained not only in nerve axons but also in boutonlike structures and 2) that transmural nerve stimulation caused relaxation and NANC-IJP, responses that were blocked by capsaicin, as observed for responses induced by applied CGRP. Thus they concluded that CGRP is involved in the mediation of vasodilator responses in this tissue (501). Somatostatin has been reported to act as a negative regulator of transmitter release from nerve terminals in the canine ileal circular muscle (524) and in the esophagus. Moreover, Rattan et al. (921) postulated an inhibitory role for CGRP in neuromuscular transmission, and in the guinea pig ureter, Santicioli and Maggi (960) reported that CGRP-induced inhibition of IJP evoked by nerve stimulation is due to activation of glibenclamide (glyburide)-, ATP-sensitive K+ channels. In large cerebral arteries of cats, CGRP hyperpolarized the membrane and IJP evoked by electrical field stimulation were markedly suppressed by capsaicin, and therefore, Saito et al. (950) suggested a role for CGRP as an I-NANC substance in inhibitory nerves. Possibly, the long-lasting hyperpolarization observed in dog basilar artery (307) may be of the same nature. In human myometrial cells, Azadzoi and Szenz de Tejada (36) found that CGRP induced a direct G protein-mediated activation of K+ channels, leading to hyperpolarization and SM relaxation. Santicioli and Maggi (960) reported that, in the guinea pig ureter, electrical field stimulation evoked a graded hyperpolarization that was blocked by TTX and prevented by capsaicin desensitization (capsaicin itself produced a slight hyperpolarization). Exogenously applied CGRP also produced hyperpolarization, but this was insensitive to TTX. Furthermore, an antagonist of ATP-sensitive K+ (KATP) channel openers, glyburide, blocked, but low K+ solution enhanced, both IJP and the CGRP-induced hyperpolarization. Thus CGRP appears to be released from the peripheral endings of capsaicin-sensitive primary afferent neurons, causing hyperpolarization due to activation of KATP channels. Distributions of inhibitory CGRP neurons in the guinea pig ileum (46) and rabbit and rat colon (710) have been elucidated. Calcitonin gene-related peptide has been detected in sensory and motoneurons in the GI tract (683, 935). Calcitonin gene-related peptide enhanced contraction in the guinea pig small intestine through an acceleration of ACh release (428, 684) and also in guinea pig proximal and distal colon and taenia coli (716), but it caused a concentration-dependent inhibition of [3H]ACh release from the rat stomach antrum. Furthermore, CGRP enhanced the contraction evoked by NANC nerve stimulation in the guinea pig small intestine (901). In the guinea pig proximal colon, Kojima and Shimo (601) likewise reported that CGRP enhanced the contraction evoked by NANC nerve stimulation (in the presence of- and -adrenergic and muscarinic inhibitors), in this case through a prejunctional mechanism via a non-CGRP-1 receptor (242, 901) located on intramural tachykininergic neurons. In the canine lingual artery, Kobayashi et al. (591) reported that, on electrical stimulation, NANC nerves released CGRP, but not NO, inducing relaxation by activation of the CGRP-1 receptor. Using the sucrose gap method, Maggi et al. (684) reported that CGRP produced a TTX-resistant membrane hyperpolarization that was partly blocked by either TEA or CPA while being unaffected by glyburide. They concluded that CGRP produces a direct relaxation of circular SMC of the guinea pig proximal colon through activation of Ca2+-dependent K+ channels and Ca2+ release and uptake from the SR.
In GI tract, distributions of PACAP, as a relaxing substance, have been elucidated in the guinea pig taenia coli (525) and stomach (549), rat colon (368), human and cat esophageal sphincter (833), and rat distal colon (576). In the trachea, the PACAP-induced relaxation was inhibited by charybdotoxin (415); presumably this agent activates Ca2+-dependent maxi-K+ channels.
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IV. CHARACTERISTICS OF ION CHANNELS IN VISCERAL SMOOTH MUSCLE CELL MEMBRANES |
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A. K+ Channels
1. Subtypes of K+ channels
The recent development of molecular biological techniques has enabled the development of a genomic and structural classification for K+ channels (see Table 1). Classical voltage-dependent K+ channels (including Ca2+-activated, delayed rectifying, and A-like K+ channels) form an ionophore with a tetramer of subunits having six transmembrane (TM) domains. Although these K+ channels could be functionally expressed as a homomultimer, the presence of variation in K+ channel properties has led to the idea that the K+ channels are actually expressed as a heteromultimer, the components being encoded by different types of K+ channel genes (217, 946). This type of K+ channel has an S4 region in its structure that gives it voltage dependency, and the channels form a super family. Genetically, the voltage-dependent K+ channels make up four families (Shaker, Shab, Shaw and Shal), which have been subdivided into many Kv subfamilies (183, 184, 372). In terms of function, these K+ channels can be divided into two large groups (delayed rectifier and transient K+ channel types). The relation between family membership and function is not a simple one. For example, Shaker and Shal (Kv4)-type K+ channels exhibit A current-like properties, whereas Shaw and Shab K+ channels exhibit delayed rectifier-like properties (197). The-subunits of Kv1.1, Kv1.2, Kv1.5, and Kv1.6 in Shaker-type channels bestow delayed rectifying properties, but these same channels show A current-like properties with a 1- or 3-subunit (274, 275, 974). The -subunits in Kv1.3 and Kv1.4 of the Shaker type bestow A current-like properties, and coexpression of the 1-subunit enhanced the time course of inactivation (770, 1220). Kv3.1, of the Shaw type, can be classified as the delayed rectifier type, whereas Kv3.3 has inactivating properties. Moreover, Kv2.1 and Kv2.2 of the Shab type, possess delayed rectifying properties, but those of the Shal type had A current-like properties. When the effects were observed of the muscarinic (m3) receptor on Kv1.2 and Kv1.5 channels cloned originally from canine colonic SMC, they were found to cause a poorly reversible decrease in the opening probability of these channels (1135). In contrast, it has been reported that possession of the 1-subunit has no effect on non-Shaker-type K+ channels (974, 1220).
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The inward rectifying K+ channel possesses a two transmembrane structure. Other members of this group include the KATP channel, Na+-sensitive K+ channel, and pH-sensitive K+ channel, as well as the inward rectifying K+ channel. Although the pH-sensitive K+ channel (RACTK1) has not been identified in intestinal SMC, this channel has been identified in cardiac and arterial SMC, as well as in collecting duct cells in the kidney (1060). These types of K+ channel have no voltage dependency, because of their lack of the S1-S4 regions found in the classical voltage-dependent K+ channels. Although these K+ channels lack the voltage sensor, their inward rectifying properties emerge when Mg2+ is present in the cytosol (inward rectifying K+ current, Ref. 830; KATP current, Ref. 630; pH-sensitive K+ current, Refs. 1060, 1061). In whole cell experiments, membrane depolarization produces transient and sustained outward currents, after the Ca2+ inward current. The transient component of the outward current is formed by the activities of two different K+ channels. One of them is a Ca2+-activated K+ channel (mainly maxi-K+ channel), and the other is an outward rectifier K+ channel. In pharmacological experiments, TEA, which at low concentrations blocks the maxi-K+ channel relatively selectively, inhibited part of the transient current, but not all of it. Although intracellular application of InsP3 had no effect on the amplitude of the transient outward current, ryanodine, an open blocker of the Ca2+-induced Ca2+ release channel in the SR, abolished the transient outward current, thus indicating a major contribution by Ca2+ release from the SR to the activation of this channel (64, 850, 950, 1229). The remaining component of the transient current is blocked by 4-AP, known to be a blocker of the A current. In canine and guinea pig gastric muscle cells, the transient K+ current was reported to be a Ca2+-activated K+ current, but its sensitivity to TEA was lower than that of the classical maxi-K+ channel (824, 985). In the circular muscle layer of the guinea pig ileum, Duridanova and Boev (257) classified voltage-dependent K+ currents into three components on the basis of drug actions: 1) at a holding potential of
50 mV, apamin inhibited 29% of the whole cell K+ current (IK) at a 0 mV membrane potential. This current disappeared after blockade of the inward Ca2+ current. 2) Glyburide inhibited 31% of the total current at 0 mV. 3) Other components formed 36-46% of the IK in cells clamped at a holding potential of 80 mV; this was inhibited by charybdotoxin (maxi-K+). Adda et al. (12) studied the expression and function of voltage-dependent K+ channel genes in human airway SMC. RNA from airway SM tissues revealed the presence of Kv1.2 and Kv1.5 transcripts, as well as Kv1.1 mRNA. The available voltage-dependent K+ current in human airway myocytes was insensitive to charybdotoxin but blocked by 4-AP. Dendrotoxin, charybdotoxin, and glybenclamide had no effect on the resting tone of muscle cells. Conversely, 4-AP increased resting tension with an EC50 equivalent to that observed for current inhibition. They concluded that human airway myocytes express mRNA for several members of the Kv1 family; the channel that underlies the predominant voltage-dependent K+ current and regulation of basal tone appears to be Kv1.5.
The sustained component of the outward current is generally called the delayed rectifying outward current. However, as mentioned previously, the properties of the sustained outward current differ between tissues. In fact, Snetkov et al. (1008) reported that, even in the same tissue (human bronchial SMC), individual cells possessed pharmacologically different K+ channels; some cells had 4-AP-sensitive (Ca2+-independent) delayed rectifying K+ channels, whereas others had TEA-sensitive (Ca2+-dependent) delayed rectifying K+ channels. This TEA-sensitive outward current (sustained component) was charybdotoxin and iberiotoxin sensitive, indicating a contribution of the Ca2+-activated (maxi-) K+ channels to the delayed rectifying outward current in these SMC.
Concerning channel phosphorylation, Carl et al. (162) reported a Ca2+-activated K+ channel of 260 pS that was activated by phosphorylation via PKA. Calyculin A also augmented this channel's activity, but to a lesser extent than PKA, suggesting modulation of the channel opening by cAMP or adenylate cyclase. Because calyculin A shortened the slow wave's duration (1160), these authors postulated that phosphorylation and dephosphorylation of this Ca2+-activated K+ channel might contribute, at least in part, to slow-wave formation. In addition, they speculated that the phosphatase that acts on the Ca2+-activated K+ channel in colonic SMC might not be of type I or type II, judging from the potencies of calyculin A and okadaic acid. In this channel, G protein-coupled channel modulation has also been reported (205, 206). In fact, phorbol 12,13-dibutyrate (PDBu), a phorbol ester and a PKC activator, inhibited the transient outward current, and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7; a PKC inhibitor) abolished this action of PDBu (581). Single-channel current recording then revealed that PKA, but not PDBu, enhanced channel activity at the unitary current level, suggesting the presence of an indirect mechanism for channel activation, rather than direct channel phosphorylation by PKC.
In vascular SMC (rabbit portal vein), Isop and forskolin each enhanced the amplitude of the 4-AP-sensitive delayed rectifying K+ channel, possibly through an activation of PKA (18). Furthermore, there is evidence that Kv1.2 and Kv1.5 channels, both of which are 4-AP-sensitive delayed rectifying K+ channels, have PKA consensus sites in their cytosolic region (NH2 and COOH terminals) (564). Moreover, the rat cardiac Kv1.2 channel was found to be augmented by Isop and PKA via phosphorylation of a single site in the NH2-terminal region (436). These findings indicate that, in VSMC, delayed rectifying K+ channels are also modulated by metabolic processes. Walsh and Kass (1146) noted that PKA caused a shift to the left in the channel's activation curve and an enhancement of current amplitude by PKC, both in cardiac delayed rectifying channels. They concluded that PKA and PKC both augmented the delayed rectifying channel, but did so independently.
The KATP channel can be activated via various receptors, such as those for CGRP, adenosine, somatostatin, and galanin. In rabbit mesenteric artery and guinea pig gallbladder, CGRP opens the KATP channel through an activation of PKA (910, 1226). In fact, the KATP channel has potent PKA and PKC phosphorylation sites in the COOH-terminal region. Indeed, muscarinic receptor stimulation inhibited KATP activity through PKC stimulation.
Kim et al. (567) reported that individual Kv1.4 channels, which are Shaker-type K+ channels, gather together to form a cluster when this channel is coexpressed with PDS-95 protein. They concluded that the PDZ-1 region of the PDS-95 protein and the TAV or TDV of the COOH terminal are essential sequences for interaction. The PDS-95 protein is a guanylate kinase and has a SH3 domain near the kinase domain. Therefore, it is plausible that interaction between Kv1.4 channel-PDS-95 protein complexes may produce additional functions by phosphorylation of the protein. A clustering of channels caused by cytoplasmic protein has also been reported for the ACh receptor, as has coexpression of 43K/rapsyn and the glycine receptor with gephyrin in neurons (574, 895).
2. Delayed rectifying K+ channels
This is a common K+ channel current observed in a variety of SMC, including vascular cells. It is activated by membrane depolarization to30 or 20 mV in a Ca2+-free medium. 4-Aminopyridine and TEA are used as blockers of this current; however, both are required in concentrations of a few millimolar for channel block, and these drugs more selectively inhibit the transient (A-like) and Ca2+-activated K+ currents, respectively. In rabbit ileal muscle, the delayed rectifying K+ current was inhibited by 1 mM TEA but not by 1 mM 4-AP, whereas the same current in the rabbit pulmonary artery was inhibited by 1 mM 4-AP but not by 10 mM TEA (849). Another blocker of the delayed rectifying K+ current is -dendrotoxin, a snake venom peptide of 60 amino acids from Dendroaspis angusticeps (DTX; Refs. 173, 1024, 1167), and this toxin has also been reported to block a transient K+ current in the hippocampus, as does 4-AP (249). Studies of amino acid mutations in the DTX-sensitive delayed rectifier K+ (RBK1) channels has shown that Ala352-Glu353 and Tyr379 in the H5 region between the S5 and S6 transmembrane domains are important residues for the toxin sensitivity of this channel (449). The activation of this current is both time and depolarization dependent, but it is not, in general, inactivated during a short depolarizing pulse. However, a very long depolarizing pulse (longer than several seconds) does induce a slow current inactivation. In addition, 4-AP blocked a cloned K+ channel (Kv1.5), classified as a delayed rectifying K+ channel, which was expressed in Xenopus oocytes and in mammlian cells (116, 289, 1036). A cloned channel (CSMK1) from canine colonic circular muscle was also inhibited by 4-AP, at submillimolar concentrations (385). This SM delayed rectifying channel has a very high homology with others of the Kv1.2 subfamily [90-91%; rat atrial K+ channel (RAK), rat brain K+ channels (RBK2, RCK5)]. In fact, block by 4-AP of this K+ channel has been demonstrated in both open and closed states. However, perhaps because of its very small unitary conductance, only a few papers have been published on the unitary current of this delayed rectifying channel. The reported unitary conductance (estimated by noise analysis) is ~5-10 pS (rabbit portal vein, Refs. 58-60; rabbit coronary artery, Ref. 1136), these values being slightly smaller than those obtained for natural and cloned delayed rectifying K+ channels from the canine colon (160-164, 385, 868). A similar value for unitary conductance has been reported for the anomalous rectifier K+ channel in tunicate egg cells (5.2 pS, Ref. 314). The unitary conductance of cloned delayed rectifying K+ channels was 14 pS (CSMK1, Kv1.2) or 10 pS (Kv1.5) in symmetrical 140 mM K+ solutions (385). These values are much the same as those obtained for native delayed rectifier K+ channels recorded from SMC of the porcine airway (13 pS) and canine colon (19 pS) (120, 161). Northern blotting of CSMK1 and Kv1.5 channel mRNAs has shown that the Kv1.5 channel is common throughout the entire GI tract as well as in several vascular tissues and the heart, whereas the Kv1.2 channel is restricted to the GI tract (385, 868). These data indicate that the delayed rectifier K+ channels observed in various cells do not make up a uniform population. In colonic SMC, the IC50 value for the action of 4-AP on the delayed rectifying K+ channel was 70 µM, but it was 300 µM in the trachea and in pulmonary artery cells (162, 785, 854). Delayed rectifying K+ channels highly sensitive to 4-AP have also been reported in rabbit portal vein and in porcine and canine airway SMC (59, 120). Cloned Kv1.2 and Kv1.5 channels had IC50 values for the action of 4-AP of 75 and 211 µM, respectively (385, 868). The dissociation constant (Kd) value obtained with 4-AP (270 µM) for cloned Kv1.5 (hPCN1) channels was similar to that found for K+ channels in colonic SMC. However, the Kd value (590 µM) for cloned Kv1.2 (NGK1) channels was quite different from that found in colonic Kv1.2 channels. This suggests that we should always take into consideration any small differences in the protein sequence as well as heteromultimeric channel formation when studying drug sensitivity.
It has been reported that there are delayed rectifying K+ channels resistant to 4-AP in the gastric antrum, taenia coli, colon, ureter, and urinary bladder of the guinea pig, rabbit ileum, rat anococcygeus muscle, canine coronary, pulmonary and renal arteries, and feline cerebral artery (for review, see Ref. 626). In fact, for inhibition of this delayed rectifying K+ current, high concentrations of TEA were required (IC50 = 3 mM in rabbit ileal longitudinal muscle, Ref. 849; 3 mM in canine tracheal muscle, Ref. 785; >10 mM in canine colonic circular muscle and cloned Kv1.2 and Kv1.5 channels, Ref. 868). After studying RCK2, a Kv1.6-type delayed rectifying K+ channel found in rat brain, Kirsh et al. (573) reported that the channel had two distinct TEA-binding sites (on inner and outer margins of the pore). Furthermore, of the other delayed rectifying K+ channels, Kv2.1 had a high sensitivity to TEA at the internal site, whereas Kv3.1 was sensitive at the outer site (572). Although the blocking of delayed rectifying K+ channels by 4-AP and TEA is voltage dependent, the different sensitivities to 4-AP and TEA suggest that different types of delayed rectifying K+ channels might be expressed in different SM tissues. Recently, Horowitz et al. (430) showed that coexpression of Kv1.2 and Kv1.5 channels in oocytes and other cells resulted in the formation of heterotetramers of both channel subunits. Heterogeneities, in terms of the properties of delayed rectifying K+ channels, between tissues and between cloned and native K+ channels might be explained by the formation of heteromers involving different subunits.
4-Aminopyridine (2 mM) or TEA (5 mM) prolongs plateau duration (guinea pig gall bladder, Ref. 1227) or produces an action potential on membrane depolarization (guinea pig pulmonary artery, Ref. 79; canine trachea, Ref. 467). Thus it is clear that the delayed rectifying K+ current serves as a repolarizer of cells in a way that controls action potential duration. Indeed, inhibition of this current (pharmacologically or by removal of cytosolic K+) prolongs action potential duration and leads to the formation of a plateau potential (849, 1146). Furthermore, as mentioned above, delayed rectifying K+ channels in cardiac cells were found to be regulated by stimulation of various receptors via PKA or PKC activation, or directly via G proteins. In vascular SMC, PKA has been reported to activate the delayed rectifying K+ channel after -adrenoceptor stimulation (18). These reports indicate that the delayed rectifying K+ channels in SMC, including visceral cells, can be modulated by agonist stimulation and membrane depolarization via multiple pathways.
3. Transient outward K+ currents
Transient outward K+ currents in SMC can be classified as either Ca2+-dependent or -independent K+ currents. Their relative sensitivity to drugs has also revealed the presence of two types of transient outward current. Charybdotoxin and TEA are rather specific blockers for the Ca2+-activated K+ current, and as low concentrations of TEA, but neither 4-AP nor apamin, effectively block the Ca2+-dependent transient K+ current, this is possibly a current produced by activation of the maxi-K+ channel. Indeed, in rabbit portal vein, periodic activations could be recorded of the maxi-K+ channel under certain conditions in the inside-out membrane patch (1185). Caffeine and ryanodine, but not InsP3, also effectively inhibited the Ca2+-dependent component of the transient outward current, together with a slight inhibition of the VOCC, suggesting an involvement of the Ca2+-induced Ca2+ release (CICR) mechanism in the Ca2+-dependent transient current (850, 950). In the guinea pig ureter, a Ca2+-independent K+ transient current can be recorded (465, 643), and this current can be blocked by 1 mM 4-AP, but not by 5 mM TEA (643). Its pharmacological and biophysical characteristics are similar to those of the A current in neurons and to those of the transient outward current found in cardiac cells (9, 528). The unitary current conductance of this transient K+ current is 14 pS under quasi-physiological ionic conditions (465). Although the transient outward current recorded in SMC such as those of the ureter and portal vein had a similar 4-AP sensitivity to that of the delayed rectifying K+ current, its inactivation kinetics differed from the delayed rectifier in these preparations. This suggests that the two channels might differ in structure (the Shaker-type K+ channel has a "ball and chain" structure at the NH2 terminal, whereas the delayed rectifier type K+ channel does not, Ref. 1220). Schwarz et al. (972) have already reported that the-subunits of both A-type (Kv1.3 and Kv1.4) and delayed rectifying (Kv1.1, Kv1.2 and Kv1.5) K+ channels are produced by alternative splicings from the same gene. The evidence indicates that these K+ channels have the same "core" sequence (6 TM and H5 regions), but different NH2 and COOH terminals. Indeed, deletion of the amino acid sequence at the NH2 terminal in the A-type channel prevented the current's inactivation, and insertion of a synthesized peptide having the same sequence as the NH2 terminal reproduced the inactivating property of this channel (1223). Sewing et al. (976) also showed that coexpression of the ,-subunit with an 1-subunit of the delayed rectifier type Kv1.5 channel transferred the delayed rectifier property to another K+ channel having an A-like channel property. These reports strongly indicate that the various inactivation rates of transient outward currents might be due to the formation of a heteromultimeric channel by different - and -subunits (951, 970).
4. Inward rectifying K+ channel
An inward rectifying K+ current has not yet been recorded in VSMC (other than in blood vessels), and this K+ current has been studied mainly in cerebral, mesenteric, and coronary arteries (for review, see Ref. 626). In middle cerebral and pial arteries, an increase in extracellular K+ causes vasodilation by two different mechanisms: an ouabain-sensitive mechanism and a Ba2+- and Cs+-sensitive one (636, 715). Furthermore, Edwards and Hirst (261) reported that the vasodilation induced by an elevation in extracellular K+ was related to the membrane hyperpolarization observed under the same conditions. This in turn resulted from activation of the inward rectifier K+ channel distributed in the distal region of the middle cerebral artery; indeed, a depolarization, rather than a hyperpolarization, occurred in the proximal region of the same artery, where the inward rectifier K+ channel was not recorded (261, 262). The resting membrane potential of the distal cerebral artery is reported to be around43 mV (262), indicating that most of the inward rectifier K+ channels are closed. However, because Ba2+ (0.5 mM), a blocker of the inward rectifier K+ channel, caused a depolarization of the membrane (262), the inward rectifier K+ channel did make some contribution to the resting membrane potential. Possibly, closure of this channel may be more important than its opening. Closure might be important for depolarization induced by vasoconstrictors or for keeping a relatively high membrane potential (depolarized condition), whereas opening of this channel would set the resting membrane potential at a relatively low level. It is interesting and well established that the property of inward rectification is not a property of the channel itself but depends on a unidirectional channel block caused by intracellular Mg2+ and polyamines (706, 1207). Recently, gene cloning has led to the identification of similar K+ channels with inward rectifying properties, such as the ATP-regulated K+ channel (ROMK1; weak rectifying K+ channel) and G protein-gated K+ channels (IKACh; GIRK1) (419, 433, 612). However, classical inward rectifying, ATP-regulated, and G protein-gated K+ channels have not yet been identified in VSMC.
5. ATP-sensitive K+ channels
The ATP-sensitive K+ channels were identified in cardiac muscle by Noma (827), and the channels have since been found in various tissues, such as pancreatic-cells, neurons, cardiac and skeletal muscle cells, and SMC, including visceral tissues. Recent cloning of ATP-sensitive K+ channels from the rat heart (cKATP; Kir 6.1) and pancreatic islets (uKATP; Kir 6.2) has revealed that this channel belongs to the superfamily of inward rectifying K+ channels, since it has two transmembrane domains with an H5 region and a weak rectifying property. The cKATP shows a high degree of homology to other subfamilies (73% homology to GIRK, 70% to IRK, 64% to ROMK), but uKATP showed a lower level of homology (43-46%) (35, 468). By Northern blotting of the mRNA of both KATP channels, it has been found 1) that cKATP is highly expressed in heart, kidney, spleen, submaxillary gland, thymus, hypothalamus, and hippocampus, but not in skeletal muscle or SM, including that of the small intestine and uterus (35); but 2) that uKATP is expressed in a wide variety of tissues, including skeletal muscle and SM (stomach, small intestine, and colon) as well as heart, kidney, liver, brain, testis, adrenals, pancreatic islets, and ovary (468). However, the same authors noted that uKATP was not expressed in an insulin-secreting cell line, in which an ATP-sensitive K+ channel has been recorded, or in other cell lines (PC12, GH3, AtT-20, and endothelial cell lines), suggesting the presence of another type of KATP channel in these cells (468). Both types of KATP channel had inward rectifying properties when Mg2+ was in the cytosol, and they had similar unitary conductances (68 pS, cKATP; 70 pS, uKATP; both in symmetrical 140 mM K+ solutions). The value of the unitary conductance for the KATP found in rabbit and guinea pig ventricular cells (~75-80 pS in symmetrical 140 mM K+ solutions; Refs. 284, 382, 844, 1093) was close to those of the cloned KATP channels. Moreover, the unitary conductances of the KATP channels found in pancreatic - and insulin-secreting cells were, respectively, 88 pS (mouse -cell) and 50-55 pS (RINm5F, Ref. 927; HIT T15, Ref. 816). On the other hand, smaller unitary conductances have been reported for guinea pig urinary bladder (10 pS), porcine coronary artery (35 pS), porcine urethra (43 pS), and rabbit portal vein (24 and 50 pS) (for review, see Ref. 626). Conversely, larger values were obtained in rabbit mesenteric artery, canine aorta, and rat ventromedial hypothalamic neurons (135 pS, Ref. 1023; 130 pS, Ref. 610; 150 pS, Ref. 34) in symmetrical K+ (140 mM) or asymmetrical high K+ conditions (60 mM/140 mM K+). Figure 5 shows the inhibitory action of ATP on the unitary K+ current recorded from dispersed SMC of the rabbit portal vein.
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Glyburide (glibenclamide) and other sulfonylurea derivatives are selective blockers of KATP channels in pancreatic
-, neuronal, cardiac, and SM cells. However, cloning of KATP channels from cardiac and pancreatic islet cells showed that the subunits forming the channel pore had no sulfonylurea binding site (35, 468). In fact, the sulfonylurea receptor has been identified as a peptide with 13 transmembrane domains (TM13) and two nucleotide binding (folds) sites, and it is thought that a combination of inward rectifier K+ channel (composed of 2 transmembrane segment, TM2; Kir 6.1, Kir 6.2) and sulfonylurea receptor (SUR1, SUR2) forms the KATP . It is reported that a combination of Kir 6.2 and SUR1 forms pancreatic KATP and that a combination of Kir 6.2 and either SUR2A or SUR2B forms the cardiac or SM-type KATP . It is of interest that a combination of Kir 6.1 and SUR2 produces a GDP-activated channel (359, 493, 1015, 1193). Figure 6 shows the topography of KATP channels (A: KATP is composed of sulfonylurea receptor and KIR channel; B: the structure of sulfonylurea receptors, Ref. 1018).
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The KATP channels open under pathophysiological conditions, such as cardiac ischemia, but they may also open under physiological conditions via receptor activation. This channel is also pharmacologically important as a target for K+ channel openers. So far, activation of receptors by Isop, CGRP, adenosine, vasopressin, somatostatin, and galanin has been reported to open KATP channels, whereas ACh and vasopressin block the channels found in various tissues, including VSMC (Isop, Ref. 798; CGRP, Refs. 110, 910; adenosine, Ref. 234; somatostatin, Ref. 524; galanin, Ref. 67; ACh, Ref. 816; vasopressin, Ref. 1143). The mechanism underlying the activation of KATP channels by CGRP and Isop has been said to involve cAMP-PKA stimulation, since forskolin, cAMP, and PKA directly open the channels, whereas okadaic acid, a phosphatase inhibitor, prevents deactivation of the channel (1226). In the case of adenosine-induced activation, Dart and Standen (234) reported that the adenosine receptor coupled with the KATP channel was the A1 , and not the A2 receptor. They therefore concluded that the cAMP-PKA system did not contribute to the activation of the KATP channel by adenosine. Rather, they considered that a Gi protein was involved in the adenosine-induced channel opening, since direct introduction of the Gi
subunit into the cytosol activated the channel through a membrane-delimited pathway.
With regard to the channel's inhibition by vasopressin in insulin-secreting and vascular cells, a direct blocking action has been suggested on the basis that vasopressin inhibits the KATP channel in the outside-out patch, but not in the cell-attached patch configuration (698, 1143). On the other hand, Bonev and Nelson (109) showed that inhibition of KATP channels by ACh (through muscarinic receptor activation) could be mimicked by the application of phorbol esters (phorbol 12-myristate 13-acetate) or diacylglycerol analogs (e.g., 1-oloyl-2-acetyl-sn-glycerol; OAG), and attenuated by either PKC inhibitors (calphostin C and bis-indolylmaleimide) or guanosine 5'-O-(2-thiodiphosphate) (GDPS). Because vasopressin is known to also stimulate PLC and thus synthesize InsP3 and DAG, vasopressin might inhibit KATP channels through PKC.
In SMC, it has been known for some time that several types of drugs, such as nicorandil, cromakalim, minoxidil, and pinacidil, hyperpolarize the membrane or relax the tissue (for review, see Refs. 28, 130, 263, 579, 626, 1176). Now, it is well accepted that K+ channel openers directly open KATP channels in various cells; this has been shown by means of single-channel current recordings from isolated cells and because cloned KATP channels could be opened by K+ channel openers (35, 468). On the other hand, Helevinsky et al. (396) considered that KATP channel openers act as a potent Cl channel inhibitor in vascular SMC (hyperpolarization of the membrane).
Activation of KATP channels results in membrane hyperpolarization, which causes suppression of action potential generation. In canine colonic SMC, cromakalim reduced the amplitude, duration, and maximum rate of rise of the slow wave, without affecting its frequency (286, 898). Although cromakalim does have direct inhibitory actions on L-type Ca2+ channels (854, 898), the inhibition of action potentials or of slow waves by K+ channel openers are considered to be indirect actions that are secondary to membrane hyperpolarization. Neither muscle relaxation nor contraction, whether induced by inhibitory nerves, -adrenoceptor, or muscarinic receptor stimulation, were modified by glyburide in the canine colon, indicating that KATP channels do not participate in nerve-mediated responses in this tissue (929). Similarly, although neurotransmission was inhibited by K+ channel openers indirectly through membrane hyperpolarization, it was not affected directly via inhibition of transmitter release (in guinea pig and rabbit mesenteric arteries, Refs. 797, 804). However, there is substantial evidence that the KATP channel does contribute to nerve-mediated responses in guinea pig small intestine and trachea and rat CA3 neurons (67, 453, 714, 1236). In the guinea pig ureter, electrical field stimulation evoked a TTX-sensitive transient hyperpolarization (IJP, Ref. 958). Because these IJP were capsaicin and glibenclamide sensitive, and because exogenous application of CGRP hyperpolarized the membrane, it was suggested that CGRP might activate KATP channels in this tissue (685, 958).
The activity of KATP channels is modulated by cytosolic factor(s), since channel activity disappears or declines ("rundown") on membrane excision in rabbit portal vein and guinea pig ventricular cells. Mg-ATP is one of this channel's modulators, because this combination produces a reappearance of channel opening by phosphorylation. On the other hand, the rundown phenomenon was not seen in the KATP channels of the rat portal vein (534, 1225). The diphosphates of various nucleotides (e.g., ADP, GDP, UDP, IDP, and CDP) augmented KATP channel activity and also reactivated the channels after the occurrence of complete channel rundown. Actually, Mg2+ are normally thought to be essential for the reactivation of KATP channels by ATP and other nucleotides, and for the prevention of channel rundown (61, 62, 535, 976, 1093). However, in the rabbit portal vein, the application of GDP without Mg2+ could open the channel even after rundown, according to Beech et al. (61) and Tung and Kurachi (1110). In contrast, in the studies of Kajioka et al. (533) and Kamouchi and Kitamura (538), GDP with or without Mg2+ failed to reactivate the KATP channels in the rabbit portal vein. The reason for this discrepancy is not clear, but the presence of such a discrepancy may be an indication that other cytosolic modulating mechanisms, in addition to GDP binding, are needed for channel reactivation. Channel phosphorylation might be such a mechanism; indeed, in cardiac cells, nucleotide diphosphates (NDPs) proved able to reactivate the dephosphorylated channel, indicating that either phosphorylation or NDP binding may be required for channel reactivation (975). Recently, Furukawa et al. (326) demonstrated that F-actin, but not G-actin, prevented rundown in cardiac cells and that channel phosphorylation (by Mg-ATP) did not reactivate the KATP channel after the rundown induced by cytochalasin D or long exposure to high Ca2+. They speculated that stabilization of the channels in the membrane by F-actin is essential for maintaining their activity. Although the nature of any such interaction between KATP channels and F-actin is not clear, this could indicate the presence of additional endogenous mechanisms for channel reactivation.
The K+ channel openers failed to open KATP channels after rundown in guinea pig cardiac and rabbit vascular cells; however, these drugs did open the same channels in the cell-attached condition, or in the presence of NDP or Mg-ATP under cell-free conditions, in guinea pig ventricular muscle and the rabbit portal vein (61, 533, 538, 975). Consequently, Shen et al. (975) and Kamouchi and Kitamura (538) proposed the idea that KATP channels have two states, switchable by endogenous modulator(s), that is, operative and inoperative states. They considered that K+ channel openers could open the channel only when it was in the operative state.
In the guinea pig urinary bladder, KATP channels can be continuously recorded in outside-out patches, and K+ channel openers augmented the channel's activity as they did in the rat portal vein (108, 109, 534, 1225). Because GDP and other NDP activated the KATP channel under cell-free conditions, other mechanism(s) might be present for the prevention of the rundown phenomenon, other than those involving NDP or channel phosphorylation (by Mg-ATP).
B. Na+ Channels
1. TTX-sensitive and -resistant Na+ channels
In excitable cells, the present classification of Na+ channels recognizes the following subtypes: I, II (IIA), III, ml, and h1 subtypes (on the basis of their cDNA cloning) (174, 565). These have been described in rat neurons, skeletal muscle, and heart cells. Saxitoxin (STX), TTX, and µ-conotoxin are the pharmacological tools used for their separation. Epithelial cells have a further type of Na+ channel, which is sensitive to amiloride, a diuretic, and which has a channel structure that is different from that of the classical voltage-dependent Na+channels, such as type I. Although the amiloride-sensitive Na+ channel has been identified in rabbit colon and lung epithelial cells, no report has yet been published concerning its existence in SMC. Because cardiac Na+ channels have a sensitivity to TTX and STX that is low by comparison with that seen in neuronal cells, the TTX-resistant Na+ channels observed in some SMC might be classified as cardiac-type Na+ channels (type h1). On the basis of data from blot hybridization analysis of the brain types I-III and glial-type Na+ channels, only type III channels are present in intestinal SMC (345, 1058). In the rat azygos vein, the TTX sensitivity of the fast Na+ current was lower than the TTX sensitivity of those in cardiac and gastric SMC, so these vascular cells may have a novel Na+ channel, like the epithelial Na+ channel (1044, 1119). Differences in TTX sensitivity seem to be determined by differences in amino acid sequence, with variation occurring in one amino acid in the SS2 region in domain I (395, 1082). Other toxins that modify Na+ channel gating have not yet been tested on SM Na+ currents. Moreover, chloramine-T inhibited the inactivation of the TTX-sensitive Na+ current in the rabbit pulmonary artery (852). As far as these effects were concerned, the pharmacological properties of the voltage-dependent Na+ channels in SMC were the same as those seen in neuronal and cardiac cells. Kao and McCullough (543) first reported, in rat uterine and guinea pig taenia coli, that a part of the action potential is accounted for by a Na+ current. Because the pioneering experiments were carried out using the multicellular sucrose-gap method, the early consensus about the nature of the ionic currents making up the action potential in VSMC was that it was due solely to activation of the VOCC. It is now clear that many VSMC generate both Na+ and Ca2+ currents (rat azygos vein, rabbit main pulmonary artery, rat portal vein, rat aorta, and rat vena cava, for review, see Ref. 626; rat ileum, Ref. 997; rat and human colons, Ref. 786; guinea pig taenia coli, Ref. 1202; rat stomach fundus and guinea pig ureter, Ref. 1607; rat stomach, Ref. 1201; rat uterus, Refs. 24, 543, 848, 1019, 1021, 1022, 1217). The Na+ current is activated within 1-2 ms, inactivated within 10 ms, and is highly sensitive to extracellular Na+ concentration. From such evidence, we can deduce the presence of a TTX-resistant Na+ current component in the action potential in a given tissue, i.e., some tissues are highly sensitive to TTX (an IC50 of 10-200 nM and found in rat portal vein, Ref. 854; rabbit pulmonary artery, Ref. 855; guinea pig ureter, Ref. 786; rat and human colons, Ref. 1187; rat portal vein, Ref. 854; rat vena cava, Ref. 746). These currents are similar to, or the same as, that observed in nerve fibers (presumably type II). Other tissues are resistant or less sensitive to TTX (an IC50 of the micromolar order; rat uterus, Refs. 25, 848; rat stomach, Refs. 786, 1200; rat azygous vein, Ref. 1244). Furthermore, a third type of Na+ channel has been identified in cultured A7r5 cells and in rat aortic and portal vein cells (2341); this epithelial-like Na+ channel had a voltage-independent property and was insensitive to TTX and amiloride, but sensitive to phenamil. In rat uterine SMC, the current density of the TTX-sensitive Na+ channel increased during pregnancy (489, 697), possibly enhanced by factor(s) in the serum. In this tissue, Sperelakis et al. (1019, 1020) demonstrated that the number of TTX-sensitive fast Na+ channels increased during gestation, the average current density (in pA/pF) increasing linearly from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, then stabilizing at 0.86 on day 21. Kusaka and Sperelakis (635) speculated that nerve growth factor (NGF) and fibroblast growth factor (FGF) might play key roles in regulating Na+ channel expression during pregnancy, and possibly to some extent in leiomyosarcoma cells. In a study in which they used a conventional microelectrode technique, Inoue et al. (487) found that superfusion with Na+-deficient solution eliminated the active responses (small action potentials) observed in Ca2+-free solution in the human pregnant myometrium, although they did not test the TTX sensitivity of such active responses. The evidence suggests that a few to 10% of the population of Na+ channels is available at the resting membrane potential of50 mV in the pregnant myometrium (1019-1021) and in other SMC (intestine, Ref. 1187; gastric fundus, Ref. 786). This "window current" may contribute to the triggering of the Ca2+ action potential at the resting membrane potential and to the depolarization phase just after membrane hyperpolarization. However, this interpretation will be inapplicable to the rabbit pulmonary artery, in which the TTX-sensitive Na+ channel was first described, because this tissue does not produce action potentials under normal conditions (855). More data are needed to clarify the physiological importance of the Na+ channels found in SMC, particularly how and where this channel current contributes to the generation of spontaneous activity (the TTX sensitivity of Na+ channels, their Kd values, and activation and inactivation voltages in VSMC are indicated in Table 2).
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From their inactivation time constants, fast and slow components have been identified in frog node of Ranvier and leiomyosarcoma cells (0.6-0.7 and 3-4 ms, Refs. 78, 635); however, the sensitivity to TTX was similar for both components in a given tissue (IC50 = 2.3 and 7.8 nM in node of Ranvier; 47.1 and 67.5 nM in leiomyosarcoma cells). In many Na+ channels, such as those in rat brain and cardiac muscle,
1- and/or 2-subunits are associated with the -subunit, and the 1-subunit has been found to accelerate the inactivation kinetics of brain and skeletal muscle Na+ channels. However, a -subunit was not always found with an -subunit in the voltage-dependent Na+ channel (for example, rat skeletal rNaSk2 channel, Ref. 1160), and expression of the 1-subunit would be sufficient to produce a functioning channel capable of showing current inactivation. Because the 1-subunit has been identified in the human uterus, the two inactivation kinetics observed in leiomyosarcoma cells may indicate the presence of two types of Na+ channels, one with and one without a -subunit. However, Moran et al. (771) reported that expression of the type II Na+ channel in Xenopus oocytes produced a Na+ current with fast and slow inactivation properties, suggesting that a single type of Na+ channel might possess two different gating mechanisms. Indeed, PKC slowed the current inactivation in rat ventricular cells (831, 909), and forskolin produced a very slow inactivating component by a direct action on the channel (858). There has been no report, so far, to indicate whether or not Na+ channels in SMC are modulated by intracellular messengers.
The site producing inactivation of Na+ channels has been said to be in the linker region between domains III and IV, because partial deletion of the amino acids in this region was found to eliminate the fast inactivation of the Na+ channel (rat brain type II channel, Refs. 890, 1171; human heart channel, Ref. 386). On the other hand, Moran et al. (771) and Freig et al. (302) reported that proline or lysine residues in the S4 region of domain II were important for the slow inactivation of the Na+ current. Because mutations in these positions do not alter the channel's activation kinetics, activation and inactivation sites can be presumed to be distinctly separated, but multiple sites may contribute to channel inactivation.
C. Ca2+ Channels
1. Subtypes of voltage-dependent Ca2+ channels and their specific characteristics
The main ionic current for action potential generation in SMC is the Ca2+ current that passes through VOCC. As deduced from a number of pieces of evidence obtained in microelectrode experiments with Ca2+ antagonists, the main Ca2+ channel distributed in the SM cell membrane is the L-type channel. In VSMC, the Ca2+ current is a major element in the formation of the rising phase of the action potential. Voltage-operated Ca2+ channels in VSMC are classified as either L type (long lasting; or high voltage activated, HVA; 20- to 28-pS channel conductance) or T type (transient; or low voltage activated, LVA; 7- to 15-pS channel conductance), with the N type (neither L nor T) being absent. In some SMC, the T-type channel has been identified (guinea pig taenia coli, Ref. 1214; human myometrium, Ref. 487; guinea pig ileal circular muscle, Ref. 258; SM cell line, Ref. 303). On the other hand, the L-type channel alone has been found in other places. Such an identification has been carried out 1) in vascular SMC (rat mesenteric artery, rabbit mesenteric artery, rat portal vein, rabbit ear artery, dog saphenous vein, guinea pig portal vein, guinea pig coronary artery, rabbit coronary artery, human cystic artery, human mesenteric artery, rabbit portal vein, and rabbit basilar artery, see review in Ref. 1276), 2) in GI tract (intestine, Refs. 1, 341, 342, 490, 644, 645, 652; stomach, Refs. 554, 761, 985, 1130, 1132, 1133), 3) in airway SMC (dog, Ref. 608; guinea pig, Ref. 418; rat and human bronchus, Refs. 696, 1179), and 4) in urinary bladder (336, 337). Furthermore, three types of Ca2+ channels have been identified on the basis of measurements of unitary current conductance in guinea pig intestinal SMC (1214): two of them are thought to be T- and L-type channels, but the third type has yet to be identified. Smirnov and Aaronson (998) and Bkaily (90) also suggested that other types of Ca2+ channel might be present in rat ileum and aorta, viz., N and R type channels; however, this has yet to be confirmed. Characteristics of voltage-dependent Ca2+ channels in VSMC are shown in Table 3 (except for vascular SMC).
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L-type channels in VSMC have the same properties as those found in cardiac and vascular SMC. Thus the channel 1) is activated at a relatively high membrane potential, 2) permeates the Ba2+ better than Ca2+, 3) is blocked by Ca2+ channel blockers, and 4) is inactivated by both membrane depolarization and intracellular Ca2+. The T-type channels found in visceral cells exhibited 1) low voltage activation, 2) resistance to DHP derivatives, and 3) rapid inactivation (guinea pig taenia coli, Ref. 1213; human myometrium, Ref. 487). In guinea pig portal vein and rat sensory neurons, the presence has been reported of DHP-sensitive, low voltage-activated Ca2+ channels (294, 498); however, no such Ca2+ channel has been identified in VSMC. In general, the L-type channel is thought to represent the main pathway for Ca2+ entry into SMC after the generation of action potentials, whereas the role of the T-type or low-voltage threshold Ca2+ channel is uncertain because this channel is normally inactivated at the resting membrane potential seen in a variety of SMC. Recently, a low voltage-gated, DHP-sensitive Ca2+ channel was reported to be activated during the silent phase between spikes in rat dorsal root ganglion (DRG) neurons (294). The open probability of the 10-pS DHP-sensitive channel showed a reverse voltage dependence, when a brief but strong depolarizing pulse was applied just before the stimulation. On the basis of simultaneous recordings of unitary and whole cell currents, these authors concluded that this 10-pS channel did not contribute to the action potential, but instead participated in the modulation of spike frequency. This idea might be tested in these SMC that possess T-type channels. Calcium channels possess heteromultimeric subunits (e.g.,
1-, 2-, -, -, and 
-subunits in the case of the L-type channel, Refs. 156, 1030). Similarly, a functional N-type Ca2+ channel in rabbit brain was found to be composed of 1-, 2-, -, and -subunits, as well as a 95-kDa subunit that was three times larger than the -subunit (1177). The present classification of the channel pore-forming subunit (1) recognizes 1s , 1A , 1B , 1C , 1D , and 1E types. Of these 1-subunits, three types (1S , 1C , and 1D) have been found to help form DHP-sensitive Ca2+ channels in skeletal muscle, brain, heart, pancreatic islet cells, and SMC. In three types of 1-subunit in DHP-sensitive Ca2+ channels in SMC, an 1C-subunit has been identified (rat aorta, Refs. 598, 599; rabbit lung, Ref. 87; canine colonic SM cell, Ref. 928). In the rat aorta and rabbit lung, the full length of the amino acid sequence of the 1-subunit has been deduced; it shows a 65% homology with that of rabbit skeletal muscle and 93% or higher homology with that of the rabbit cardiac channel (87, 598). Northern blotting analysis has shown that this 1-subunit (1C) is also present in uterus, stomach, lung, small intestine, and large intestine (598). The rat brain C-type 1-subunit has been further divided into to rbC-I and rbC-II subtypes (alternative splicing variants), the major splice in the case of canine colonic SMC being the rbC-II, whereas that of the rabbit aorta, rabbit heart, rat heart, rabbit lung, and human brain was of the rbC-I type (291, 928, 1009). In rabbit intestine and trachea and rat A7r5 cells, the two types of variant were equally transcribed (291). Other subtypes of the 1-subunit have been given the names P type (1A) and N type (1B) according to the pharmacological and biophysical properties of their expressed channel (for review, see Ref. 1030). An 1E-type subunit has been identified in the rat brain and classified as a low voltage-activated channel, but the inactivation rate of the current recorded from the expressed channels showed different kinetics from that of the previously described T-type channel (1016).
The -subunit is considered to be located beneath the 1-subunit. There are four genes for the -subunit, namely, 1 , 2 , 3 , and 4 . Alternative spliced forms of 1 and 2 (1a , 1b , 1c , 2a , and 2b) also exist. Collin et al. (207) identified a 3-subtype in the colon, small intestine, and lung in humans as well as in brain and ovary, but not in the heart, liver, or kidney. On the other hand, Hullin et al. (445) demonstrated that 2- and 3-isoforms could be identified in trachea and aorta. However, 1- and 4-isoforms have not yet been detected in SMC (54, 900). A further subunit, the -subunit, has been identified in skeletal muscle and lung, but not in brain, heart, kidney, liver, or stomach (115, 515, 899). This subunit is thought to have four transmembrane regions and is expected to associate with an 1-subunit. Tissue-specific expression of -subunit RNA has not been tested for in other SM tissues. Apparently, 2- and -subunits are derived from a single gene and bound covalently by a disulfide linkage (177, 272, 516). Two transmembrane regions in the 2-subunit and a single transmembrane region in the -subunit are thought to be present (177). Recently, Gurnett et al. (376) proposed a different idea for the topological location of the 2d-subunit; in their scheme, the 2d-subunit was considered to have a single transmembrane region in the -area, to judge from the N-glycosylation sites and results with a specific antibody. There are two alternative spliced forms of the -subunit (skeletal muscle type a and brain type b). In cardiac cells, the presence of a 2-subunit has been confirmed, as it has in skeletal muscle and brain (1177); however, this subunit has not yet been identified in SMC.
Pore formation by the Ca2+ channel 1-subunit is well documented (177), and now the S5-S6 region of each domain is recognized as forming the channel wall. Other subunits, when associated with the 1-subunit, produce modulation of the channel's properties. Experiments performed after coexpression of various subunits with 1 have shown that they positively modulate the channel's kinetics and incorporation into the membrane (171, 172, 208, 376, 673). Itagaki et al. (494) reported that the amplitude of the Ba2+ current through the aortic 1-subunit (1C type) was higher when it was coinjected with the skeletal muscle -subunit, but that this did not change the inactivation time constant. Welling et al. (1169) also reported that coexpression of an SM 1C-subunit with either a skeletal 1- or an SM 3-subunit enhanced the Ca2+ current in the Chinese hamster ovary membrane, the 1-subunit inducing a much greater effect than the 3-subunit. On the other hand, the 3-subunit moderately or markedly enhanced the endogenous Ca2+ channels in Xenopus oocytes, which had DHP-insensitive Ca2+ channels or expressed cardiac 1-subunits (171, 207, 673). Furthermore, the skeletal muscle -subunit reduced the amplitude of the Ba2+ current passed by the skeletal muscle 1-subunit but increased the current passed by the cardiac 1-subunit (673, 1123). These pieces of evidence suggest that all types of -subunit probably modulate the activity of the 1-subunit and that a given channel's particular combination of 1- and -subunits would be very important in determining its potency.
Because coexpression of a -subunit with a cardiac 1-subunit produced no effect on the channel current (1166), the -subunit is thought to contribute to the stable expression of the 1-subtype (515). However, Wei et al. (1166) also reported that, when coexpressed, -subunit and 1- (cardiac type) and 1a-subunits (skeletal muscle type) acted cooperatively to change the Ca2+ current generated by an 1b-subunit. Because the -subunit has been cloned only from skeletal muscle, and this type of -subunit is not present in the stomach (515, 899), determination of the physiological relevance of the -subunit to SMC needs further investigation.
Like the -subunit, the 2-subunit has been reported to enhance the Ca2+ current without inducing a change in kinetics (494). On the other hand, Gurnett et al. (376) found that an 2- or -subunit alone induced no augmentation of the current passing through 1A- to 4-subunits. They also reported that no synergistic action was seen even when 2- and -subunits were independently expressed in the same cell from their separate cRNA, whereas current augmentation was induced after coexpression of an 2d-subunit from the same cRNA.
In the rat myometrium during pregnancy, Tezuka et al. (1082) investigated the mRNA levels for the 1- and -subunits of the L-type VOCC to determine whether alterations are associated with term or preterm labor. They concluded that mRNA levels for the VOCC subunits increase before both term and preterm labor but decline during periods when VOCC are likely to be at their peaks. The increase in the levels of mRNA for VOCC most likely reflects a change in the expression of VOCC during term and preterm labor that may facilitate the increase in uterine contractility required for this process. The so-called "progesterone withdrawal" or "progesterone blockade" appears to be responsible for regulating the levels of mRNA for VOCC in the myometrium in preparation for labor.
2. Modulation of Ca2+ channels by agonists and second messengers
Like the known regulatory actions of various receptor agonists on Ca2+ mobilization, such as activation of ligand-gated ion channels and modulation of intracellular Ca2+ store sites, the direct and indirect regulation of voltage-dependent Ca2+ channels by receptor agonists has been extensively investigated. Channel modulation by agonists can occur through one of two different types of mechanism, viz., direct modulation via G proteins or indirect modulation via either second messengers or channel phosphorylation. There is molecular biological evidence for the presence in the1- and -subunits of various sites at which phosphorylation can be induced by PKA and PKC (564). In aortic and lung 1-subunits, there are said to be, respectively, five or two putative PKA phosphorylation sites (87, 598) located in the COOH-terminal region (S1574, S1626, S1699, S1847, and S1927 in rat aorta; S1622, S1923 in rabbit lung; in the lung, other serine residues were also present in positions corresponding to those in the aortic subunit). In cardiac and skeletal muscles, it has been deduced that there are four or six predicted PKA phosphorylation sites in the COOH-terminal region and one in the NH2-terminal region or in the linker region between domains II and III (175, 741, 938, 939). However, all these predicted sites may not always be phosphorylated by PKA; in fact, in skeletal muscle, the most important serine residue is believed to be S1854 (639, 939). Furthermore, Hell and co-workers (397, 398) noted that there were two different-length forms of the neuronal 1C- and 1D-subunits (210 and 247 kDa vs. 190 and 187 kDa) and that PKA only phosphorylated the longer subunit of 1C . They considered that the shorter 1C-subunit might be created when the long form was truncated by proteolysis, whereas the shorter sizes of the other classes of 1-subunits (1A , 1B , 1D , and 1E) might be expressed by alternative splicings (397). Thus cAMP-dependent phosphorylation sites could be eliminated by posttranslational truncation of the 1C-subunit (1237).
On the basis of electrophysiological experiments, cAMP-dependent and/or -adrenergic modulation of the L-type Ca2+ channel has been well documented in cardiac muscle cells. However, the purified 1-subunit of cardiac cells is not phosphorylated by PKA (967, 1209), suggesting that the phosphorylation site for PKA in the cardiac L-type Ca2+ channel is on the -subunit rather than the 1-subunit. In SMC, injection of cAMP or superfusion with membrane-permeable cAMP (dibutyryl cAMP) either had no effect on the L-type current (guinea pig urinary bladder, Ref. 588; rabbit intestine, Ref. 846) or increased the Ca2+ current (pig coronary artery, Ref. 310; trachea, Ref. 1168). Moreover, in guinea pig taenia coli, both results have been reported to be induced by Isop and cAMP application (784, 1190). In pig coronary artery, -adrenergic stimulation and forskolin both enhanced the Ca2+ current through a cAMP-dependent process (310), whereas in the trachea and guinea pig taenia coli, enhancement of the Ca2+ current by -adrenoceptor activation did not involve a cAMP-dependent pathway (784, 1168). Recent molecular biological studies have suggested that the cAMP-dependent phosphorylation site on the -subunit is Thr-165 in skeletal muscle and neuronal 1- and 2a-subunits, a residue which is not present in the SM 3-subunit (1237). Therefore, coexpression of different types of -subunit may be a critical factor for cAMP-dependent phosphorylation of L-type SM Ca2+ channels. Interleukin-1, tumor necrosis factor-, and lipopolysaccharide also enhanced voltage-dependent Ca2+ channels in the SMC of the rat tail artery. However, these enhancements were not modulated by PGs but inhibited by dibutyryl cGMP (1173). The underlying mechanisms have not yet been clarified (1186 in vascular SMC). A nonreceptor tyrosine kinase (pp60c) injected into the SM cells of the rabbit ear artery enhanced the VOCC and, moreover, peptide A, tyrphostin-23, and genistein (inhibitors of pp60c) inhibited the Ca2+ inward current (1172). These authors concluded that this modulation of the voltage-dependent Ca2+ current was dependent on tyrosine phosphorylation, but not on activation of PKC.
It is well known that the coupling of various receptor agonists with the Gq protein-PLC system causes augmentation of the L-type channel in visceral and vascular SMC. Current augmentation is thought to occur through PKC activation and/or by direct activation via PTX-sensitive and -insensitive G proteins, but not through InsP3/inositol tetrakisphosphate (InsP4) formation (850, 851, 1129, 1184). Direct application of DAG or PDBu, a phorbol ester, has been shown to produce augmentation of the Ca2+ current, and H-7 or staurosporine prevented the current augmentation induced by PDBu (671, 851, 1129). However, Oike et al. (851) reported that histamine-induced augmentation of the L-type current was not affected by such protein kinase inhibitors in rabbit vascular cells, suggesting that PKC-mediated current activation is not the main process producing agonist stimulation. In guinea pig taenia coli, calyculin A (a phosphatase type 1 and type 2A inhibitor) augmented the L-type current, and this effect was inhibited by H-7 (1113). It is now thought that there are several PKC phosphorylation consensus regions in the 1- and -subunits (445, 564, 1030). In the case of the neuronal 1C-subtype, different sizes of the protein (190 and 210 kDa) have been reported (398), the smaller form being the result of truncation by proteolysis. Although PKA phosphorylation sites are deleted by such proteolysis at the COOH-terminal region, phosphorylation sites for PKC, CaM-dependent protein kinase, and PKG were preserved in both sizes of the subunit (377, 398). Stea et al. (1029) reported that, whereas the Ca2+ current passed by 1B- and 1E-subunits coexpressed with 1b was enhanced by PMA, that passed by 1A- and 1C-subunits was not (1030). The PKC-dependent modulating site was determined, by using a chimeric 1B/A-subunit, to be in a cytoplasmic region between domains I and II. These results indicate 1) that PKC phosphorylation sites in the 1b-subunit have no role in the current augmentation induced by PKC and 2) that PKC phosphorylation sites in the 1-subunit do not always modulate the channel's activities. As the presence of 1C , but not of other 1-subunits, has been deduced in SMC, current augmentation by stimulation of PLC-coupled receptors may not directly involve regulation via the 1- and/or -subunits. That being the case, other subunits, such as 3 and/or 2 , may affect the channel's activity during PKC-mediated channel modulation. However, the possibility cannot be excluded that alternative-spliced variants of the 1C-subunit might be affected by PKC.
It is interesting that all types of Ca2+ channel 1-subunits possess an EF-hand sequence in the COOH-terminal region; Ca2+ binding here might affect the channel's modulation (37). Study of the first-order structures of the L-type Ca2+ channel (1C-, 1S-, and 1D-subunits) has shown that there is only one amino acid difference between them in the 29-amino acid sequence of EF hand, whereas 12-amino acid differences were present between L- and non-L-type channels. Because L-type channels show strong inactivating properties on exposure to high intracellular Ca2+ (Ca2+-induced inactivation of the Ca2+ current, Ref. 846), differences in the Ca2+-binding sequences of L- and non-L-type Ca2+ channels might be important for channel inactivation by ion flux or depolarization (37).
3. Actions of Ca2+ agonists and antagonists
From the structure of the channels and the actions of Ca2+ antagonists, the Ca2+ channels in SM have been identified as L-type and1C-type channels (87, 598, 599, 925, 1009). Exceptionally, Smirnov and Aaronson (998) classed as N type the Ca2+ channels in rat ileal SMC, from their low sensitivity to DHP. The actions of Ca2+ antagonists and agonists fit well with the modulated receptor hypothesis proposed by Hille (411), and with the modal model of Hess et al. (404). The Ca2+ antagonist binding sites have been determined by physiological, biochemical, and molecular biological methods in neuronal and cardiac Ca2+ channels (177, 619, 796, 921, 1018, 1041, 1076). Concerning the DHP-binding site of the L-type Ca2+ channel, Regulla et al. (923) reported that 10 amino acids near the EF-hand structure in the COOH-terminal region formed the binding site for DHP antagonists in the skeletal muscle 1S-subunit. Later, it was proposed that the major functional interaction between DHP derivatives and the 1S-subunit occurred in its external region between S5 and S6 of domain IV (799, 1076). Electrophysiological evidence tends to confirm this idea, since DHP derivatives are effective in inhibiting the Ca2+ current only when applied externally. There are big differences between subunits that are DHP sensitive (1S-, 1C-, and 1D-subunits) and those that are DHP insensitive (1A-, 1B-, and 1E-subunits) in terms of their amino acid sequence in the SS1 region of domain IV, suggesting that a region near SS1 of domain IV (S5-6) may be a DHP-binding site. In SMC of the rabbit intestine and portal vein, D-600 has been reported to inhibit Ca2+ channels from the extracellular side (649, 845). In contrast, in porcine coronary artery, as well as in neocortical neurons and cardiac ventricular cells, D-890 (a completely charged form of D-600) inhibited the Ca2+ current from the inside of the membrane (239, 403, 589). The binding site for D-600 has been predicted to be at the junction between the extracellular and intracellular regions of S6 in domain IV (1041). Because the COOH-terminal region near S6 in domain IV is well conserved in various 1-subunits, including those of the N- and P-type channels, and because there is no difference between the amino acid sequences of cardiac and SM 1-subunits, it is hard to explain the electrophysiological discrepancy between the findings in these tissues in terms of the structure of the phenylalkylamine-binding site of the 1-subunit. Consequently, modulation by other regions of the 1-subunit or by other subunits should be considered. The effects of synthesized calciseptine (found naturally in the venom of the black mamba) on voltage-dependent Ca2+ channels was observed by Teramoto et al. (1080). Calciseptine voltage dependently and concentration dependently inhibited the inward current and shifted to the left the steady-state inactivation curve. Calciseptine blocked the open probability of the 25- and 12-pS Ca2+ channels (more potently in the 25-pS channel) without affecting the amplitude of the single-channel conductance. Thus they concluded that the actions of calciseptine are similar to those of DHP derivatives and that it acts from the outside of the membrane. Figure 7 shows the effects of nifedipine on the L- and T-type voltage-dependent Ca2+ channel recorded from the guinea pig portal vein.
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Unitary current analysis has shown that Ca2+ antagonists shift the channel to mode 0 (no channel opening) from mode 1 (short channel opening), a so-called reduction of "channel availability," whereas Ca2+ agonists shift the channel to mode 2 (long channel opening) without a change in the mean open time (404, 517). In the rabbit ileum, Inoue et al. (490) reported that DHP modified both channel availability and mean open time; this was similar to an observation made in guinea pig ventricular cells by Lacerda and Brown (637). A transition from mode 1 to mode 2 was observed on application of
-adrenoceptor agonists, cAMP, or PKA in cardiac cells (404), suggesting that channel phosphorylation by PKA changed the channel state (mode). In rat ventricular cells, a very strong depolarization alters the channel mode without cAMP or -adrenoceptor stimulation (896). Moreover, in the same study, such long channel opening was occasionally recorded under normal conditions. In rabbit ileal SMC, Inoue et al. (490) also reported that long channel opening could be observed with a modest level of membrane depolarization at low frequencies of stimulation, suggesting the possible existence of a third mechanism, other than strong membrane depolarization and channel phosphorylation.
There are two conductive channels (large and small unitary conductances) that are blocked by DHP in SMC (156, 478, 1178). The large conductive channel has characteristics similar to those of the typical L-type channel, whereas the small conductive channel has a low threshold for channel activation but shows little channel inactivation (156, 488), as also observed in rat DRG neurons (294). The latter property distinguished this channel from the other T-like currents recorded in SM and cardiac cells and other neurons. Although high concentrations of DHP derivatives inhibited other ionic currents (447, 1079, 1208), inhibition of the above small conductive channel by DHP derivatives occurred at relatively low concentrations. Because there is as yet not a clear understanding of the structure of DHP-resistant channels (T type) or of the DHP-sensitive, low-conductive channel, we must await further data before drawing firm conclusions about the differences between these channels.
D. Cl Channels
Ion-sensitive microelectrode measurements put intracellular Cl concentration at 40-50 mM, and the calculated Cl equilibrium potential from these values is 35 to 20 mV (guinea pig vas deferens, cecum, and ureter, Refs. 14-17). These results indicate a contribution by an active Cl transport system to the accumulation of Cl in these SM cells, as well as accumulation by passive transport through Cl channels. In patch-clamp experiments, a Ca2+-dependent Cl current has been identified in several SMC (rabbit portal vein, Refs. 424, 1148, 1150, 1151; rabbit colon, Refs. 1048, 1049; rat intestine, Ref. 841; rabbit pulmonary artery, Refs. 423, 424; rabbit esophagus, Ref. 18; A10 cell line, Ref. 425; guinea pig trachea, Ref. 515; pig trachea, Ref. 668).
In SMC, genes expressing two types of Cl channel have been identified by Northern blotting analysis, namely, the ClC-2-type channel and the ICln channel (492, 1085). The ClC-2-type Cl channels were first identified in rat heart and brain, although they have since been found in various tissues including stomach, intestine, and A10 cell lines derived from the rat aorta (1085). These authors showed that the expressed ClC-2 channels were markedly, but slowly, activated by membrane hyperpolarization; in this, they resembled the hyperpolarization-activated current. Like ClC-1, which is the major skeletal muscle Cl channel (1033, 1034), the ClC-2 channel usually shows inwardly rectifying properties, although some ClC-2 channels have been found with slight outward rectifying properties (324). The amino acid sequences have been determined for ClC channels, including ClC-0 (the Torpedo marmorata Cl channel, Ref. 522), ClC-K1, ClC-K2 (the specific kidney Cl channels, Refs. 6, 1112), and ClC-3 (a ClC-K1-like Cl channel, Ref. 556). They have 12 putative transmembrane-spanning regions among 13 strongly hydrophobic sites, suggesting a very unique channel structure, a so-called "double-barreled" structure (743). These ClC channels have several PKA and PKC phosphorylation sites. Recently, Furukawa et al. (324) found that a truncated form of the ClC-2 type Cl channel (ClC-2; an alternative spliced form of ClC-2) in the rabbit heart and cerebellum had poor volume dependency. Moreover, although ClC-2 could be expressed in colon and cerebellum, ClC-2 did not express in the colon (324). Another volume-dependent Cl channel, named the ICln channel, has been identified in various epithelia and in nonepithelial cells, including SMC (492, 611, 891). This ICln has neither PKA and PKC phosphorylation sites nor a long hydrophobic amino acid chain forming the common transmembrane structure, but is believed to form a channel by dimerization (301). The expressed ICln Cl channels in Xenopus oocytes showed marked outward-rectifying properties and Ca2+ insensitivity. One of the specific characteristics of ICln is that this channel has a nucleotide-binding domain with the same sequence as the cystic fibrosis transmembrane conductance regulator (CFTR) channel, suggesting cAMP or ATP modulation of channel activity (891).
The physiological role of Cl channels in VSMC is one that is related to changes in cell volume, as suggested for other cells. Recent work on epithelial cells has indicated that stabilization of F-actin prevented, whereas destruction of F-actin activated the large-conductance Cl channel (971). This indicates that actin and other cytoskeleton proteins link closely to Cl channels and help regulate the channel's activity. With regard to the role of Cl channels in determining membrane excitability, it has been noted that Cl channel blockade by many drugs produces an inhibition of channel activity and hyperpolarizes the membrane. In skeletal muscle, blockade of the ClC-1 channel produces muscle stiffness, and lack of the Clc-1 gene leads to a failure to express functional Cl channels in myotonic mice (1033), suggesting that Cl channels have an important role in muscle relaxation. On the other hand, Sun et al. (1047) reported that the PTX-sensitive, G protein-coupled Cl channel in rabbit colonic SMC was activated by substance P, a neurokinin-1 (NK-1) receptor agonist. They speculated that activation of this Cl channel might participate in the initial membrane depolarization induced by NK-1 receptor stimulation and thus activate voltage-dependent Ca2+ channels.
E. Nonselective Cation Channels
There are two major ionic currents in vascular SMC that permeate several cations nonselectively. One is a receptor-operated nonselective channel, and the other is a hyperpolarization-activated channel. Receptor-operated and G protein-coupled or intracellular second messenger-mediated channels are discussed in detail in section V. In this section, we merely mention the hyperpolarization-activated channels, the presence of which has been reported in several SMC with spontaneous excitability (rabbit jejunum, Ref. 73; rabbit portal vein, Ref. 539). This current is activated at hyperpolarized potentials that are more negative than 60 mV and in a time-dependent fashion. This current is thought to contribute to some extent to the quick recovery from the afterhyperpolarization back to the resting membrane potential and to the slow depolarization that acts as a trigger for action potential generation.
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V. RECEPTOR-OPERATED ION CHANNELS IN VISCERAL SMOOTH MUSCLE CELLS |
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It has long been known that, in many types of VSM, neurotransmitters released from peripheral autonomic nerves elicit contractions accompanied by a transient depolarization that subsequently evokes Ca2+-spike discharges, although in some cases the opposite change, hyperpolarization, is observed. In other types of SMC, on the other hand, locally produced bioactive substances (such as those produced by inflammation, mechanical stresses, hypoxia, and metabolic perturbations), and even some circulating hormones secreted from distant organs, can produce long-lasting membrane depolarizations and contractions (106). These depolarizations are attributed mainly to induction of cationic (Na+ and Ca2+) or anionic (usually Cl) conductances, and in some cases to suppression of K+ conductance, thereby increasing the rate of Ca2+ entry into the cell through voltage-dependent and -independent pathways. Subsequent attempts to characterize the electrophysiological nature of the above events using the patch-clamp technique have revealed that the pathways involved could lead to the opening of receptor-operated cation channels (ROCC), Ca2+-dependent Cl channels, or the closing of the M channel (M current), with the subsequent activation of VOCC.
The ROCC so far identified in SMC are quite diverse in their mode of activation, biophysics, pharmacology, and perhaps physiological functions. On the basis of these differences, they can be further subdivided into at least four distinct families (i.e., purinoceptor-coupled cation channels, G protein-coupled voltage-dependent cation channels, G protein-coupled voltage-independent cation channels, and Ca2+-activated cation channels; see Tables 4-6).
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A. Purinoceptor-Coupled Channels
Purinoceptors can be subdivided into two distinct classes, i.e., metabotropic and ionotropic types. The metabotropic purinoceptors (P2t , P2u , P2y , and uridine nucleotide-sensitive receptors; 7 transmembrane segment type) 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; it is capable of synthesizing InsP3 by hydrolysis of phosphatidylinositol 4,5-bisphosphate through activation of PLC. Gi/o is coupled with the activated P2t receptor; it reduces the amount of cAMP through an inhibition of adenylate cyclase (818). In contrast, ionotropic purinoceptors (P1 family type; 2 transmembrane segment type) are directly coupled with cation channels without mediation of G proteins. Genes expressing such ionotropic receptors have recently been identified and termed P2x1-P2x6 and P2x7 (previously termed P2z receptor) (97, 125, 133, 187, 209, 1053, 1115). These receptors may consist of multiunit subtypes (552). In addition, Chang et al. (185) isolated the cDNA encoding a novel P2 receptor from the rat aortic SMC library and functionally characterized it. They found it to be a member of the G protein-coupled receptor family that includes P2u and P2y receptors; this cloned P2 receptor was found to be coupled to PLC, and not to adenylate cyclase, in C6 rat ganglioma cells transfected with the cloned P2 expression vector. The rank order of agonist potency, as judged by intracellular Ca2+ mobilization responses, was UTP > ADP = 2-methylthio-ATP > adenosine 5'-O-(2-thiodiphosphate) > ATP = adenosine 5'-O-(3-thiotriphosphate) (ATPS). These results indicate that the novel metabotropic P2 receptor has pharmacological characteristics distinct from any of the P2 receptor subtypes thus far identified and suggest the existence of a novel regulatory system by which extracellular nucleotides may modulate potential difference. As agonists for the P1 receptor, ATP, ,-methylene-ATP, and 2-methylthio-ATP are commonly used, whereas the antagonists used are suramin, FPL-67156 (mainly for P2x), reactive 2 (low-affinity binding site for the P2x and high-affinity binding site for the P2y), as well as EPL-66096 and EPL-67085 (for the P2t receptor). In the rest of this section, we focus mainly on receptor-operated ionotropic responses.
1. Cation channels involved in the response to purinoceptor activation (purinoceptor-operated channel)
Benham's group (72, 75) was one of the first to clearly demonstrate a molecular entity to which such purinoceptors link. They showed, using the patch-clamp technique in rabbit ear artery SMC, that ATP activates a rapidly developing and desensitizing cationic current with a moderate inward-rectifying property and a reversal potential close to 0 mV. In activating this current, the order of potency was,-methyleneadenosine 5'-triphosphate (AMP-PCP) = ATP > ADP >> adenosine, suggesting that the receptor responsible was of the P2x subtype. The single channels underlying the ATP-activated cationic current are nonselective cation channels (NSCCATP) that discriminate poorly among cations but are not permeable for anions (however, see the alternative view of Thomas and Hume, Ref. 1087). Calculations from reversal potentials using a modified Goldman-Hodgkin-Katz equation suggested that NSCCATP have a severalfold higher selectivity for Ca2+ and Ba2+ over the monovalent cations Na+, K+, and Cs+ (e.g., the permeability ratio PCa/PNa is ~3; for other examples see Table 4). This further gave an estimation that ~6% of the ATP-induced cationic current would be carried by Ca2+ under physiological conditions. However, the unitary conductance of NSCCATP was much smaller with Ca2+ as the sole charge carrier (~5 pS with 110 mM Ca2+) than it was in physiological saline (~20 pS, with 130 mM Na+ plus 1.5 mM Ca2+). These two seemingly paradoxical observations can be interpreted as indicating the presence of a binding site (or sites) inside the NSCCATP pore with a higher affinity for Ca2+ than for Na+; such site(s) would preferentially trap Ca2+ and slow its permeation, as has been suggested for high-threshold VOCC (56, 75). Organic and inorganic blockers of VOCC, such as nifedipine (10 µM) and Cd2+ (500 µM), on the other hand, were ineffective at inhibiting the current representing NSCCATP (note that Cd2+ was later shown to reduce its amplitude, as were Zn2+, Mn2+, and La3+, Refs. 448, 800). Nakazawa and Matsuki (801) also found in the rat vas deferens an ATP-induced inward current that desensitizes rapidly and is not affected by presence of nicardipine or Cs+. The current conductance measured using the cell-attached patch-clamp procedure was 20 pS, and it reversed its polarity at ~0 mV. These results were exciting, because they seemed the first direct proof of the hypothesis of receptor-operated Ca2+ channels (106).
Subsequent exploration has revealed that, despite some differences (Table 4), ATP-activated currents (or channels) with much the same properties are ubiquitous among other types of SMC (including rat vas deferens, guinea pig urinary bladder, rabbit portal vein, guinea pig ileum, and rat aorta; Refs. 304, 476, 385, 801, 966). Further characteritics of NSCCATP were elucidated in these studies: 1) activation of NSCCATP can occur on a millisecond scale (latency for activation is shorter than 100 ms, Ref. 304; as short as 18 ms, Ref. 476); 2) binding of at least two ATP molecules seems necessary to open NSCCATP (i.e., positive cooperativity), with the half-effective concentration being of the micromolar order (4.6 µM, Ref. 641; 2.3 µM, Ref. 476; 1.1 µM, Ref. 1115); and 3) the temperature dependence of NSCCATP is low, with a Q10 value comparable to that estimated for passive diffusion in aqueous media (1.25, Ref. 966). These properties are reminiscent of those of directly ligand-gated channels, suggesting that NSCCATP is a member of a channel family that requires neither G proteins nor intracellular second messengers for activation (56, 75). It is consistent with this view that the activity of NSCCATP could be recorded from outside-out membrane patches with high concentrations of Ca2+ buffers and no added nucleotides on the cytoplasmic side (90, 800).
Recently, a cDNA encoding the P2x receptor has been cloned from rat vas deferens (1115) and human urinary bladder (282). Expression of such a clone in Xenopus oocytes was alone sufficient to reproduce the full profile of the native NSCCATP , such as agonist selectivity, single-channel conductance, kinetics, ionic selectivity, and pharmacological properties. This confirmed the previous idea that the P2x receptor is a macromolecular complex consisting of receptor and channel domains. Another striking prediction from this study was that the cloned channel has only two membrane-spanning domains, as in the inward rectifier K+ channel family. Furthermore, the putative pore, which is likely to be a part of the second transmembrane domain, contains a sequence (Thr-Met-Thr-Thr-Ile-Ile-Gly-Ser-Gly) that closely resembles a well-conserved motif of the pore region of the voltage-gated K+ channel. These results strongly suggest that the cloned P2x receptor is entirely different from the so-far cloned heteromultimeric ligand-gated channels that are activated in a membrane-delimited fashion (e.g., nicotinic, GABAergic, glycine, and glutamate receptors). Unexpectedly, the characteristic architecture of the P2x receptor shows some resemblance to the mechanosensitive channels found in Caenorhabditis elegans and also to the epithelial amiloride-sensitive Na+ channels, although these show no homology in terms of primary amino acid sequence (see Ref. 1052).
The concept of significant Ca2+ permeability through NSCCATP (based on reversal potential measurements) involves a number of not inconsiderable problems. It relied on the unrealistic assumption that it is possible to ignore any interaction between permeating ions (i.e., the independence principle for ion permeation). Furthermore, junction potentials, particularly those that arise between the cytosol and the patch pipette, and which change time dependently, were not seriously considered; this may have led to erroneous estimations of permeability ratios (805). To avoid these complications, a more straightforward approach has been attempted, involving simultaneous measurements of the ATP-induced current and the concomitant rise in [Ca2+]i using the fluorescent Ca2+ indicator indo 1 (69, 966). In rabbit ear artery SMC, it was found that exogenously applied ATP (1-10 µM) is as potent as NE (10 µM) or caffeine (10 mM) in elevating [Ca2+]i (by up to ~500 nM) at membrane potentials at which voltage-dependent Ca2+ entry is unlikely (143). This rise in [Ca2+]i seems likely to result from direct Ca2+ entry through NSCCATP rather than from a liberation of stored Ca2+, since store depletion with NE or caffeine failed to affect it, whereas elimination of external Ca2+ or the use of highly depolarized conditions (which should reduce the Ca2+ driving force) abolished it. Nevertheless, the ability of this ATP-induced current to elevate [Ca2+]i appears to be quite low. Inspection of the relationship between [Ca2+]i increment and the charge transported through NSCCATP indicates that a 1-pC entry produces only a 0.5 nM increment in [Ca2+]i in ear artery SMC. This is much smaller than the value typically reported for VOCC, which pass Ca2+ highly selectively (e.g., 3 nM/pC in guinea pig ureter, Ref. 2). A similar conclusion was reached for the urinary bladder by comparing the relative abilities to elevate [Ca2+]i shown by ATP-induced and voltage-dependent Ca2+ currents (VOCC) in one and the same preparation; the former involved a cation entry ~19 times larger than in the latter (966). These calculations can be further translated to indicate that ~10% of the ATP-induced current is carried by Ca2+ under physiological conditions. Moreover, direct Ca2+ entry through NSCCATP may be significantly attenuated when the membrane is allowed to depolarize under unclamped conditions, and in contrast, the role of voltage-dependent Ca2+ entry would then become more important. However, this is unlikely to be the case in the guinea pig urinary bladder. In this preparation, Schneider et al. (968) measured the increment in [Ca2+]i resulting from ATP-induced Ca2+ entry and found that, under current-clamped conditions, the ATP-induced current is just as effective in elevating [Ca2+]i as under a voltage clamp. The size of this increase in [Ca2+]i was found to be considerably larger than when a depolarization of similar magnitude to that induced by ATP was generated electrically.
Despite such supporting evidence, a contrary conclusion has been drawn from contractile studies in other SMC, in which ATP-mediated transmission does seem to play a physiological role. Blakeley et al. (91) reported that in the rat vas deferens, where ATP is the excitatory transmitter generating the fast EJP, nifedipine selectively abolished the fast component of the nerve-evoked twitchlike contraction by inhibiting the superimposed spike activity without affecting the amplitude of the fast EJP. Katsuragi et al. (551) demonstrated that both the ATP-induced [Ca2+]i increment and the concomitant tension development in guinea pig urinary bladder can be completely blocked by pretreatment with nifedipine or nitrendipine, although they are unaffected by -conotoxin, a blocker of presynaptic VOCC. Furthermore, a mild inhibition of ATP-induced responses caused by use of a lower concentration of nifedipine was reversed by the dihydropyridine agonist BAY K 8644. These findings are difficult to reconcile with the notion of an important direct Ca2+ entry through NSCCATP and instead favor the idea that the primary action of ATP is to depolarize the membrane and thus activate VOCC, thereby inducing contraction. We do not fully understand the background behind these disparate findings made in either single cells or in whole tissue experiments. Recently, Katsuragi et al. (550) observed the effects of ATP on guinea pig ileal longitudinal muscle segments; they concluded that the release of ATP by receptor stimulation may result mainly from an activation of PLC, coupled with a PTX-insensitive G protein, and the subsequent accumulation of InsP3 in SMC.
Another interesting effect of ATP-induced Ca2+ entry through NSCCATP has recently been reported from the rat portal and human saphenous veins (669, 872). In these SMC, the magnitude of the ATP-induced [Ca2+]i rise was greatly decreased after the use of procedures that inhibit CICR, such as pretreatment with caffeine, ryanodine, or tetracaine, whereas the inhibitor for the InsP3 receptor, heparin sodium, was found to be ineffective. These results give rise to the idea that a modest Ca2+ entry via the receptor-gated pathway could serve to initiate a subsequent large Ca2+ release from the ryanodine-sensitive stores through the CICR mechanism. Such an amplification (by way of internally stored Ca2+) of the effect on [Ca2+]i of Ca2+ entry is an attractive idea; such a mechanism may be more generally involved in agonist-induced Ca2+ mobilization than presently envisaged. A further complication may need to be added to the story of the effects mediated by ATP; in the rabbit pulmonary artery, pretreatment with ATP at a concentration too low to activate any detectable membrane currents dramatically potentiated the Ca2+-dependent Cl current activated by 1-adrenoceptor-mediated Ca2+ release (400). The mechanism underlying this synergistic effect remains unclear, but an examination of the possible involvement of second messengers such as arachidonic acid or cADP ribose might prove rewarding.
The purinoceptors distributed in SM cells may not be limited to the P2x subtype. Thus, in pig cultured aortic SMC, exogenously applied ATP causes a large Ca2+ release from the internal stores, thereby activating a Ca2+-dependent Cl conductance, but not a cationic conductance (251). Although no pharmacological characterization was attempted in this study, it is conceivable that the Ca2+ release might result from a stimulation of the production of InsP3 via a G protein-coupled subtype of the P2 receptor, the structure of which is completely different from that of P2x (e.g., P2y or P2u; Refs. 255, 384). Similar observations have been reported in studies of rat cultured aorta (1137) and in studies of freshly isolated SMC from the rabbit pulmonary artery (400) and rat aorta (876). Interestingly, the latter study suggested that expression of the P2 receptors linked to cationic channels (P2x) and InsP3 production (P2y) may vary during the course of cell culture; freshly isolated cells may express the former exclusively, but the latter gradually comes to predominate with repeated passages of reseeding and culturing. In contrast, the Ca2+ release from the internal stores in this cell, caused by P2u receptor stimulation (UTP being used as the agonist), remained constant, regardless of the progress of cell culture. It is an important implication of this study that those SMC with higher excitability might preferentially express the effectors participating in fast Ca2+ mobilization (such as VOCC, ryanodine-sensitive stores, and directly ligand-gated channels with fast kinetics, like NSCCATP) but that these might be replaced in cells in a less excitable or undifferentiated state by InsP3-producing receptors and InsP3-sensitive stores (704, 876).
Finally, we should turn to the presence of ATP-activatable cationic conductances, the properties of which are clearly different from those so far described. The ATP-induced cationic conductance recorded from rat myometrium is selective for monovalent cations and exhibits no discernible desensitization in the continued presence of ATP. Even though activation of this current is thought to involve the P2x receptor (on the grounds that ,-methylene-ATP is effective, but adenosine and ADP are not) and not to require second messengers, the receptor involved may be different from the ear artery P2x receptor (i.e., NSCCATP). The evidence for this is that only the free form of ATP, in submillimolar concentrations, seems able to induce this conductance; millimolar concentrations of divalent cations such as Co2+, Mg2+, Ca2+, and Ba2+ depressed the current severely in a manner predictable from the chelation of ATP by these metals. Another noteworthy property is that the current is significantly downregulated by treatment with estrogen or after gestation, suggesting that it has a functional significance during the nonpregnant period as well as in the menstrual cycle. These results may imply the presence of a heterogeneity among SM P2x receptors. To date, seven subtypes (P2x1-P2x7) have been cloned from rat vas deferens, PC12 pheochromocytoma cells, rat dorsal root sensory neurons, and autonomic ganglia. Although both P2x1 and P2x3 are activated by submicromolar concentrations of ATP or ,-methylene-ATP and desensitize rapidly, the P2x2 subtype is insensitive to ,-methylene-ATP or ,-methylene-ATP, requires much higher concentrations of ATP than the former two subtypes for its activation, and shows only slow desensitization (similar features have also been found for P2x4 , P2x5 , and P2x6 subtypes). Furthermore, the possibility has recently been suggested that P2x2 and P2x3 subunits may form a heteropolymer and generate a cationic conductance with hybrid properties, namely, activation by ,-methylene-ATP and slow desensitization kinetics (562, 563). Thus it seems that molecular characterization of SM P2x receptors will be an intriguing subject for future investigation.
Exogenously applied to rabbit portal vein SMC, ATP can activate, in addition to the typical NSCCATP , a long-sustained cationic current (1184). This long-sustained cationic current was activated only weakly by ,-methylene-ATP, and it did not desensitize appreciably, as shown by the response to subsequently applied ATP. Activation of this current was completely blocked by pretreatment with PTX, and its amplitude was significantly reduced by internal dialysis with GDPS. This suggests that a PTX-sensitive G protein is involved in its activation. A similar PTX-sensitive G protein-mediated potentiation of VOCC could be demonstrated in the same preparation.
In summary, the purinoceptors found in SMC exhibit considerable heterogeneity and, accordingly, their related functions may be divergent. Even if we look just at the cation channels, their roles in Ca2+ mobilization seem to differ considerably depending on the type of SMC in which they are found, presumably because the type and density of Ca2+ stores as well as of Ca2+-permeable channels may differ.
2. K+ channels involved in the response to purinoceptor activation
Adenosine 5'-triphosphate also activates other types of purinoceptors that participate in inhibitory responses in SMC. For example, in the GI tract of many species, bulk application of ATP into the bath or iontophoretic application of ATP evoked a sustained or a rapidly developed hyperpolarization, respectively, each being mainly due to an increase in K+ conductance. Yamanaka et al. (1203) compared the effects of ATP and nicorandil [a K+ channel (KATP) opener and nitro compound] on circular SMC of the guinea pig small intestine and found that both agents hyperpolarized the membrane and increased ionic conductance. In the presence of a maximally hyperpolarizing concentration of nicorandil, the membrane was still further hyperpolarized by ATP. However, these hyperpolarizing actions seem to have different underlying mechanisms: 1) that induced by nicorandil was Ca2+ dependent, but this was not the case for ATP; 2) the hyperpolarization induced by nicorandil was not prevented by apamin, but that induced by ATP was; and 3) local anesthetics inhibited the hyperpolarization induced by either agent, but with different potencies. Moreover, the IJP evoked by field stimulation were blocked by apamin but, in the presence of ATP, the amplitude of the IJP was markedly reduced, whereas in the presence of nicorandil it was only slightly reduced. Thus exogenously applied ATP and nicorandil (KATP) may well activate different K+ channels. Zagorodnyuk et al. (1222) investigated the effects of ATP on isolated SMC prepared from the human intestine; they concluded that an increase in K+ conductance is responsible for the generation of NANC-IJP, and that is due to an enhancement of the Ca2+-dependent K+ channel (see also in urinary bladder). However, the IJP and ATP-induced hyperpolarization were both insensitive to TEA and to 4-AP, whereas apamin slightly decreased the amplitude of both the IJP and the ATP-induced hyperpolarization. Further detailed investigations are required to help us better understand both the ATP-induced hyperpolarization and the ionic features of IJP in the intestine. However, if these hyperpolarizations are due to activation of the Ca2+-dependent K+ channel, it is tempting to postulate that, in these tissues, ATP may activate metabotropic purinoceptors (via Gq/11 activation) and that an increase in cytosolic Ca2+, caused by release of Ca2+ from the SR, may activate these channels, as reported in many other tissues. On the other hand, Dart and Standen (234) reported that adenosine activated K+ channels in the pig coronary artery through activation of the A1 receptor. This K+ channel possessed a small current amplitude (>2 pA, at60 mV in 143 mM K+) and was blocked by inhibitors of K+ channel (KATP) openers, but not by charybdotoxin or apamin. Therefore, they suggested that activation of the A1 purinoceptor may induce an activation of KATP in this tissue.
B. G Protein-Coupled Cation Channels
In comparison with fast ligand-gated NSCC (NSCCATP), the majority of agonist-activatable cation channels (ROCC) in SMC have much slower kinetics of activation and desensitization, and they are thus likely to be associated with G protein-coupled receptors. The properties of these ROCC are diverse, apparently in line with the variety of target organs in which they are distributed. Smooth muscles have been divided into functionally different groups, depending on the time course and pattern of their contractile responses (termed "phasic" and "tonic" muscle by Somlyo and Somlyo, Ref. 1010). In general, the phasic type of muscle (e.g., intestinal SM) is spontaneously active in terms of the firing of action potentials and contracts to spasmogenic stimuli in a rapid and transient manner. In contrast, tonic muscle, such as that found in large blood vessels, is electrically quiescent or less excitable (showing a graded electrical response) and contracts with a slow and maintained time course. The molecular basis of these differences could be explained by a differential contribution by VOCC (or action potentials), or by two internal stores, or by distinct properties of the contractile system in the two types (1014). We have noticed, in addition, that the distribution of G protein-coupled ROCC may also differ between these two muscle types; there is a preferential distribution of voltage-dependent cation-permeable channels in phasic muscle, but of voltage-independent ones in tonic muscle. As described below, these channels also appear to differ in their other properties, such as [Ca2+]i sensitivity and, presumably, Ca2+ permeability (Tables 5 and 6).
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Briefly, G proteins are composed of - and ,-subunits. The former come from the Gs family (Gs-1 to Gs-4 and Golf), the Gi family (Ggust , Gt-1 , Gt-2 , Gi-1 to Gi-3 , Go-1 , Go-2 , and Gz), the Gq family (Gq , G11 , G14-16), or the G12 family (G12 and G13). The ,-subunit is composed of 1-5 and 1-10 (for reviews, see Refs. 18992449). There are more than 50 low-molecular-weight soluble G proteins (mol wt 20,000-30,000); these proteins do not contain subunits and are placed into ras, rho, rab, and other families. It is now clear that - and ,-subunits, by their association and dissociation, not only modify the metabotropic process (via activation of phospholipase A2 , PLC, and others) but also directly and indirectly regulate ion channel activity (Ca2+, K+, Na+, Cl, or nonselective receptor-operated ion channels).
Many subtypes of the G protein constituent subunits are present in SMC, and a number of subtypes of the relevant agonist receptors are also distributed in excitable and nonexcitable cells. Thus it is reasonable to suppose that a large variety of receptor-G protein-coupled ionic responses, produced either directly or indirectly through the synthesis of second messengers, may underlie the range of VSMC responses. For example, among muscarinic receptors, M1 , M3 , and M5 receptors are coupled with the Gq/11 family and synthesize InsP3 , whereas M2 and M4 receptors are coupled with the Gi/o family and induce an increase or decrease in K+ channel activity and a decrease in Ca2+ channel activity, as well as a decrease in the amount of cAMP. If we look at adrenoceptors, we find that the 1-adrenoceptor subtypes (1A , 1B , and 1D) are coupled with Gq/11 , whereas the 2-adrenoceptor subtypes (2A , 2B , and 2C) are coupled with Gi/o , and the -adrenoceptor subtypes (1 , 2 , and 3) are coupled with Gs (activation of adenylate cyclase). Furthermore, among the 5-HT receptor subtypes, 5-HT1A , 5-HT1B , 5-HT1D , 5-HT1E , and 5-HT1F are coupled with Gi/o , whereas 5-HT2A , 5-HT2B , and 5-HT2C are coupled with Gq/11 . Moreover, 5-HT4 , 5-HT6 , and 5-HT7 are coupled with Gs , but 5-HT3 is directly coupled with NSCC. Among purinoceptors, a variety of relationships between individual receptor types and coupled G proteins have been described (1184). Thus activation of a given receptor modifies an ionic channel through activation of the receptor's coupled G protein. This may involve 1) direct regulation of ion channels without the synthesis of second messengers (e.g., ROCC), 2) regulation of ion channels through an increase in the Ca2+ concentration in the cytosol via the actions of InsP3 and initiated by activation of Gq/11 (e.g., K+ channels, Cl channels), or 3) regulation of ion channels through an increase or decrease in the amount of cAMP induced via the actions of Gs or Gi/o (e.g., K+ channels, Ca2+ channels). Section VB3A includes a discussion of the G protein-coupled processes described in section VB1.
In relation to G protein-coupled responses, there are three recognized classes of PLC isozymes, referred to as , , and (654). Sternweis and Smrcka (1034) and Noh et al. (826) reviewed that a variety of studies have shown that the -isozymes (PLC-1, -2, -3, and -4) are activated by the - or ,-subunit of heterotrimeric G proteins. Thus ligands, such as AT II, that bind to seven transmembrane receptors are thought to activate the PLC- isozymes. In contrast, the PLC- isozymes (PLC-1 and -2) appear to be activated by tyrosine phosphorylation. These forms of PLC play a major role in the InsP3 generation of growth factor receptors, such as the receptor for FGF or PDGF. Growth factor receptors contain intrinsic tyrosine kinase activity that leads to PLC- activations. The activation of the -isozymes of PLC is, at present, not well understood.
1. Voltage-dependent, [Ca2+]i-sensitive receptor-operated nonselective cation channels
Most of the ROCC that may fall into this group have been discovered in spontaneously active SMC, such as those of the GI tract and portal vein (Table 5). Arguably, tracheal ROCC from several species might be somehow related to this group, if we use a looser definition. The phasic contractile activity (or spontaneous contractions) of gut SM is thought to be closely associated with the amplitude and frequency of the spike discharges superimposed on slow membrane potential oscillations (slow waves) (135, 136). This is because the spike activity reflects the rate of voltage-dependent Ca2+ entry into the cell through high-threshold VOCC, which itself results in a periodic increase in [Ca2+]i and the resulting contractions (e.g., Ref. 414). Spasmogens capable of augmenting phasic activities are often able to depolarize the membrane, thereby altering the frequency and pattern both of the slow waves and of the spike activity (269, 440). One of the major electrophysiological changes underlying such depolarizing actions in SMC is increased cation entry (Na+, Ca2+) via receptor-gated pathways, as postulated from reversal potential and radioactive ion flux measurements in whole tissue studies (106, 107). The first clear proof for the presence of a receptor-inducible cationic conductance was provided by a patch-clamp experiment in rabbit jejunal longitudinal SMC. In these SMC, iontophoretic application of ACh to single cells was found to depolarize the membrane to close to 0 mV, with an associated decrease in membrane resistance, and to elicit spike discharges and contractions; all these features were consistent with the results of previous microelectrode experiments (105). Switching to the voltage-clamp mode revealed that ACh elicited an inward current passing cations nonspecifically (as deduced from the reversal potential) in a potential-dependent manner (Icat). An Icat with almost identical properties was later recorded from guinea pig ileal SMC (481) in a study in which single-channel recordings were made under whole cell clamp conditions. The results of this study suggested that the macroscopic inward current activated via muscarinic receptors is not a mixture of individual Na+-, K+-, or Ca2+-selective currents but probably reflects simultaneous openings of 20- to 30-pS nonselective cation channels (NSCCs) having nonspecific cationic permeability, as well as voltage-dependent kinetics that would account for the kinetics of the macroscopic current. Subsequently, a similar type of muscarinic Icat channel (or channels) was found in the canine pyloric sphincter (1134); in gastric (984), colonic (651), and guinea pig gastric SMC (569); and (unexpectedly) in chromaffin cells as well (471). These findings suggest that this type of NSCC may be ubiquitous throughout the whole gut (Table 5). It was originally reported that, in rat anococcygeus muscle, sympathetic stimulation or iontophoretically applied NE can depolarize the membrane through two distinct ionic mechanisms (111, 112). The initial fast depolarization was attributed to Ca2+-activated Cl conductance (112); this was later confirmed in single-cell experiments. Subsequently, it was reported that a similar response can be recorded in vascular SMC, such as those of the guinea pig pulmonary and rat mesenteric arteries (153). The second component of the NE-induced depolarization is now thought to be induced by activation of cationic currents via the 1-adrenoceptor (1-adrenergic Icat) (rabbit portal vein, Refs. 153, 1150; rabbit ear artery, Refs. 22, 23, 1149, 1150). The NSCC underlying the 1-adrenergic Icat has been found to have a unitary conductance of ~25 pS (in saline), to be modestly dependent on the membrane potential, and to be also activated by muscarinic agonists (417, 482). These properties imply that some similarities may exist between the muscarinic Icat and the 1-adrenergic Icat (482).
A muscarinic receptor-inducible cationic component, which was unfortunately not clearly separated from the coexisting Cl conductance, has been reported in canine and guinea pig tracheal SMC (512). Although its details remain unclarified, this current component may be voltage-dependent (512) and permeable to divalent cations (see below), and it thus could be placed in the same group as the muscarinic Icat and 1-adrenergic Icat .
In the following sections, more details are given of the properties of these Icat .
A) INVOLVEMENT OF G PROTEINS IN ACTIVATION. Activation of the gut muscarinic Icat occurred with a latency of ~500 ms, required several seconds to reach maximum, and desensitized only slowly (over several tens of seconds) despite continued receptor stimulation (74, 474, 475). A similar temporal profile of activation has been described for the 1-adrenergic Icat in rabbit portal vein (latency ~900 ms, rise time ~4 s, half decay time 11 s; Ref. 1150). These kinetics are much slower than those typically reported for directly ligand-gated ion channels, such as NSCCATP , but may be faster than the responses mediated by second messengers, such as InsP3 and cAMP. An analog of this type of activation kinetics is seen in the muscarinic K+ channel (KACh) that is thought to couple directly to a PTX-sensitive trimeric G protein (Gk) in a membrane-delimited manner (620). In general, occupation of G protein-coupled receptors by agonist results in an increased rate of GDP release from the -subunit, to which GTP binds in exchange, thus promoting the dissociation of the GTP-bound form of the -subunit from the ,-subunit. Although subsequent interaction of activated G proteins with target effectors was initially believed to be mediated solely by the GTP-bound form of the -subunit, it is now known that the ,-subunit can also regulate a number of cellular effectors, including KACh (919; see sect. III).
The possible involvement of G proteins in the muscarinic activation of Icat has been examined in the light of such G protein kinetics, using strategies similar to those employed to investigate KACh (477, 478, 604, 606). The following list summarizes several supporting observations made in these studies. 1) Intracellular perfusion with GDPS, a nonhydrolyzable inactive form of GTP, or with preactivated PTX incubated with NAD+ and the sulfhydryl reagent, dithiothreitol, completely inhibited the activation of Icat , when the muscarinic receptor was repeatedly stimulated. 2) Noisy and sustained cationic currents (IGTP
S) could be induced by continuously dialyzing GTPS, an active nonhydrolyzable form of GTP, into the cell. Activation of IGTPS was greatly promoted by application of muscarinic agonists. 3) Detailed comparison has revealed that IGTPS and the muscarinic Icat are almost identical in terms of many of their pharmacological and biophysical properties (170, 484). 4) Second messengers such as InsP3 , cAMP, and Ca2+ were ineffective at activating Icat (478). These four results are compatible with the idea that the muscarinic activation of Icat occurs via a PTX-sensitive G protein, probably without mediation by second messengers, as postulated for KACh . However, the possibility cannot entirely be excluded that cell membrane-localized production of unknown signaling molecules (e.g., arachidonic acid or fatty acids) may play the fast second messenger role in activating Icat , as reported for stretch-activated K+ channels (859). Indeed, in guinea pig ileal SMC, it has been reported that muscarinic activation stimulates the production of arachidonic acid, thereby increasing Ca2+ influx into the cell (569). Thus it will be necessary in the future to reexamine the precise role of G proteins in muscarinic Icat activation on a more fundamental basis, using, for example, single-channel recording or molecular characterization of muscarinic NSCC.
As for the Icat in other SMC, no experiments have been conducted to test explicitly the involvement of G protein. However, some recent studies have indicated that, in some tissues at least, GTPS can activate cationic currents that closely resemble an agonist-induced Icat (guinea pig stomach, Ref. 1172; rabbit portal vein, Ref. 1185).
B) CA2+ PERMEABILITY. Originally, the Ca2+ permeability of muscarinic NSCC was thought to be high, since sizable inward currents flow during the application of muscarinic agonists with just Ca2+, Ba2+, or Mn2+ in the bath (474, 475, 479). The relative permeability ratios calculated from the reversal potentials suggested that Ca2+ may be a few times more permeable than monovalent cations through this channel (70, 474). Moreover, the rapid removal of external Na+ from the bath reduced the amplitude of the muscarinic Icat by 90%, suggesting that ~10% of the current would be carried by Ca2+ under physiological ionic conditions (474, 475). However, these conclusions may now be invalid, since the assumption of independent ion permeation is unlikely to be tenable. In addition, [Ca2+]i measurements made using fluorescent Ca2+ indicators have clearly revealed that, after store depletion, muscarinic agonists are unable to elevate [Ca2+]i by more than a few tens of nanomolar (869). These observations strongly suggest that any direct contribution made by Ca2+ entry through muscarinic NSCC to the initiation of contractions may be only minor. Instead, membrane depolarization resulting from stimulated Na+ entry through the NSCC channel, which would in turn increase Ca2+ influx through VOCC, may have more physiological importance. The data from whole tissue experiments are indeed consistent with this view. It has been shown that the contractile responses induced by muscarinic stimulation are exclusively sensitive to blockade by organic Ca2+ antagonists, in most gut SMC (124). However, the possibility cannot entirely be excluded that local increases in [Ca2+]i , caused by Ca2+ entering through the NSCC and undetectable by global [Ca2+]i measurements, might play an important role if they occur in close proximity to the NSCC. Such local roles might include regulating local membrane conductances (808) or possibly the activities of pumps, carriers, and enzymes. In this regard, the use of membrane-specific fluorescent dyes (e.g., C18-fura 2, Ref. 279) may help future workers explore the cellular destinations of the Ca2+ that enters through muscarinic NSCC, as well as enabling a more accurate estimation of the degree of Ca2+ permeability.
A relatively high selectivity for Ca2+ over monovalent cations (PBa/PNa = 4.6) has been suggested for the 1-adrenergic Icat (1150). However, no [Ca2+]i measurements have yet been made to test this notion. A divalent cation permeability is likely to exist, too, in the canine tracheal muscarinic Icat (Fig. 8A), and agonist-stimulated Ca2+ entry through a pathway distinct from VOCC or Na+/Ca2+ exchange has been suggested by fluorescence measurements (791). The significance of these observations remains to be determined.
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C) VOLTAGE DEPENDENCE. As originally found in the rabbit jejunum, the chord conductance of the muscarinic Icat declines considerably as the membrane is hyperpolarized. This gives a typical U-shaped current-voltage relationship, with an apparent inward peak at around
60 to 20 mV (74, 433, 569, 651, 984). Detailed analysis of the voltage-dependent inactivation of Icat in the guinea pig ileum has suggested that the availability of muscarinic NSCC could be determined by the position of an equilibrium between at least two distinct states: one open (A-R*) state and one closed (A-R) state. In this scheme, the rate of closing [i.e., the transition from the open to the closed states (a)] might more critically govern the overall voltage dependence than the rate of opening (b)
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60 to 50 mV) match closely the resting membrane potentials (Vrest) measured in whole tissue experiments. This suggests that small excursions from Vrest could lead to profound potentiation or attenuation of the depolarizing ability of ACh. In support of this view, Inoue and Isenberg (479) observed that a slight electrical membrane hyperpolarization (~10 mV) greatly retarded the rate of ACh-induced depolarization, which was, however, dramatically accelerated upon termination of the electrical hyperpolarization. A similar effect was also observed when the membrane was chemically hyperpolarized with the K+ channel opener pinacidil (Fig. 1A in Ref. 485). These results suggest that the physiological importance of the voltage-dependent property of the muscarinic Icat may lie in its involvement in positive- and negative-feedback mechanisms. It may serve as a self-reinforcing mechanism for cholinergic EJP by accelerating and prolonging the rising and maintained phases, respectively. It may also serve to finely shape the time course and magnitude of ACh-induced depolarization in concert with spike discharges, slow membrane oscillations (slow waves), or -adrenoceptor-mediated hyperpolarizations (138); in this, it might act together with [Ca2+]i-dependent potentiation. One such example has been suggested in canine colonic SMC, where the enhanced amplitude and prolonged duration of the slow waves induced by ACh have been attributed to the potentiation of a small Icat through voltage-dependent as well as [Ca2+]i-dependent mechanisms (651).
The molecular mechanism responsible for voltage-dependent gating of the muscarinic Icat is unlikely to involve a permeation block by external Mg2+ (cf. N-methyl-D-aspartate-activated NSCC; Refs. 711, 830), since removal of external Mg2+ does not affect this property (74, 1231). However, it must be conceded that a thorough chelation of trace amounts of Mg2+ with EDTA was not attempted in these studies. Taken at face value, these studies suggest that voltage dependency is probably an intrinsic property of NSCC.
It is worth noting, however, that at least two factors can profoundly affect the voltage dependency of Icat . Zholos and Bolton (1230) have found that higher intensity muscarinic receptor stimulation produces a marked negative shift of the steady-state activation curve for Icat without affecting the slope factor, whereas desensitization produces the opposite effect. These observations, together with the finding that photolytically released GTP and GDPS mimic the effects of more intensive muscarinic activation and desensitization, respectively, led the authors to propose that the concentration of active G protein -subunits (i.e., of the GTP-bound form) might critically determine the state of voltage dependence of the channel. Although no molecular information is yet available, it would be intriguing to examine whether such GTP dependence could result from the multimeric nature of a G protein/NSCC complex, as has been proposed for the cardiac KACh channel (620). Another important factor that might affect the voltage dependence of Icat is the possible interaction of cations in the course of permeation. Although not definitely determined in the previous studies, it is likely that Cs+ is the most potently conductive cation through the muscarinic NSCC in the guinea pig ileum (1153, 1231). The magnitude of Icat with Cs+ as the sole charge-carrying cation is, at 60 to 50 mV, severalfold larger than that seen with Na+, which is always accompanied by a marked negative shift in the channel's voltage dependence. We investigated this property in some detail by changing the molar ratio of Na+ to Cs+ while keeping the total cationic concentration constant (Fig. 9). The amplitude of Icat measured with various concentrations of Na+ and Cs+ shows a concave relationship with no obvious nadir, a significant deviation from the linearity expected from the independence principle. This nonlinear relationship is suggestive of interactions between permeating cations inside the channel pore by which the voltage dependence of Icat might be affected. It is now becoming increasingly probable that an interaction between permeant cations is a common feature of nonselective cation channels, such as the InsP3 receptor, ryanodine receptor RyR, NSCCATP , and vasopressin-activated NSCC (see below). This being so, cation-to-cation interaction in muscarinic NSCC deserves further, detailed investigation.
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Some modulatory effects of divalent cations on the voltage dependence of Icat have also been noted. Calcium was found to inhibit a Cs+-carrying Icat in the guinea pig ileum in a fashion not predictable from the surface-charge neutralizing effects (1231; see below). The
1-adrenergic Icat was originally thought to be less voltage dependent (although some inward rectification has been noted in the ear artery: Amédée et al., Ref. 23). However, a clear voltage dependence has been detected by selective recording of cationic conductance in the rabbit portal vein, using either tail-current analysis (482) or slow-ramp protocols (399). As was the case for the muscarinic Icat , the voltage dependence of the 1-adrenergic Icat followed a Boltzmann-type sigmoid curve (399). However, as noted by Inoue and Kuriyama (482), the Vh values are variable, due presumably to the condition of the individual cells examined. It is conceivable that metabolic conditions in the cell, such as the level of phosphorylation of NSCC, might contribute to such a variability in voltage dependence.
Figure 8B illustrates our unpublished preliminary data on the voltage dependence of the ACh-induced Icat , evaluated by the use of a slow-rising ramp voltage in the canine trachea; a clear suppression of the current-voltage curve at more negative potentials can be seen.
D) [CA2+]I DEPENDENCE. The magnitude of the muscarinic Icat decreases greatly when high concentrations of Ca2+ buffers, such as EGTA or BAPTA, are loaded into the cell (480, 651, 671). This does not mean that Ca2+ is the primary activator for muscarinic NSCC, since maneuvers that elevate [Ca2+]i cannot activate Icat in the absence of muscarinic agonists. For example, Ca2+ entry through VOCC, which does not in itself induce a discernible Icat , can cause a marked augmentation of the amplitude of Icat , when accompanied by muscarinic receptor stimulation (480, 569, 651, 663). The relationship between the degree of this potentiation and the Ca2+ charge transported through VOCC indicates that half-maximal potentiation would occur at 2-4 pC (480). This value is comparable to the charge carried by a single action potential (2.5-3 pC, assuming a cell input capacitance of 50 pF and a spike amplitude of 50-60 mV). A potentiation of Icat was also observed when [Ca2+]i was elevated by using caffeine to release stored Ca2+. Conversely, the amplitude of Icat was depressed, both when the store was depleted by pretreatment with caffeine or ryanodine (480, 870) and when InsP3-dependent Ca2+ release, which occurs concomitantly with the activation of Icat , was prevented by intracellular application of heparin sodium (651, 870). These observations point strongly to the physiological importance of Ca2+ entry through spike discharges and Ca2+ release from internal stores as steps in a positive-feedback mechanism, the effect of which is to increase the rate and prolong the duration of the ACh-induced depolarization. Presumably, the prolongation of action potentials or slow waves by ACh (651) or the occurrence of inward current oscillations during muscarinic activation (605) may result, at least in part, from this mechanism.
A more obligatory role for Ca2+ has been proposed in respect of muscarinic NSCC activation in canine gastric SMC (984), on the basis that the Icat evoked by ACh is greatly diminished in Ca2+-free solution or after pretreatment with caffeine, and then resembles the current evoked by caffeine in both magnitude and appearance. Such a tight link to the internal store implies that release of Ca2+, rather than a G protein-mediated pathway, might play a primary role in activating Icat in this muscle.
Quantification of the steady-state [Ca2+]i dependence of the muscarinic Icat has been attempted by dialyzing into the cell various ratios of EGTA to Ca2+ (guinea pig ileum, Ref. 470). The relationship between Icat conductance and [Ca2+]i constructed in this way could be described by a Michaelis-Menten-type curve with half-maximal and maximal levels of [Ca2+]i of 100-200 nM and 1 µM, respectively. These concentrations more or less cover the physiologically attainable [Ca2+]i range, thus confirming that [Ca2+]i-mediated Icat potentiation may indeed occur under physiological conditions.
The [Ca2+]i dependence of the 1-adrenergic Icat was originally thought to be negligible, since inclusion of even high concentrations of EGTA cannot prevent the activation of this current (26, 482). However, it was later shown that buffering of the Ca2+ in the pipette to a level lower than the physiological [Ca2+]i (14 nM) results in a larger and more sustained Icat (399). Conversely, in Ca2+-free solution, the 1-adrenergic Icat was first potentiated but eventually abolished after prolonged exposure (1150). The exact background behind these phenomena is as yet unknown.
E) DEPENDENCE ON HIGH-ENERGY PHOSPHATES. It is generally recognized that the amplitude of ionic currents decreases gradually over time, during continued cell dialysis from the patch pipette. This phenomenon is called rundown and is thought to reflect the washout of diffusible intracellular constituents needed to maintain the channel's activity (806). A relevant example is the decline in the amplitude of Icat found to begin on switching from nystatin-perforated to conventional whole cell recordings, with the muscarinic receptor being stimulated repeatedly (479). The rate of decline is appreciably accelerated when there is no inclusion of high-energy nucleotides, but considerably slowed by the addition of millimolar Mg-ATP to the solution in the patch pipette (483). This suggests a critical requirement for high-energy phosphates in the maintenance of Icat . Recently, Bakhramov (38) made an extensive screening of compounds that would be expected to affect the intracellular energy state, using a 5-min intracellular dialysis protocol. Interestingly, creatine phosphate was found to be highly effective in upregulating Icat , whereas creatine was ineffective. A somewhat puzzling observation was that millimolar Mg2+ added in the pipette did not facilitate this upregulation, but rather suppressed it significantly. On the other hand, dialysis of ATP at concentrations lower than 1 mM resulted in an Icat of increased amplitude, whereas concentrations higher than 1 mM caused a dose-dependent inhibition. The inhibitory effect of ATP apparently counteracted the potentiating effect of creatinine phosphate but could not be mimicked by either of the nonhydrolyzable analogs AMP-PNP and ATPS. These results can be best taken to indicate the presence of at least three regulatory sites for Icat in the cell: two inhibitory sites (for Mg2+ and ATP) and one facilitatory site (for creatine phosphate). The latter two might involve some phosphorylation/dephosphorylation reactions, but the actual details remain unclear.
With regard to the possible role of phosphorylation in maintaining the activity of the muscarinic NSCC, a recent study of the guinea pig ileum is worth mentioning. In these cells, tyrosine kinase inhibitors (such as genistein, tyrphostin A25, and lavendustin) suppressed the muscarinic Icat dose-dependently (IC50 = ~5 µg/ml or 18.5 µM for genistein), whereas a tyrosine phosphatase inhibitor, orthovanadate (30 µM), augmented it (483, 486). Inhibitory effects of almost the same magnitude as those exerted by tyrosine kinase inhibitors have been demonstrated in a similar type of tissue (intestinal SM of the guinea pig taenia coli). These inhibitory effects were exerted on carbachol-induced contractions, which depend exclusively on external Ca2+ (124) and are accompanied by an increased level of phosphotyrosine (276). These observations suggest that tyrosine kinase activity may be of great functional importance in regulating muscarinic receptor-stimulated Ca2+ influx, or muscarinic NSCC, at least in intestinal SM. In addition, the converse of the effect exerted by tyrosine kinase inhibitors [i.e., activation of a large-conductance NSCC (139 pS with 150 mM Na+)] has been reported from cultured coronary arterial SMC, but the physiological relevance of this channel, and therefore of such activation, is unclear (745).
The mechanism underlying the rundown of the muscarinic Icat may be related, in part, to the washout of diffusible Ca2+-binding proteins such as CaM, since the introduction of this protein into the patch pipette greatly reduced the rundown of Icat in guinea pig gastric SMC (569). This preventive action of CaM may be linked tightly to Ca2+ influx across the plasma membrane, since Icat was inhibited dose-dependently when the external Ca2+ concentration was reduced in a stepwise manner. Furthermore, a CaM antagonist, W-7, produced a marked suppression of Icat , although it was almost ineffective when Icat was activated in a way that bypassed the receptor (viz., by internally perfusing GTPS). The most common modes of action of CaM involve either a direct interaction with the target or an alteration in the phosphorylation/dephosphorylation balance of the target proteins via stimulation of Ca2+/CaM-dependent protein kinase II (126) and of a phosphatase (i.e., calcineurin). In this context, the results of this study (569) may most simplistically be interpreted to indicate that a certain concentration of the Ca2+/CaM complex is required to maintain the activity of muscarinic NSCC and that this maintenance occurs in both phosphorylation-dependent and -independent ways. If correct, this interpretation may help to partly explain the molecular basis of [Ca2+]i dependence.
F) MECHANOSENSITIVE MODULATION. The muscarinic Icat has recently been reported to be subject to mechanosensitive modulation, i.e., hypotonic exposure augments the amplitude of Icat in parallel with cell swelling (518, 1153). This potentiating effect is unlikely to be the consequence of an indirect effect of hypotonic swelling, such as dilution of cellular constituents, since an increased rate of superfusion can produce a similar potentiating effect (Fig. 10). Although the details of this potentiation still remain unclear, it may be a more ubiquitous type of regulation of receptor-operated, as well as voltage-gated channels (e.g., maxi-K+ channel, Ref. 570) than has been previously envisaged (771). In fact, in some preliminary experiments, we have observed that the 1-adrenergic Icat is also potentiated by hypotonic exposure (Y. Waniishi and R. Inoue, unpublished data).
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G) PHARMACOLOGY, INCLUDING BLOCKADE BY INORGANIC CATIONS. Some detailed information has been recently provided about the pharmacological profile of the muscarinic Icat (170, 568, 651). Many frequently used K+ channel blockers (TEA, 4-AP, procaine, quinine, and quinidine), VOCC blockers (verapamil, nicardipine, Cd2+, and Ni2+), Ca2+-releasing agonists or inhibitors (caffeine, procaine), and Cl
transport (or channel) blockers (fenamates, DIDS) have all been found to be effective at inhibiting the muscarinic Icat . The site of inhibition is unlikely to reside on the muscarinic receptor, since the GTPS-induced Icat exhibits similar pharmacological sensitivities (170, 568). Among these blockers, quinine and its stereoisomer quinidine seem to be relatively selective for the muscarinic Icat , since their IC50 values (0.25-1 µM) are significantly lower than the typical values reported for their actions on other classes of channel (214). In contrast, the IC50 values for the other blockers coincide almost exactly with the concentrations in which they are normally used. For example, the concentrations of TEA, 4-AP, procaine, and caffeine that effectively inhibit Icat fall within the range 1-10 mM, and those of verapamil and nicardipine fall in the micromolar range. These results suggest either that the inhibitory actions of standard K+ and Ca2+ channel blockers may not reflect a strict discrimination between different channel structures, or alternatively that the basic design of the channel pore region might not differ greatly among the different types of cation-permeable channels. In this context, the reader should be reminded that the amino acid sequence of the putative pore of NSCCATP shows a striking similarity to the common motif for the pore region of the voltage-gated K+ channel (1115). In any case, special care should be taken when examining the contribution or role of ROCC with the aid of these pharmacological tools.
Fenamates, such as mefenamic acid and flufenamic acid, are derivatives of diphenylamine-2-carboxylate (DPC) and have been proposed as potent inhibitors of the Ca2+-activated NSCC in rat exocrine pancreatic cells. Chen et al. (190) investigated the effects of these compounds, together with those of other DPC derivatives, on the muscarinic Icat in the guinea pig ileum. Among them, flufenamic acid was found to be relatively selective for Icat (IC50 = 32 µM). With regard to its inhibitory efficacies, the ratio for flufenamic acid (muscarinic Icat over voltage-dependent Ca2+ current) was found to be 7.9 at a concentration that causes a >80% reduction in this Icat (100 µM). In contrast, the ratio for quinine (at 10 µM) was found to be slightly inferior at 4.5. Interestingly, a recent investigation in the rabbit portal vein revealed that fenamates exert potentiating effects on the 1-adrenergic Icat (1192). The possession of such dual actions by fenamates suggests that DPC derivatives might be useful as a starting point in the design of more selective antagonists and agonists for the voltage-dependent type NSCC in SM.
In addition to those of organic blockers, complex effects of divalent cations on voltage-dependent Icat have been reported. In the guinea pig ileum, standard VOCC blockers, such as Zn2+, Cd2+, Ni2+, Co2+, and Mn2+, have been found to inhibit the muscarinic Icat with IC50 values of 38 (Inoue, unpublished data), 98, 131, 700, and 1,000 µM, respectively (473, 474, 1153). This inhibition is likely to occur through occupation of a single binding site, probably located almost outside the transmembrane electrical field (i.e., Hill coefficient of ~1.0, almost voltage-independent inhibition). A similar type of inhibition has been reported for the muscarinic Icat in the canine colon (Cd2+, Ni2+, ~100 µM; Ref. 651), and for the 1-adrenergic Icat in rabbit portal vein (Cd2+, 100 µM; Ref. 482). In contrast, the effects of Ca2+ are more variable. It was originally reported by Inoue (473) that, in Na+-rich external solution, millimolar concentrations of Ca2+ potentiate the muscarinic Icat in the guinea pig ileum in a way that is probably independent of [Ca2+]i . However, this observation was later questioned by the finding that the effect of Ca2+ on the muscarinic Icat in Cs+-rich external solution is solely inhibitory (1231). Similar conflicting results have very recently been presented for the hydrogen ion (479, 1232). A study of the literature reveals that potentiating and inhibitory effects of Ca2+ may occur in neuronal-type and muscle-type nicotinic NSCC, respectively. The most plausible mechanisms to account for these effects could well be 1) a permeation block caused by competition for occupancy of the channel (pore) between Ca2+ and monovalent cations and 2) a direct facilitatory modulation of cationic conductance by Ca2+ (229, 1127). A similar situation may exist for the muscarinic Icat , since significant interaction seems to occur between permeant cations in the NSCC pore. It is consistent with this idea that a recent patch-clamp study in the rabbit portal vein has revealed that the 1-adrenergic Icat is indeed dually regulated by external Ca2+; micromolar Ca2+ augmented the current, whereas millimolar Ca2+ inhibited it (399). Obviously, more information will be needed before we can completely untangle such complex aspects of ion permeation through the muscarinic as well as the 1-adrenergic NSCC.
In summary, the Ca2+ permeability of voltage-dependent NSCC may not be particularly important in directly providing Ca2+ for the contractile system. Instead, their enhancement of voltage-dependent Ca2+ entry through their depolarizing actions seems likely to have a major functional importance. Because the membrane potential, the [Ca2+]i , and the mechanical load are all changing dynamically in spontaneously active SM, the presence of immediate positive-feedback controls over the NSCC (voltage dependence, [Ca2+]i dependence, and mechanosensitivity) may be of great benefit for the effective tuning of their kinetics; this probably works in close cooperation with other Ca2+-handling effectors, such as VOCC, Ca2+ stores, and perhaps Na+/Ca2+ exchange (485).
2. Voltage-independent, [Ca2+]i-insensitive receptor-operated NSCC
Potent vasoconstrictors such as endothelin (ET) and arginine vasopressin (AVP) can produce a long-maintained contraction of tonic SM, such as rat aortic SMC, which appears to be associated with either a monophasic transient increase in [Ca2+]i (694, 1141) or a biphasic rise in [Ca2+]i , consisting of transient and long-sustained phases (548, 983, 1141). The transient [Ca2+]i rise is thought to reflect mainly liberation of stored Ca2+ via production of InsP3 , whereas the long-sustained rise in [Ca2+]i reflects a Ca2+ entry from the extracellular space that is partly sensitive to dihydropyridine inhibition (407, 944). Van Rentergem and co-workers (1120, 1121) explored the mechanisms underlying the contractile and [Ca2+]i responses to these vasoconstrictors by means of the patch-clamp technique in an established culture line of vascular SMC, namely, A7r5 cells. In addition to the inhibitory effects on VOCC, two common electrophysiological changes were observed in response to the ET agonist ET-1 or AVP. These changes were 1) an early hyperpolarization accompanied by cessation of spontaneous spike activities, and 2) a subsequent slow depolarization with superimposed increased spike discharges. The hyperpolarization probably reflects a secondary activation of Ca2+-dependent K+ channels due to liberation of stored Ca2+ via the InsP3-dependent mechanism, whereas the depolarization appears to be associated with an increased permeability to monovalent as well as divalent cations through NSCC. The AVP-induced cationic current could still be recorded when the cell was dialyzed with 10 mM EGTA, but it was not activated by the Ca2+ ionophore A23187 or by InsP3 alone. This suggests that activation of AVP-activated NSCC is unlikely to be mediated by an elevation in [Ca2+]i due to InsP3-mediated Ca2+ release, although such Ca2+ insensitivity has not been tested in the case of the channels activated by ET-1. The activity of the ET-1-induced cationic current seems to be independent of the membrane potential, since the reported current-voltage relationship is almost linear. These results suggest that the NSCC activated by ET-1 or AVP may constitute a new family of ROCC, capable of passing divalent cations and independent of voltage or [Ca2+]i . Similar actions of ET-1, namely, depolarizing the membrane and inducing inward currents, have been reported from rat aortic and mesenteric arterial SMC in short-term primary culture. In these cells, it was found that ET-1, as well as its homolog safratoxin S6b, and AVP (but not phenylephrine) can activate nifedipine-insensitive cationic currents that pass Na+, Li+, and Cs+, and which are inhibitable by submillimolar Ni2+ or Co2+ and abolished by removal of external Ca2+. In this study, however, a possible contribution of a Ca2+-inducible conductance (such as a Cl current) was not eliminated; the ET-1-induced current showed a transient time course, rather than a sustained one, and it was abolished by replacing external Ca2+ with Sr2+ or Ba2+, or by inclusion of 10 mM BAPTA in the pipette.
The AVP-induced cationic currents in A7r5 cells can be further divided into two distinct components: one highly selective for Ca2+ (PCa/PCs >17) and the other a cationic one (1120). The Ca2+-selective component was found to exhibit a high permeability to Sr2+ (but only poorly to pass Mn2+) and to be inhibited by multivalent cations known potently to block Ca2+-selective channels such as VOCC, as well as ICRAC (see below). The observed sequence of inhibitory efficacy was La3+ > Cd2+ > Co2+ > Ni2+ ~ Mn2+. Another interesting property of this current component is that the current amplitude tends to saturate at high external Ca2+ concentrations, in a manner that fits with Michaelis-Menten kinetics (apparent Kd = 9.7 mM). This property is suggestive of the presence of one or more high-affinity binding sites for Ca2+ in the channel pore, from which the high selectivity for Ca2+ might result. It has been estimated that the rate of [Ca2+]i increase due to this current component under unbuffered conditions would be as high as several hundreds of nanomolar per second at physiological concentrations of external Ca2+.
A recent study in which molecular techniques were combined with patch-clamp and [Ca2+]i measurements examined the effects of extremely low concentrations of ET-1 (<0.1 nM) on mouse fibroblast cells overexpressed with recombinant human ETA receptors (Ltk cells) and on freshly dissociated SMC from the rabbit aorta (276). These low concentrations of ET-1, which may be physiological, have been reported to produce membrane depolarizations and vasoconstrictions that are partly insensitive to DHP, without stimulating InsP3 production (702). The novelty of this approach lies in the overexpression technique, which achieves an amplification of responses linked to ETA receptors that are normally very tiny and not suitable for electrophysiological analysis. The results clearly indicated that, in Ltk cells overexpressed with ETA receptors, a maintained [Ca2+]i rise can be elicited by ET-1 at concentrations as low as 0.1 nM, whereas at higher concentrations (1-10 nM), the same agent can also elicit a rapid [Ca2+]i rise, which probably reflects Ca2+ release. Correspondingly, in voltage-clamp experiments, 0.1 nM ET-1 induced a sustained Ca2+-permeable cationic current insensitive to voltage or [Ca2+]i . The ET-1-induced cationic current and concomitant rise in [Ca2+]i in transfected Ltk cells was not affected by the VOCC blocker nifedipine (10 µM) but was almost completely suppressed by a general blocker for NSCC, mefenamic acid (300 µM). A cationic current exhibiting similar pharmacological and biophysical properties could be recorded from freshly dissociated aortic SMC. These results, taken collectively, suggest that activation of voltage- and [Ca2+]i-independent, highly Ca2+-permeable NSCC may serve as a key Ca2+-entry pathway for inducing persistent vasoconstriction under physiological conditions. This would be in sharp contrast to the voltage-dependent, [Ca2+]i-sensitive NSCC found in spontaneously active SM (however, note that another important effect of ET is to sensitize the contractile machinery, see sect. VC).
This presumed physiological function of vasoconstrictor-activated NSCC, namely, as a Ca2+-entry route, would imply that selective inhibition of these channels might be of therapeutic importance for certain pathological conditions such as hypertension, where vasoconstrictor peptides may play essential roles (852).
The activation mechanism for vasoconstrictor peptide-activated NSCC remains unclear. Although it probably does not involve [Ca2+]i or InsP3 directly, we cannot rule out the involvement of PKC (852) or some other unknown intracellular mechanisms that transduce cell-surface signals to the channel proteins. For example, coupling of G protein to these NSCC, which has already been mentioned above, might also exist as a general rule, linking G protein-coupled receptors and ROCC in vascular SMC. This view is supported by the observation that activation of AVP-activated NSCC in A7r5 cells is completely prevented by dialyzing GDPS into the cell, via a PTX-insensitive mechanism.
C. Cytosolic Ca2+-Activated Nonselective Cation Channels
Two types of NSCC have been reported to be directly activated by an elevation in [Ca2+]i in SM. In rat portal vein SMC, various agents and events that can elevate [Ca2+]i , such as NE, ACh, caffeine, Ca2+ influx through VOCC, and the slow continuous leakage of stored Ca2+ caused by ryanodine, all induced resolvable openings of NSCC with an extremely large conductance (~200 pS in saline) under whole cell recording conditions (670). These channel openings occur concomitantly with the main conductance activated during agonist-mediated Ca2+ mobilization in this preparation, namely, Ca2+-dependent Cl currents (875), and are completely abolished when [Ca2+]i elevation is prevented by the inclusion of a high concentration of EGTA in the pipette or by replacing external Ca2+ with Ba2+. The most noteworthy feature of this NSCC is its extraordinarily high selectivity for Ca2+: the PCa/PNa calculated from reversal potential measurements is as high as 21, whereas the unitary conductance measured in high-Ca2+ containing solution (91 mM outside; ~75 pS) is substantially smaller than that measured in saline. Such an apparent discrepancy between high Ca2+ selectivity and low Ca2+ conductance is suggestive of the preferential sojourn of Ca2+ inside the cation channel pore, a phenomenon already mentioned above. However, the physiological significance of the activation of this NSCC is uncertain, since its opening appears to occur with an extremely low frequency, even with maximum intensity agonist stimulation (no more than triple simultaneous openings have been observed in the whole cell preparation). As a possible function, a role has been proposed for this channel as a route for refilling the internal stores after agonist stimulation (670).
Another type of Ca2+-dependent NSCC has been reported to exist in ear artery SMC (1149). This channel exhibits a basal activity that is further enhanced by the Ca2+-releasing agents caffeine and NE or by the Ca2+ ionophore ionomycin. The unitary conductance of this NSCC is low (28 pS in saline), and its Ca2+ permeability seems extremely low (no discernible current flows with 89 mM Ca2+ alone). These properties are in sharp contrast to those of the Ca2+-dependent NSCC found in the rat portal vein and may be closer to those of channels found in tissues other than SM, including cardiac myocytes, neuroblastoma cells, pancreatic acinar cells, and lacrimal glandular cells (1064). The physiological role of such ear artery NSCC may not in essence differ from the generally presumed function of depolarizing the membrane and increasing voltage-dependent Ca2+ entry. However, the interesting question has been raised in this study (2403) as to why the probability of recording Ca2+-activated NSCC in the ear artery seems higher with a low Cl concentration in the pipette than in Cl-rich conditions. We made an analogous observation when studying the 1-adrenoceptor-activated NSCC in the rabbit portal vein, namely, that the potentiating effects of fenamates on this type of NSCC (see above) tend to decrease under Cl-rich conditions (K. Yamada and R. Inoue, unpublished data). Moreover, the importance of intracellular Cl activity in regulating ion channel activity has recently been recognized, e.g., in KATP (719, 1072). This form of regulation will be an intriguing subject for future research on the NSCC in SM.
Although their place in this section may seem questionable, since changes in [Ca2+]i would not be a primary regulator, we should mention here that Ca2+-sensitive NSCC having a conductance (211 pS in symmetrical K+ saline) as large as the conductance of those in the rat portal vein have been identified in the rat cerebral artery (719). These NSCC are spontaneously active and only weakly dependent on [Ca2+]i as well as on the membrane potential, and they are poorly permeable to Ca2+, and not stretch activatable. The functional significance of these channels may lie not in depolarizing the membrane, but rather in passing outward currents at membrane potentials more positive than approximately 40 mV (the reversal potential is about 42 mV), thereby limiting excessive membrane depolarization during action potential activity.
D. Unclassified Cation Channels
Conductances observable during caffeine application are generally thought to be triggered by the release of Ca2+ from the internal stores and thus to be Ca2+ dependent. However, Guerrero et al. (374) have discovered a novel class of 80-pS NSCC that open in direct response to caffeine (20 mM) via a [Ca2+]i-independent mechanism. These channels can be activated with 10 mM BAPTA in the pipette, but not by application of ACh or by depolarizing pulses that elicit comparable elevations in [Ca2+]i . A relatively high Ca2+ permeability (~20% of the current being carried by Ca2+) has been deduced from a comparison of the charge transported through the channel and the [Ca2+]i rise measured in the presence of ryanodine, after correcting for endogenous buffering and removal capacities (375). Activation of this channel is unlikely to involve the intracellular accumulation of cAMP due to phosphodiesterase inhibition, since a stable and membrane-permeable analog of cAMP, 8-bromo-cAMP, failed to mimic the effects of caffeine.
E. Muscarinic Receptor-Inactivated K+ Current: M Current
The M current was first described in the bullfrog sympathetic ganglion (129) as a novel K+ conductance that was found to be inhibitable by muscarinic activation and distinguishable from other voltage-dependent K+ conductances. It was later identified in a broad range of tissues including hippocampus, sympathetic neurons, a neuroblastoma-glioma cell line, NG108-15, and SM (894). The M current is activated at potentials more positive than 70 to 60 mV and exhibits a sigmoidal dependence on the membrane potential. This property enables the current to act as if a net "inward" current were flowing during muscarinic suppression that would increase at more positive potentials and decrease at more negative potentials. The best-characterized example of M current in SM is found in the toad stomach (987), and a similar K+ current component has been detected in guinea pig stomach SMC (641). The reversal potential of the toad stomach M current was found to accord closely with the K+ equilibrium potential when external K+ concentrations were varied, thus suggesting its strict selectivity for K+ over other monovalent cations (987). The steady-state activation curve for this current is well fitted by a Boltzmann-type sigmoid relationship with a half-maximum activation voltage of 49 mV and a slope factor of 9 mV (987).
Voltage-jump experiments have revealed that the current appears to activate or inactivate upon depolarization or hyperpolarization, respectively, to a new level appropriate for the absolute level of the membrane potential, with time constants ranging from tens to hundreds of milliseconds (10, 986, 990). This kind of gating behavior is reminiscent of other types of ligand-gated channels, such as nicotinic NSCC at the end plate and muscarinic K+ channels in the heart (11), and shows a striking quantitative similarity to the voltage-dependent NSCC (e.g., Ref. 479). Indeed, a current-clamp experiment in the toad stomach (987) revealed that ACh is able to depolarize the membrane rapidly, with a time course that cannot be distinguished from that caused by NSCC activation in other SMC (74, 479, 569, 651, 1134). However, the activity of the toad stomach M current was not affected by removal of Ca2+ from the bath or inclusion of high concentrations of EGTA in the pipette (986). This means that the M current is not affected by changes in [Ca2+]i , which contrasts with a remarkable dependence of voltage-dependent NSCC on [Ca2+]i . In contrast, the presence of a millimolar concentration of Ba2+ in the bath, which is also known to affect a number of voltage-dependent K+ currents (214), strongly inhibits this current. It is now generally agreed that a variety of neurohormones, such as substance P and luteinizing hormone-releasing hormone, modulate the M current through their own receptors. Likewise, the toad stomach M current has also been found to be suppressed by tachykinins (substance P, substance K) via receptors distinct from the muscarinic ones (989). In addition to such inhibitory actions, it has been found that the toad stomach M current has a unique property: it can be augmented by the -adrenergic agonist Isop (986, 988). This upregulating effect of -adrenoceptor activation is probably due to increased cAMP production through stimulated adenylate cyclase activity, since forskolin or the membrane-permeant analog 8-bromo-cAMP is also effective in augmenting the current. The physiological significance of this mechanism is unclear but may lie in -adrenoceptor-induced relaxation of this muscle, since increased M-current activity may lead to membrane hyperpolarization, thereby lessening the membrane excitability and reducing the likelihood of the generation of spikes.
The molecular mechanism underlying the muscarinic suppression of the M current remains unclear, although an involvement of a PTX-insensitive G protein has been suggested (259, 691). To date, modulatory effects on this current exerted by InsP3 and a PKC activator, phorbol ester, have been demonstrated (201, 202, 259, 688, 692). However, neither of these has been proved to be obligatory (130). A recent cell-attached recording of single M-channel activity (three distinct conductances of 19, 12, and 7 pS have been reported; Ref. 981) has revealed that the channel activity can be suppressed even when muscarinic agonists are applied outside the patch, suggesting a contribution to muscarinic suppression by one or more remote signal(s), presumably diffusible second messengers. Clapp et al. (202) observed a modulation of the actions of ACh on the M current by a synthetic DAG, 1,2-dioctanoyl-sn-glycerol (DiC68). They concluded that ACh and DiC8 each suppressed both the endogenous and Isop-induced M currents without altering the time course of M current deactivation. This suggested that these agents act by decreasing the number of channels available to be opened, thus providing evidence that muscarinic regulation of M currents is mediated by DAG. Interestingly, a very recent study in neuroblastoma/glioma NG108-15 cells has demonstrated that a NAD+ metabolite (cADP ribose) imitates, while streptozotocin (which reduces the level of NAD+) attenuates, the inhibitory effect of ACh on single-channel activities representing the M current, suggesting a possible second messenger role for cADP ribose in M current (channel) inhibition (406).
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VI. MOBILIZATION OF CALCIUM AND MAINTENANCE OF CALCIUM ION HOMEOSTASIS IN VISCERAL SMOOTH MUSCLE CELLS |
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Basically, the induction of a contraction (activation of cross bridges between contractile proteins) is initiated by the formation of a Ca2+-CaM complex triggered by an increased Ca2+ concentration in the cytosol. In this section, we review the ways in which Ca2+ homeostasis is maintained in VSMC; that is, by the influx and efflux of Ca2+ across the sarcolemmal membrane (SL) and by the release from and uptake of Ca2+ into the cytosolic Ca2+ store sites (namely, the SR). In VSMC, the total surface area of the SR is thought to be the equivalent of a few percent of the total SL surface, a value much smaller than that for skeletal muscle (327). Some of the SR is distributed just beneath the SL (in close contact with caveoles or the SL), and some is deep in the cytosol. A useful review of Ca2+ homeostasis in VSMC was published by Himpens and Missiaen (413).
A. Ca2+ Concentration in the Cytosol of VSMC
In VSMC, in studies using the luminescent Ca2+ indicator aequorin, the [Ca2+]i was first measured by Fay et al. (287) in toad stomach, and later by Morgan and Morgan (775). Since those pioneering studies, many investigators using various dyes have measured the [Ca2+]i from different tissues and species. In resting and active tissues, Ca2+ concentrations are mainly measured using aequorin, quin 2, fura 2, furaptra, fluo 3, rhod 2, or indo 1 (96, 373, 1105, 1107, 1142). Under resting conditions, an average value for [Ca2+]i is thought to be 100-150 nM. However, this value is affected by various factors (e.g., temperature, stretch, cytosolic and external ionic environments, and experimental protocol), and it also differs according to the tissue and species (335, 336, 776). Bukoski et al. (134) estimated that, in resistance arteries, the [Ca2+]i was 79 nM in the resting state (measured using fura 2), whereas Jensen et al. (519), using the same fura 2 procedure, reported a value of 114 nM in Wistar-Kyoto rat mesenteric artery at the resting membrane potential of 61.2 mV. In the guinea pig coronary artery, Ganitkevich and Isenberg (338, 339) found a resting [Ca2+]i of 155 nM, when the cell was voltage-clamped near the resting membrane potential. In the guinea pig urinary bladder, it has been reported that, at a holding potential of 62 mV, a short (1 s) caffeine application (10 mM) increased [Ca2+]i within 1 s from the resting 118 to 1,490 nM, but upon washout of caffeine, the [Ca2+]i did not return immediately to the resting level; in fact, it was reduced to 47 nM (this phenomenon is termed "undershoot"; Ref. 336). According to Itoh et al. (501), in the rabbit mesenteric artery the resting value was ~110 nM. In Ca2+-free EGTA-containing solution, this value fell to 70-80 nM, and the addition of a K+ channel opener caused a further reduction (to 50-60 nM; Refs. 508, 1056). In rat portal vein SMC, exogenously applied ATP (10 µM) increased the cytosolic Ca2+ from the resting value of 92 to 557 nM; however, when NE (10 µM) was given as pretreatment, only 23.6% of the control increase (i.e., that seen with ATP alone) was induced by subsequently applied ATP (872).
The amount of [Ca2+]i required to activate contractile proteins may also be estimated; this is done by measuring the contraction evoked by Ca2+ in MC permeabilized ("skinned") by saponin, -toxin (a Staphylococcus exotoxin), or -escin (a saponin ester). Saponin is commonly used to prepare a completely permeabilized cell because it forms small pores through the cell membrane (SL), and the latter two agents are used to only partially permeabilize the SL (smaller pores through which substances of more than 1,000 mol wt cannot penetrate) without damaging agonist receptors (326, 462). Using these permeabilization procedures, many investigators have studied the part played by Ca2+ in mechanical responses. According to such studies, the minimum concentration of Ca2+ that will produce a contraction is ~200 nM, and a maximum contraction was evoked by 0.8-1.0 µM. Between these extremes, the mechanical response evoked is in proportion to the concentration of Ca2+. Comparison with the values given earlier shows that the Ca2+ concentration required to reach the threshold for contraction is just above the resting cytosolic concentration. In a combined study, Jensen et al. (520) measured [Ca2+]i from the rat mesenteric artery, using both the Ca2+ electrode (1194, 1195) and fura 2. They found that, at the resting membrane potential (57 mV), the value given by the Ca2+ electrode was 115 nM and that obtained with fura 2 was 129 nM. Under treatment with 1 µM NE, the membrane potential was 36 mV and the Ca2+ concentration measured using the above two procedures was 708 or 537 nM, respectively. This result sits well with the opinion of Fay et al. (288) that, in vivo, the response to a wide range of physiological stimuli would involve a rise in [Ca2+]i to no more than 600-800 nM, because of the existence of powerful feedback mechanisms that resist further change beyond this level (57, 1175, 1191). Thus, between the resting and active states, [Ca2+]i may vary from 100-150 nM to 600-800 nM.
Although the [Ca2+]i has been estimated during resting and active states, and such estimates are informative and useful, it is also necessary to know whether the [Ca2+]i is or is not distributed homogeneously during active states in SMC. It should be also mentioned here that it is questionable whether [Ca2+]i measured by dyes accurately reflects the concentrations in the cytosol and, furthermore, whether or not the SR contains similar densities of the ryanodine and InsP3 receptors. In this context, an interesting notion was put forward by Yamaguchi et al. (1196). They argued that when the peripheral layer of the cytosol has a high [Ca2+]i , its fluorescence can sometimes confound attempts to measure [Ca2+]i with conventional optics. Thus a given level of mechanical response and of electrical activity in the SL induced by mobilization of Ca2+ in the cytosol may not give rise to consistent measurements due to nonhomogeneous distributions of Ca2+ during the active state of the cells. Yamaguchi et al. (1196) measured the Ca2+ distribution in airway SMC cytosols after the application of Isop or caged cAMP, using fura 2 with confocal microscopy. They concluded that Isop decreased [Ca2+]i in the inner cytosol but increased it in the peripheral cytosol by mechanisms that depended on both extracellular SL and SR Ca2+ release channels. If correct, this means that a heterogeneous distribution of CICR- and InsP3-induced Ca2+ release (IICR) receptors in SMC presumably exists. Furthermore, when Rembold (924) studied Ca2+ movements in arterial cells from measurements of the intensities of aequorin- and fura 2-Ca2+ transients, as well as myosin light chain phosphorylation and force, they concluded 1) that the focal increase in [Ca2+]i induced by histamine occurred in different cellular regions from those in which Ca2+ restoration took place, and 2) caffeine inhibited SM contraction by localizing the increase in [Ca2+]i to a region distant from the contractile apparatus. They further suggested that there can be transient and sustained focal increases in [Ca2+]i . Aequorin was more accurate than fura 2 at detecting increased [Ca2+]i in a small region of the cytoplasm during its release after refilling of the Ca2+ stores and on caffeine stimulation. Etter et al. (280) applied a new Ca2+ indicator, FFP18, which is more water soluble than previously used indicators, and reported that images of the intracellular distribution of FFP18 show that more than 65% is located on or near the plasma membrane. Calcium transients measured using FFP18 during membrane depolarization-induced Ca2+ influx revealed that near-membrane [Ca2+]i rises quickly and reaches millimolar levels at early times (rises within 20 ms, peaks at 50-100 ms at near-membrane sites). In contrast, levels of only a few hundred nanomolar are measured by fura 2. They therefore postulated that the existence of such large, rapid increases in [Ca2+]i directly beneath the surface membrane may explain how numerous Ca2+-sensitive membrane processes are activated at times when the changes in bulk cytoplasmic Ca2+ concentration are too small to activate them. A region just beneath the SL is called a "fussy" zone, because it may retain Ca2+ or it may form a barrier against diffusion. Significantly, some SR vesicles are distributed just beneath the SL membrane. In fact, in a variety of SMC including rabbit and guinea pig portal veins, spontaneous transient outward currents (STOC), which are thought to reflect a periodic local release of Ca2+ stored in the superficially localized SR (involving bursts of mainly Ca2+-dependent K+ currents; for details see below), were recorded from both whole cell and cell-free patch membranes (71, 80882471185).
Lesh et al. (657, 658) demonstrated, using an immunohistochemical procedure, that the ryanodine receptor, RyR (a CICR-mechanism activator; see below) is mainly distributed on the more superficial SR vesicles in the guinea pig taenia coli, but not in the aorta. Xiong et al. (1185) found, in the guinea pig portal vein, that SR vesicles were attached to SL fragments that were sensitive to caffeine (which stimulates Ca2+ release via the CICR path in the SR) or heparin sodium (an inhibitor of IICR from the SR). This was deduced from measurements of electrical currents using the cell-free patch-clamp procedure. They further concluded that because caffeine was more effective than heparin at blocking STOC or the increased openings of K+ unitary currents in such a cell-free preparation, the caffeine-sensitive Ca2+ channels (RyR) are presumably preferentially distributed near the surface membrane.
The functional connection between the SR and the SL in VSMC has been discussed mostly to try to explain SR refilling from the extracellular space. Thus it has been proposed that there is a direct path between the SR and SL (117, 168), or a continuous vectoral release of Ca2+ from the SR lumen to the extracellular space (188, 189). Janssen and Sims (513) observed Ca2+ mobilization in canine tracheal myocytes during the ACh-induced inward current and contraction and concluded that the current and contraction are mediated via Ca2+ released from an internal store that can be depleted by prolonged removal of extracellular Ca2+, or by repeated applications of either caffeine or the Ca2+-ATPase inhibitor CPA. At least two Ca2+ influx pathways appear to contribute to the refilling of the internal store: one is not activated by depolarization or ACh, and the second involves direct contact with the SR and thus conducts extracellular Ca2+ directly into the SR, bypassing the cytosol. Van Breemen et al. (1115) summarized their own view that, in SMC, the superficial SR accumulates a portion of the Ca2+ that enters the cell through the SL, and thus functions as a buffer barrier to Ca2+ entry into the cytosol ("superficial buffer barrier"; SBB). Evidence for this notion is as follows: 1) contraction is related more to the rate than to the extent of Ca2+ entry, 2) refilling of the SR from the luminal space is mediated by Ca2+ influx and Ca2+ pumping by the SR Ca2+ pump, 3) the superficial SR unloads Ca2+ to the lumen by a multistep process that involves the opening of Ca2+- and InsP3-sensitive channels followed by Ca2+ extrusion via Na+/Ca2+ exchange, 4) the SBB generates a peripheral Ca2+ gradient, and 5) some agonists generate InsP3 , which then short-circuits the SBB to increase the effectiveness of Ca2+ influx in raising [Ca2+]i , and consequently increases the SM contraction. More findings related to the refilling of Ca2+ into the store sites after their depletion to the lumen are discussed below.
The main channels involved in Ca2+ release from the SR are normally classified as ryanodine- and InsP3-sensitive Ca2+ channels. On the other hand, after considering the nonhomogeneous distributions of the ryanodine- and InsP3 receptors, Iino et al. (459) and Iino (456) put forward the idea that the Ca2+ compartments responsible for the release of Ca2+ should be classified as either Sa (contains CICR and IICR receptors) or Sb (contains IICR alone) on the basis both of their modes of Ca2+ release and the resulting contractions observed in intact and skinned muscle tissues. They estimated that Sa makes up 40% of the total Ca2+ components in the taenia coli, ~50% in the pulmonary artery, <20% in the portal vein, and <10% in the myometrium. However, these estimates were not completely supported by other investigators (750).
It is of interest that caffeine, a releaser of Ca2+ through the CICR mechanism, does not produce a contraction in the myometrium. Indeed, Morgan et al. (774) found that caffeine has no effect on resting [Ca2+]i in cultured human myometrial cells. Moreover, Lynn et al. (677) found, in the same preparation, that the CICR mechanism was accelerated by a combined application of ryanodine and caffeine, but not directly activated by caffeine alone. Similarly, Lynn and Gillespie (676), using 45Ca2+ technique in skinned muscle preparations from the human uterine artery, found that the CICR mechanism in this tissue was sensitive to ryanodine and ruthenium red, but not to caffeine. It thus would appear that there is a tissue specificity in respect of the CICR mechanism in VSMC. Concerning the actions of caffeine, Guerrero and co-workers (374, 375) observed, using simultaneous measurements of the transmembrane ionic current and [Ca2+]i , that activation by caffeine of nonselective cation channels in the SL was not a consequence of the emptying of internal Ca2+ stores. Rather, they proposed that caffeine activates these SL channels either by direct interaction or alternatively by a linkage between the RyR on the SR and the ROCC on the SL.
B. Factors That Increase [Ca2+]i in VSMC
1. Influx of Ca2+ through the SL
In the rabbit coronary artery, Matsuda et al. (707) estimated that, with the average peak current at +20 mV being 217 pA in 110 mM Ba2+, the probability of Ca2+ channel opening is 0.13, average cell surface area is 1.87 × 103 µm2 , and the single-channel conductance is 0.53 pA at +20 mV. From the above values, the estimated channel density is 1.71 channels/mm2. Nelson and co-workers (809, 810) made similar calculations using data obtained by means of the patch-clamp procedure,
, phosphorylation sites of protein kinase A; 
, phosphorylation sites of protein kinase C. (From Y. Kurachi, personal communication.)
) and 21.8 pS (
). II: effects of nifedipine on activity of 2 types of unitary Ba2+ channel current. A: unitary Ba2+ current evoked by a depolarizing pulse (duration 150 ms) to 
