I. Introduction

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Contraction of smooth muscle is regulated by the cytosolic Ca²⁺ level ([Ca²⁺]_i)^b, and the sensitivity to Ca²⁺ of the contractile elements in response to changes in the environment surrounding the cell. The first sequence of events in regulation includes the binding of endogenous substances, such as neurotransmitters and hormones, to their specific receptors. This activates various types of guanosine 5'-triphosphate (GTP) binding proteins, which are coupled to different ion channels and enzymes, and modulate their activities. These enzymes include both phospholipase C, which metabolizes phosphatidylinositol and produces inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol, and adenylate cyclase, which metabolizes adenosine 5'-triphosphate (ATP) to produce cyclic adenosine 3',5'-monophosphate (cyclic AMP). Some receptors, such as that for the atrial natriuretic peptide, are directly coupled to guanylate cyclase, which metabolizes GTP to produce cyclic guanosine 3',5'-monophosphate (cyclic GMP).

The second regulatory sequence includes changes in [Ca²⁺]_i. Calcium influx is the major pathway to increase [Ca²⁺]_i. This mechanism includes voltage-dependent L-type Ca²⁺ channels, nonselective cation channels, the Ca²⁺-release activated Ca²⁺ influx pathway, and the reverse mode of the Na⁺/Ca²⁺ exchanger. Calcium release from the sarcoplasmic reticulum (SR) also increases [Ca²⁺]_i. A decrease in [Ca²⁺]_i is mediated by Ca²⁺ sequestration by the SR, and extrusion by membrane Ca²⁺ pumps and Na⁺/Ca²⁺ exchanger. Second messengers such as IP₃, diacylglycerol, cyclic AMP, and cyclic GMP alter [Ca²⁺]_i by affecting these mechanisms. Distribution of Ca²⁺ in the cytoplasm is not uniform. Calcium ion in the cytosolic compartments regulates contractile elements, whereas Ca²⁺ in the subplasmalemmal compartments regulates Ca²⁺-dependent mechanisms in the plasmalemma (ion channels, ion pumps, and enzymes). Calcium concentrations in these compartments are regulated independently.

The third regulatory sequence includes changes in myosin light chain kinase activity. This enzyme is activated by Ca²⁺ and calmodulin and phosphorylates myosin regulatory light chain (MLC). Phosphorylated myosin interacts with actin to induce contraction. Phosphorylated MLC is dephosphorylated by MLC phosphatase. The amount of phosphorylated MLC is therefore dependent on the balance between MLC kinase and MLC phosphatase. However, during continuous stimulation, [Ca²⁺]_i, the amount of phosphorylated MLC and shortening velocity gradually decrease, whereas isometric force increases monotonically. This indicates that nonphosphorylated myosin is also involved in the maintenance of contraction. Agonists and second messengers modify the MLC kinase/MLC phosphatase ratio independently of [Ca²⁺]_i. This mechanism, known as Ca²⁺ sensitivity of MLC phosphorylation, changes contractile force even in the presence of a constant level of [Ca²⁺]_i. Both cyclic AMP and cyclic GMP change the MLC kinase/MLC phosphatase balance and induce relaxation.

All of these mechanisms are supported by energy supplied mainly from oxidative phosphorylation and partly from aerobic glycolysis. Oxidative phosphorylation supplies ATP mainly to contractile elements, whereas aerobic glycolysis supplies ATP mainly to membrane ion pumps. Although smooth muscle develops approximately double the force per cross-sectional area of skeletal muscle, it consumes 100- to 500-fold lower ATP than does skeletal muscle. This difference is explained by the lower ATPase activity of the smooth muscle myosin molecule.

Within the past decade, considerable progress has been made in the understanding of Ca²⁺ movements and distribution in smooth muscle cells. Simultaneous measurements of [Ca²⁺]_i and contraction in intact smooth muscle cells and tissues using various types of intracellular Ca²⁺ indicators have allowed analysis of Ca²⁺ sensitivity of contractile elements (see Karaki, 1989a, 1990, 1991). Permeabilization of the cell membrane enabled the measurement of contraction in the presence of the constant concentrations of Ca²⁺, ATP, and other substances in the cell. Calcium-imaging techniques have revealed uneven distribution of Ca²⁺ in the cell and localized increases in the form of Ca²⁺ sparks and waves. Comparison of the increase in [Ca²⁺]_i and contraction suggested the roles of localized Ca²⁺ in regulation of different mechanisms located in different parts inside the cell.

This review article is focused on topics related to mechanisms regulating [Ca²⁺]_i and physiological roles of Ca²⁺ in smooth muscle. Effects of pharmacological agents on movements and distribution of Ca²⁺ will also be discussed. Readers should refer to review articles by Abdel-Latif (1986) and Nishizuka (1995) on the receptor-linked signal transduction, by McDonald et al. (1994), Kuriyama et al. (1995) and Knot et al. (1996) on ion channels, by Murphy (1994), Somlyo and Somlyo (1994), and Strauss and Murphy (1996) on regulation of contractile elements, and by Ishida et al. (1994), Paul (1995), and Hellstrand (1996) on energy supply.

	II. Calcium Movements

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Before directly measuring [Ca²⁺]_i using the intracellular Ca²⁺ indicators, contraction was considered to be a good indicator of [Ca²⁺]_i in smooth muscle, because Ca²⁺ was believed to be the only regulator of contraction. In vascular smooth muscle, two types of stimulants are widely used to identify the changes in [Ca²⁺]_i: high K⁺-induced membrane depolarization and activation of the -adrenoceptor by norepinephrine or phenylephrine (Weiss, 1977; Karaki, 1987). Both of these stimuli induced sustained contractions, but with different characteristics. High K⁺-induced sustained contraction was totally abolished by removing external Ca²⁺ and, also, by agents blocking the Ca²⁺ channels, including cinnarizine (Godfraind and Kaba, 1969), -diethylaminoethyl diphenylpropyl acetate (SKF525A) (Kalsner et al., 1970), verapamil (Peiper et al., 1971), and La³⁺ (Goodman and Weiss, 1971a, b; Van Breemen et al., 1972). From these results, it was proposed that high K⁺ increases transmembrane Ca²⁺ influx, increases [Ca²⁺]_i and induces contraction. In contrast, norepinephrine-induced contraction was resistant to removal of external Ca²⁺. It induced a transient contraction followed by a small sustained contraction in the absence of external Ca²⁺. Calcium channel blockers and La³⁺ also inhibited the sustained phase more strongly than the transient phase. However, a part of the norepinephrine-induced sustained contraction was not inhibited by La³⁺ or Ca²⁺ channel blockers at the concentrations needed to completely inhibit high K⁺-induced contraction. These results suggest that the norepinephrine-induced transient contraction is due to Ca²⁺ release from intracellular storage site (Hiraoka et al., 1968). The mechanism of the norepinephrine-induced sustained contraction was controversial. It was suggested that this contraction is due mainly to transmembrane Ca²⁺ influx because it is strongly inhibited in the absence of external Ca²⁺ (Somlyo and Somlyo, 1968; Hudgins and Weiss, 1968; Hiraoka et al., 1968; Weiss, 1977). Another possibility was that this contraction is due to Ca²⁺ release from storage sites because both the transient and sustained phases were less sensitive to Ca²⁺ channel blockers than was the high K⁺-induced sustained contraction (Bohr, 1963; Van Breemen et al., 1972). To further examine the mechanisms to increase [Ca²⁺]_i, it was necessary to directly measure [Ca²⁺]_i.

The amount of Ca²⁺ bound outside the cell membrane (approximately 1 mmol/kg of wet tissue) is much greater than the amount of free Ca²⁺ in the cytoplasm (approximately 10 nm to 1 µM) and/or the amount of Ca²⁺ entering the cell during a contractile stimulation (500 pmol of membrane-bound Ca²⁺/cm² of cell membrane compared to 0.3 pmol of Ca²⁺ influx/cm² of cell membrane) (Bolton, 1979). Since it was not possible to discriminate between Ca²⁺ bound to the membrane surface and Ca²⁺ in the cytoplasm using radioactive ⁴⁵Ca²⁺, it was difficult to detect changes in transmembrane Ca²⁺ influx in smooth muscle. Thus, various stimulants did not change total ⁴⁵Ca²⁺ uptake in different types of smooth muscle preparations (see Lullman, 1970; Weiss, 1974, 1977).

1. Slowly exchanging calcium fraction. To remove that ⁴⁵Ca²⁺ present in the extracellular space, Briggs (1962) incubated rabbit aortic strips with solutions containing ⁴⁵Ca²⁺ for 30-60 min followed by a 10- to 15-min washout period with identical non-radioactive solutions. Using this method, it is possible to remove rapidly exchanging Ca²⁺ and measure the slowly exchanging Ca²⁺ fraction. It was found that high K⁺, epinephrine and norepinephrine increased the amount of ⁴⁵Ca²⁺ remaining after the washout period (Briggs, 1962; Seidel and Bohr, 1971). Ouabain-induced contractions in the rabbit aorta were also shown to be accompanied by an increased ⁴⁵Ca²⁺ uptake (Briggs and Shibata, 1966). This method was also applied to intestinal smooth muscle of the guinea pig taenia coli by Urakawa and Holland (1964), and it was found that various stimulants, including high K⁺, Ba²⁺, carbachol and histamine, increased ⁴⁵Ca²⁺ uptake (for references see Karaki and Urakawa, 1972). Thus, the amount of Ca²⁺ in the slowly exchanging fraction appears to correlate with contraction. However, the time course of the increase in ⁴⁵Ca²⁺ was slower than that of contraction, and the total amount of ⁴⁵Ca²⁺ increased to as much as 500 µmol/kg in 30 min. Furthermore, the decrease in ⁴⁵Ca²⁺ following removal of stimulant was much slower than the decrease in muscle tension (Karaki and Urakawa, 1972). These results suggest that this method measures ⁴⁵Ca²⁺ in a cellular fraction in which Ca²⁺ gradually accumulates during contraction. Since the amount of ⁴⁵Ca²⁺ in this fraction is larger than that in the intracellular space fraction (measured with the lanthanum method as described later), a part of this fraction may exist in the membrane surface. Neither the precise location nor the physiological role of this Ca²⁺ fraction has been defined.

2. Lanthanum-inaccessible fraction. Due to their higher charge density, La³⁺ ions were predicted to have greater affinity than Ca²⁺ for any accessible anionic group that binds Ca²⁺ (Lettvin et al., 1964). Based upon anatomical evidence indicating that La³⁺ is restricted to the extracellular compartment (Laszlo et al., 1952), it was found that La³⁺ replaced ⁴⁵Ca²⁺ at superficial membrane sites and prevented ⁴⁵Ca²⁺ uptake to less accessible Ca²⁺ sites in smooth muscle preparations (Weiss and Goodman, 1969; Goodman and Weiss, 1971a, b; Weiss, 1974). Van Breemen et al. (1972) attempted to remove only the extracellular ⁴⁵Ca²⁺ by washing the tissue in a physiological salt solution (PSS) containing 2-10 mM LaCl₃ after completion of ⁴⁵Ca²⁺ uptake and before tissue ⁴⁵Ca²⁺ analysis. With this "lanthanum method," they showed that during contraction of rabbit aorta with a high K⁺ solution, Ca²⁺ uptake was increased from the resting level of approximately 50 µmol/kg of wet tissue to 150 µmol/kg of wet tissue. They also found that replacement of Na⁺ in PSS by Li⁺ increased both ⁴⁵Ca²⁺ uptake and muscle tension. However, there was no change in ⁴⁵Ca²⁺ uptake during contractions induced by 10 µM norepinephrine. Norepinephrine increased ⁴⁵Ca²⁺ uptake only when muscle strips were preincubated with Ca²⁺-free PSS (Deth and Van Breemen, 1974) or in muscles depolarized by high K⁺ (Karaki and Weiss, 1979, 1980a, b). These results suggest that ⁴⁵Ca²⁺ uptake increased only under "nonphysiological" conditions and appeared to support the ideas that 1) both phases of norepinephrine-induced contraction in the rabbit aorta are due mainly to Ca²⁺ release (Van Breemen et al., 1972; Bohr, 1973; Cavero and Spedding, 1983) and 2) access of extracellular Ca²⁺ is essential for refilling the intracellular release site (Deth and Van Breemen, 1977).

3. Suggested calcium movements in smooth muscle. Based on these observations, Bolton (1979) and Van Breemen et al. (1979), independently, suggested that the mechanisms of the increase in [Ca²⁺]_i in smooth muscle can be explained by two different Ca²⁺ influx pathways: receptor-linked and voltage-dependent Ca²⁺ channels (fig. 1). High K⁺ induces membrane depolarization which, in turn, opens the voltage-dependent Ca²⁺ channel. This channel is inhibited by agents blocking Ca²⁺ channels including verapamil, nifedipine and La³⁺. In contrast, norepinephrine releases Ca²⁺ from storage sites to induce initial transient contractions and subsequently opens the receptor-linked Ca²⁺ channel that is controlled by receptors for contractile agonists. In the aorta, this channel is less sensitive to Ca²⁺ channel blockers than is the voltage-dependent Ca²⁺ channel. Opening of either of these channels results in a continuous Ca²⁺ influx to induce sustained contraction. Existence of two types of Ca²⁺ channels seemed to be indicated by the findings in rabbit aorta that both the rates and total amounts of ⁴⁵Ca²⁺ uptakes, stimulated by maximally effective concentrations of both high K⁺ and norepinephrine, are additive when the two agents were present at the same time (Karaki and Weiss, 1979, 1980a, b; Meisheri et al., 1981). As discussed later, however, it now appears that high K⁺ and norepinephrine open the same L-type Ca²⁺ channel and that norepinephrine may also open a receptor-regulated nonselective cation channel which conducts Na⁺, K⁺, and Ca²⁺. High K⁺ and norepinephrine showed an additive effect on ⁴⁵Ca²⁺ uptake not only because norepinephrine activated both the L-type Ca²⁺ channel and nonselective cation channel but also because high K⁺ activated mitochondrial Ca²⁺ uptake. Furthermore, changes in Ca²⁺ sensitivity of contractile elements were not considered at the time.

View larger version (45K): [in this window] [in a new window]   Fig. 1.   Calcium movements predicted mainly from contraction. High K+ depolarizes the membrane, opens the voltage-dependent Ca2+ channel, increases Ca2+ influx, and elicits sustained contraction (1). Because the voltage-dependent Ca2+ channel is inhibited by Ca2+ channel blockers, contractions elicited by high K+ are inhibited by this type of blocker. In contrast, norepinephrine elicits Ca2+ release from the SR and initiates contraction (2). Because the amount of Ca2+ stored in the SR is limited, contraction due to Ca2+ release is transient. Ca2+ channel blockers do not inhibit Ca2+ release. Norepinephrine also opens the receptor-linked Ca2+ channel, increases Ca2+ influx, and elicits sustained contraction (3). Calcium channel blockers only weakly inhibit the receptor-linked Ca2+ channel. Thus, norepinephrine-induced contraction is less sensitive to Ca2+ channel blockers than is high K+-induced contraction. This schema can now be revised as shown in figure 7.

C. Measurements of Cytosolic Free Calcium Level

1. Aequorin. Aequorin is a Ca²⁺ binding protein first extracted from the jelly fish, Aequorea aequorea, by Shimomura et al. (1962). This protein emits light at 465 nm in the presence of Ca²⁺. Ridgway and Ashley (1967) injected this photoprotein into barnacle single muscle fibers and measured [Ca²⁺]_i by monitoring changes in aequorin light. This method was applied to a single smooth muscle cell by Fay et al. (1979). Morgan and Morgan (1982, 1984a, b) loaded the 21-kDa photoprotein into smooth muscle cells of ferret portal vein by transiently increasing the membrane permeability using a high concentration of EGTA, and measured [Ca²⁺]_i and contraction in isolated smooth muscle strips. They found that high K⁺ induced a sustained increase in [Ca²⁺]_i during sustained contraction, and both increases were inhibited by a decrease in extracellular Ca²⁺ concentrations (Morgan and Morgan, 1982, 1984a, b; De Feo and Morgan, 1985). This supports the view that the high K⁺-induced contraction is due to an increase in [Ca²⁺]_i resulting from activation of Ca²⁺ influx. In contrast, stimulation of the -adrenoceptors by phenylephrine induced a rapid rise of [Ca²⁺]_i to a maximum from which it decreased rapidly to a lower level and then declined more slowly, staying only slightly above basal [Ca²⁺]_i. At the same time, muscle tension rapidly increased to a maximum level and remained elevated as long as stimulation continued. During the phenylephrine-induced sustained contraction, removal of external Ca²⁺ decreased [Ca²⁺]_i to a level lower than basal [Ca²⁺]_i and partially inhibited the contraction. From these results, it was postulated that the contractions induced by phenylephrine and high K⁺ are due to elevation of [Ca²⁺]_i above baseline, and that phenylephrine may increase the effectiveness of Ca²⁺ on the contractile apparatus (Morgan and Morgan, 1984b). Receptor agonists produced a larger force at a given [Ca²⁺]_i than did high K⁺ during the period of force maintenance also in ferret aorta (Suematsu et al., 1991b), rabbit aorta (Takuwa and Rasmussen, 1987), guinea pig aorta (Jiang et al., 1994), swine carotid artery (Rembold and Murphy, 1988a; Rembold, 1990) and canine and bovine trachea (Gerthohoffer et al., 1989; Takuwa et al., 1987).

2. Fluorescent indicators. A new fluorescent Ca²⁺ indicator, quin2, was synthesized by Tsien (1980). This was soon followed by the second generation of indicators including fura-2 and indo-1 (Grynkiewicz et al., 1985). These indicators are not membrane-permeable. To increase permeability, an acetoxymethyl radical is attached to these indicators. After loading smooth muscle cells with the acetoxymethyl esters of these indicators, the acetoxymethyl moiety is cleaved by endogenous esterases and the indicator is trapped in the cell.

D. Mechanisms of Calcium Mobilization

1. Voltage-dependent calcium channels. There are six subtypes of voltage-dependent Ca²⁺ channels: L-, N-, P-, Q-, R-, and T-type. In smooth muscle, only the L-type Ca²⁺ channel is considered to be a major Ca²⁺ influx pathway (Vogalis et al., 1991; Ganitkevich and Isenberg, 1991; Kuriyama et al., 1995; Knot et al., 1996; Hofmann and Klugbauer, 1996). This channel is activated by membrane depolarization and inhibited by Ca²⁺ channel blockers (see Godfraind et al., 1986). Agonists open this channel by depolarizing the cell membrane through activation of the nonselective cation channel (Pacaud and Bolton, 1991), inhibition of the K⁺ channel and/or activation of the Cl channel (Kremer et al., 1989; Pacaud et al., 1991; Miyoshi and Nakaya, 1991; Iijima et al., 1991). Furthermore, agonists may open the L-type Ca²⁺ channels directly or indirectly through GTP-binding proteins in the absence of membrane depolarization (Nelson et al., 1988; Worley et al., 1991; Welling et al., 1992a, b, 1993; Tomasic et al., 1992; Kamishima et al., 1992).

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Changes in [Ca2+]i and contraction induced by high K+ and norepinephrine in the rat aorta without endothelium. Changes in [Ca2+]i and contraction were measured simultaneously in the tissues loaded with a fluorescent Ca2+ indicator, fura-2. (A and B): Effects of 72.7 µm KCl and 1 µm norepinephrine, respectively. Addition of a stimulant increased both [Ca2+]i and muscle tension. Addition of 10 mM verapamil almost completely inhibited [Ca2+]i stimulated by high K+ or norepinephrine. High K+-induced contraction was also strongly inhibited (A). However, norepinephrine-induced contraction was only partially inhibited (B). Decrease in external Ca2+ by 4 µM ethyleneglycoltetraacetc acid (EGTA) decreased [Ca2+]i below the resting level and further inhibited the norepinephrine-induced contraction. However, a small portion of the contraction was resistant to EGTA (B). (C): Effects of norepinephrine in the presence of verapamil. Ten minutes after the addition of 10 mM verapamil, 1 µM norepinephrine was added, which elicited a transient increase in [Ca2+]i followed by a small sustained increase. These changes were followed by rapid increase in muscle tension followed by sustained contraction that was smaller than that observed in the absence of verapamil in (B). (D): Effects of norepinephrine in the presence of EGTA. Five minutes after the addition of 4 mM EGTA, 1 µM norepinephrine was added. Norepinephrine elicited only a small transient increase in [Ca2+]i, accompanied by a rapid increase in muscle tension followed by a small sustained contraction that was smaller than that observed in the presence of verapamil in (C). (Modified from Ozaki et al., 1990c and Karaki et al., 1991).

2. Nonselective cation channel and calcium release-activated calcium channel. Although the larger part of the agonist-induced Ca²⁺ increase was inhibited by Ca²⁺ channel blockers, a part of the increase was not. Verapamil did not completely inhibit the norepinephrine-induced increase in [Ca²⁺]_i at concentrations which completely inhibited the high K⁺-induced increase in [Ca²⁺]_i (Karaki et al., 1988a). Similar results were obtained with other Ca²⁺ channel blockers in other types of smooth muscles stimulated with other agonists (Sakata et al., 1989; Ozaki et al., 1990c; Sakata and Karaki, 1992; Hori et al., 1992). In the presence of verapamil, norepinephrine elicited a transient increase in [Ca²⁺]_i followed by a small sustained increase in the rat aorta (fig. 2). Since the transient increase in [Ca²⁺]_i was inhibited by inhibitors of SR function such as ryanodine and thapsigargin, this increase may result from Ca²⁺ release from the SR by a mechanism that is insensitive to verapamil. In contrast, the small sustained increase in [Ca²⁺]_i, which was insensitive to verapamil, was inhibited by micromolar concentrations of La³⁺ (Harada et al., 1994, 1996). Since the Ca²⁺ channel blockers are believed to selectively inhibit the L-type Ca²⁺ channel (see review by Godfraind et al., 1986; Catterall, 1993; Kuriyama et al., 1995), and since La³⁺ inhibits both the L-type and non-L-type Ca²⁺ channels (Weiss, 1974, 1977, 1996; Ruegg et al., 1989; Hescheler and Schultz, 1993; Krautwurst et al., 1994; but see Inoue and Chen, 1993), these results suggest that the norepinephrine-induced increase in [Ca²⁺]_i is due to Ca²⁺ influx through both the L-type and non-L-type Ca²⁺ channels. Enoki et al. (1995a, b) also showed that endothelin-1-induced Ca²⁺ influx, which was insensitive to Ca²⁺ channel blockers, was inhibited by a putative inhibitor of nonselective cation channel, mefenamic acid. Electrophysiological studies have also shown that receptor agonists activate the L-type Ca²⁺ channel and also the nonselective cation channel which is permeable to Ca²⁺ (Nelson et al., 1988; Kuriyama et al., 1995; Knot et al., 1996). In cultured A10 smooth muscle cells, it was suggested that receptors are directly coupled to the non-L-type Ca²⁺ entry pathways (Simpson et al., 1990).

3. Sodium-calcium exchange. Bohr (1964) and Reuter et al. (1973) originally reported the contraction in rabbit aorta under conditions which implicate a Na⁺/Ca²⁺ exchange mechanism (Na⁺ pump inhibition or Na⁺-free solution), although some of these effects were found to be evoked by the release of endogenous catecholamines possibly due to Ca²⁺ influx into adrenergic nerves (Karaki and Urakawa, 1977; Bonaccorsi et al., 1977; Karaki et al., 1978; Rembold et al., 1992). Experiments using a membrane-enriched microsomal fraction and smooth muscle cells revealed the presence of Na⁺-dependent Ca²⁺ influx and efflux in smooth muscle of swine stomach (Raeymaekers et al., 1985), bovine trachea, porcine aorta and bovine aorta (Slaughter et al., 1987, 1989) and rat aorta (Nabel et al., 1988). Lowering external Na⁺ concentration or increasing [Na⁺]_i elevated [Ca²⁺]_i in guinea pig taenia coli (Pritchard and Ashley, 1986, 1987), rat aorta (Matlib et al., 1986), swine carotid artery (Rembold et al., 1992), human mesangial cells (Mene et al., 1990), cultured vascular smooth muscle (Batlle et al., 1991), the A10 cells (Gillespie et al., 1992a), and the A7r5 cells (Vigne et al., 1988; Bova et al., 1990; Gillespie et al., 1992b; Borin et al., 1994). The molecular structure of the Na⁺/Ca²⁺ exchanger was also clarified (Nicoll and Philipson, 1991).

4. Calcium release from the sarcoplasmic reticulum. Measuring [Ca²⁺]_i in the SR in saponin-permeabilized cultured A7r5 aortic smooth muscle cells using a fluorescent Ca²⁺ indicator, furaptra, Sugiyama and Goldman (1995) found that the K_d of the SR for Ca²⁺ was 49 µM and resting SR Ca²⁺ was 75-130 µM. In smooth muscle, Ca²⁺ is released from the SR (Stout and Diecke, 1983; Yamamoto and Van Breemen, 1986; Iino, 1987; Sato et al., 1988a). There are two types of mechanisms to release Ca²⁺ from the SR in smooth muscle, Ca²⁺-induced Ca²⁺ release (CICR) (Endo, 1977; Ogawa, 1994; Zucchi and Ronca-Testoni, 1994) and IP₃-induced Ca²⁺ release (IICR) (Ferris and Snyder, 1992; Mikoshiba, 1993; Putney and Bird, 1993). CICR is activated by Ca²⁺ (Itoh et al., 1981; Saida, 1982; Iino, 1989), whereas IICR is activated by IP₃ (Suematsu et al., 1984; Somlyo et al., 1985; Islam et al., 1996). IICR is regulated not only by IP₃ but also by Ca²⁺. IICR is enhanced by Ca²⁺ below 300 nM and, above this concentration, Ca²⁺ inhibited IICR (Iino, 1990; Iino and Endo, 1992; Iino and Tsukioka, 1994). Calcium influx through the L-type Ca²⁺ channels also activates CICR in guinea pig aorta and urinary bladder and rat portal vein and mesenteric artery (Ito et al., 1991a; Ganitkevich and Isenberg, 1992; Gregoire et al., 1993). Calcium influx mediated by the reverse-mode action of the Na⁺/Ca²⁺ exchanger, which was undetectable by fura-2, released Ca²⁺ from the thapsigargin-sensitive intracellular stores including IP₃-releasable pools in cultured guinea pig ileum longitudinal muscle cells (Ohata et al., 1996). CICR is selectively activated by caffeine and selectively inhibited by ryanodine (Ito et al., 1986; Hisayama and Takayanagi, 1988), whereas IICR is inhibited by heparin (Kobayashi et al., 1988; Ghosh et al., 1988; Chopra et al., 1989; Ganitkevich and Isenberg, 1990; Komori and Bolton, 1990).

5. Calcium pumps in plasmalemma and the sarcoplasmic reticulum. In smooth muscle, there are two types of Ca²⁺ ATPase, plasmalemmal Ca²⁺ ATPase and SR Ca²⁺ ATPase (Wuytack et al., 1982; Raeymaekers et al., 1985; Verbist et al., 1985; Raeymaekers and Wuytack, 1996). The plasmalemmal Ca²⁺-ATPase activity was four times higher than the (Na⁺ + K⁺)-ATPase activity in human myometrial smooth muscle (Popescu and Ignat, 1983). Since Ca²⁺ extrusion through the Na⁺/Ca²⁺ exchange mechanism would ultimately be limited by the (Na⁺ + K⁺)-ATPase activity, this result suggests that plasmalemmal Ca²⁺-ATPase plays a more important role in Ca²⁺ extrusion than does Na⁺/Ca²⁺ exchange. In cultured rat aortic smooth muscle cells, 12-O-tetradecanoylphorbol-13-acetate (TPA) increased the maximum Ca²⁺ efflux rate without changing the affinity for Ca²⁺ (Furukawa et al., 1988, 1989). In Ca²⁺-ATPase purified from bovine aortic smooth muscle, it was also shown that phorbol ester stimulated the ATPase activity which was accompanied by phosphorylation of the ATPase, suggesting that the plasmalemmal Ca²⁺-pump in vascular smooth muscle is activated by protein kinase C (C kinase). Sodium nitroprusside and 8-bromo-cyclic GMP also stimulated the Ca²⁺ pump activity although forskolin and dibutyryl cyclic AMP were ineffective (Yoshida et al., 1991; Furukawa et al., 1988).

6. Mitochondria. Mitochondrial inhibitors decrease ATP production and contraction in intestinal smooth muscle. However, neither ATP contents nor contractions were decreased by these inhibitors in vascular smooth muscle, possibly because ATP is supplied not only by mitochondria but also by glycolysis (Karaki et al., 1982; Nakagawa et al., 1985). Inhibition of oxidative phosphorylation by nitrogen gas, dinitrophenol or sodium azide elicited a release of Ca²⁺ from mitochondria to induce transient contraction in rat aorta (Karaki et al., 1982), rabbit colon (Kowarski et al., 1985) and rat myometrium (Sakai et al., 1986). These results suggest the possible involvement of mitochondrial Ca²⁺ release in smooth muscle contraction. Inhibition of mitochondrial Ca²⁺ uptake may also elicit contraction. Takeo and Sakanashi (1985) estimated the mitochondrial Ca²⁺ uptake activity of the coronary artery to be 250 nmol Ca²⁺/mg protein/10 min. Kowarski et al. (1985) analyzed subcellular Ca²⁺ concentrations in rabbit main pulmonary artery smooth muscle cells by electron probe X-ray microanalysis and estimated the mitochondrial Ca²⁺ to be 2.2 mmol/kg dry weight, and this was not changed after the muscle was exposed to norepinephrine. In contrast, the central SR can accumulate larger amounts of Ca²⁺, and norepinephrine released Ca²⁺ from the SR. The relative sizes of the central SR and mitochondrial Ca²⁺ pools in relaxed tissue were about 20:1. In rabbit portal vein, smooth muscle was loaded with Na⁺ for 3 h in a K⁺-free, ouabain-containing solution, after which rapid Na⁺/Ca²⁺ exchange was induced by Na⁺-free solution (Broderick and Somlyo, 1987). This procedure induced a large transient contraction accompanied by a large increase in [Ca²⁺]_i which was taken up by mitochondria.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Changes in the fluorescence of reduced pyridine nucleotides (PNred) (A) and oxidized flavoproteins (FPox) (B) during spontaneous contraction in guinea pig taenia coli. PNred and FPox fluorescence are shown by relative intensity of fluorescence taking the basal fluorescence as 100%. FPox started to increase before the initiation of contraction. (C): The effects of external Ca2+ concentration on PNred fluorescence, FPox fluorescence, and tension development in the 45.4 mM K+-depolarized taenia coli. Responses induced by 10 mM Ca2+ was taken as 100%. The Ca2+ sensitivity of each response was in the order of FPox > PNred > muscle contraction. (Modified from Ozaki et al., 1988).

E. Calcium Distribution and Function

1. Noncontractile calcium compartment. Most of the data obtained from simultaneous measurement of [Ca²⁺]_i and contraction confirm that there is a positive correlation between these two parameters and that smooth muscle contraction occurs following an increase in [Ca²⁺]_i. However, some small dissociations were identified. In some types of smooth muscle, agonists induced larger contractions than predicted from the increase in [Ca²⁺]_i. This kind of dissociation may be explained by Ca²⁺ sensitization of contractile elements. In contrast, relaxants related to cyclic AMP and cyclic GMP decrease contractile force without decreasing [Ca²⁺]_i or with only a small decrease in [Ca²⁺]_i, possibly by an attenuation of Ca²⁺ sensitivity of the contractile elements. However, some kinds of dissociations are explained neither by the changes in Ca²⁺ sensitivity nor by artifacts of [Ca²⁺]_i measurements.

View larger version (46K): [in this window] [in a new window]   Fig. 4.   Two Ca2+ compartments model (modified from Abe et al., 1996). The major Ca2+ compartment in the smooth muscle cell is the contractile compartment. In addition, there is a small Ca2+ compartment between plasmalemma and the SR that does not contain contractile elements (noncontractile compartment). Communication between these two compartments is restricted, and aequorin cannot move freely between these compartments. Calcium ion in this compartment also cannot reach the contractile compartment because of a diffusion barrier and sequestration by the SR. Inhibition of SR Ca2+ pump by cyclopiazonic acid increased [Ca2+]i in the noncontractile compartment with little effect on the contractile Ca2+ compartment. In contrast, depletion of the SR by ryanodine and caffeine inhibited the agonist-induced transient increase in [Ca2+]i in contractile compartment with little effect on [Ca2+]i in the noncontractile compartment. Rates of decrease in contraction and [Ca2+]i were affected neither by cyclopiazonic acid nor by ryanodine and caffeine.

3. Role of localized calcium. The Ca²⁺-sensitive processes at cell membranes including ion channels, ion pump and enzymes are activated in situ or in vitro by Ca²⁺ 10-100 times higher than [Ca²⁺]_i measured during stimulation in intact cells (see Etter et al., 1996). It has been suggested that increased [Ca²⁺]_i in the subplasmalemmal restricted diffusion space could 1) facilitate the coupling of Ca²⁺ influx into SR Ca²⁺ release (CICR), 2) provide a mechanism for the regulation of stored Ca²⁺ that does not affect the contractile state of smooth muscle, and 3) locally activate the specific signal transduction pathway before or without activating other Ca²⁺-dependent pathways in the central cytoplasm of the cell (Rasmussen et al., 1987; Karaki, 1989a, 1990; Kargacin, 1994; Etter et al., 1994, 1996; Van Breemen et al., 1995).

a. REGULATION OF ION CHANNELS, PUMP, AND EXCHANGER. Calcium ion activates large-conductance K⁺ channels, Cl channels and nonselective cation channels, whereas it inhibits delayed rectifier K⁺ channels and inactivates Ca²⁺ channels (see Carl et al., 1996

View larger version (24K): [in this window] [in a new window]   Fig. 5.   The superficial buffer barrier system suggested by Van Breemen et al. (1995). Calcium entering the cell is partially sequestered by the superficial SR from a restricted subplasmalemmal space. This process is modulated by various agonists. Vasodilators, which raise cyclic nucleotide levels, will enhance buffering and decrease Ca2+ entry in the deeper myoplasm, while Ca2+-mobilizing agonists, which increase IP3, will shortcut the superficial buffer barrier and enhance the flow of Ca2+ into the myoplasm. The combination of basal IP3 production and cytoplasmic IP3 phosphatase may generate an IP3 gradient near the plasmalemma, which would activate IP3 receptors and subsequently ryanodine receptors in the junctional regions. The resulting vectorial Ca2+ release would raise Ca2+ near the Na+/Ca2+ exchange to facilitate extrusion of Ca2+ coupled to Na+ influx. This mode spatially separates Ca2+ unloading at the junctional regions from Ca2+ buffering in the restricted subplasmalemmal space. The resulting peripheral Ca2+ cycle generates a variable Ca2+ gradient in the subplasmalemmal space. (Reprinted with permission from Elsevier Science.)

	III. Changes in Calcium Sensitivity

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Simultaneous measurements of [Ca²⁺]_i and contraction showed that receptor agonists and phorbol esters induced greater contractions than high K⁺ at a given [Ca²⁺]_i (see section II.C.). Changes in Ca²⁺ sensitivity are observed not only in tonic-type smooth muscle such as large arteries but also phasic-type smooth muscle such as gastrointestinal muscle. In tonic muscle, agonists induce a sustained increase in Ca²⁺ sensitivity. In phasic muscle, in contrast, temporal changes in Ca²⁺ sensitivity are observed (Ozaki et al., 1991b, 1993). Agonists transiently increase Ca²⁺ sensitivity followed by a decrease, resulting in a phasic contraction. The differences between phasic and tonic types of smooth muscle are summarized by Himpens (1992), Ozaki and Karaki (1993) and Sanders and Ozaki (1994).

A technique developed to make small holes in the smooth muscle cell membrane using the saponin analog, -escin, or Staphylococcus aureus -toxin made it possible to precisely regulate the cytosolic concentrations of Ca²⁺ as well as other substances with molecular weights less than 1000 without disrupting receptor/signal transduction pathways and the contractile machinery. In these preparations, Ca²⁺ induced contraction in the presence of ATP. This contraction was augmented by norepinephrine and other receptor agonists in the presence of fixed concentration of Ca²⁺. Since GTP was necessary for the agonist-induced augmentation of Ca²⁺-induced contraction, and since GTPS showed effects similar to those of agonists, it was proposed that agonists increase the Ca²⁺ sensitivity of contractile elements by activating a GTP-binding protein (Nishimura et al., 1988, 1990; Kitazawa et al., 1989, 1991b). Phorbol esters also augmented Ca²⁺-induced contraction although GTP was not necessary for the effects of phorbol esters. Augmentation of Ca²⁺-induced contractions elicited by receptor agonist or phorbol ester was inhibited by the C kinase inhibitor, calphostin C or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) (Nishimura et al., 1992; Takizawa et al., 1993; Katsuyama and Morgan, 1993; Jiang et al., 1994; Satoh et al., 1994). These results suggested that C kinase activation is necessary to induce Ca²⁺ sensitization. On the other hand, Oishi et al. (1992) reported that C kinase inhibitor did not prevent the Ca²⁺ sensitization induced by acetylcholine in stomach smooth muscle. Moreover, the desensitization of the C kinase activity by long exposure to phorbol ester completely inhibited the Ca²⁺ sensitization induced by phorbol esters but not that induced by receptor agonists (Hori et al., 1993b). This result suggests that Ca²⁺ sensitivity of contractile elements may be increased by pathways dependent on and independent of C kinase. This result was confirmed by others (Itoh et al., 1994c; Rapoport et al., 1995; Fujita et al., 1995; Jensen et al., 1996). Recently, isoforms of C kinase in arteries were immunologically examined and Ca²⁺-dependent -isoform of C kinase and/or Ca²⁺-independent - and -isoforms of C kinase were found to be necessary for the phorbol ester-mediated contractions (Khalil et al., 1992; Ohanian et al., 1996). Furthermore, Jensen et al. (1996) reported that although both phorbol ester-induced and GTP-binding protein-coupled Ca²⁺ sensitization of force are mediated by increased MLC phosphorylation, it is likely that -, -, -, and -isoforms of C kinases do not play an essential role in the GTP-binding protein-coupled mechanism.

Smooth muscle contraction is explained by Ca²⁺-dependent activation of MLC kinase and phosphorylation of MLC (Kamm and Stull, 1985; Hartshorne, 1987). Observed variations in the relation between [Ca²⁺]_i and contraction is explained at least partly by variations in the relationship between [Ca²⁺]_i and MLC phosphorylation but not between MLC phosphorylation and contraction (Rembold, 1990). There are four proposed mechanisms for changes in the Ca²⁺ sensitivity of phosphorylation (fig. 6).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Mechanisms of agonist-induced Ca2+ sensitization in smooth muscle. Stimulation of a receptor increases [Ca2+]i, activates MLC kinase (MLCK), phosphorylates MLC, and induces contraction. This process is modulated by four different mechanisms. The first mechanism is the inhibition of Ca2+-calmodulin-dependent protein kinase II (CaMKII), which phosphorylates MLC kinase and inhibits its activity (1). The second mechanism is inhibition of MLC phosphatase (PPase) (2). Arachidonic acid, produced by receptor-mediated activation of phospholipase A2, may directly inhibit phosphatase (2A). C kinase and tyrosine kinase may also inhibit phosphatase by inhibiting the endogenous inhibitor of phosphatase (2B). The third mechanism is to increase free calmodulin concentration (3). The fourth mechanism is to activate actin independently of MLC phosphorylation (4).

The first mechanism is that increases in [Ca²⁺]_i activate Ca²⁺-calmodulin-dependent protein kinase II which phosphorylates MLC kinase thus decreasing its activity (Stull et al., 1990; Tansey et al., 1992). Agonists would somehow inhibit this negative feedback pathway. However, studies in both airway and vascular smooth muscles showed that increased [Ca²⁺]_i increased phosphorylation of MLC kinase independent of the stimulation, suggesting that this possibility is not likely under conditions of physiological muscle stimulation (Tang et al., 1992; Van Riper et al., 1995).

Somlyo and co-workers (Somlyo et al., 1989; Kitazawa et al., 1991a, b) have asserted a second hypothesis for altering the Ca²⁺ sensitivity of MLC phosphorylation by agonist-induced phosphatase inhibition. It was also reported that GTPS may increase the Ca²⁺ sensitivity of contractile elements by directly inhibiting protein phosphatase (Kubota et al., 1992). There are two mechanistic hypotheses for phosphatase inhibition. The first mechanism is that agonists activate phospholipase A₂ to cleave arachidonic acid from membrane phospholipids which, in turn, inhibits MLC phosphatase. Arachidonic acid enhanced Ca²⁺-induced contractions in -toxin permeabilized smooth muscle and inhibited MLC phosphatase in vitro (Gong et al., 1992). It has also been reported that arachidonic acid release is associated with inhibition of dephosphorylation of MLC in intact smooth muscle tissue (Gong et al., 1995). The second mechanism is that receptor agonists activate rho, a small GTP binding protein, which may directly or indirectly inhibit MLC phosphatase (Hirata et al., 1992; Fujita et al., 1995; Noda et al., 1995; Itagaki et al., 1995; Kokubu et al., 1995; Otto et al., 1996; Gong et al., 1996). In permeabilized smooth muscle, C3 exoenzyme isolated from Clostridium botulinum, which is known to selectively inactivate rho p21 by ADP ribosylation, inhibited the augmentation of Ca²⁺-induced contractions elicited by GTPS or receptor agonists. It has been reported that phosphorylation of the large subunit of MLC phosphatase decreased phosphatase activity and that there was an endogenous protein kinase that phosphorylated the large subunit (Trinkle-Mulcahy et al., 1995; Ichikawa et al., 1996). Recently, Matsui et al. (1996) reported that a novel rho-associated serine/threonine kinase (rho kinase) phosphorylated the myosin-binding subunit of MLC phosphatase in vitro. Kimura et al. (1996) also reported that rho kinase phosphorylated myosin-binding subunit of MLC phosphatase and inhibited its activity. Furthermore, over-expression of rho or activation of rho in NIH 3T3 cells increased phosphorylation of both subunit of MLC phosphatase and MLC. In swine vascular smooth muscle, Nishimura et al. (1996) reported the messenger RNA expression of rho A and rho kinase. These findings indicate that receptor agonists may activate the rho/rho kinase pathway, phosphorylate the large subunit of the phosphatase, and inhibit phosphatase activity. Phorbol esters also decrease the rate of relaxation and MLC dephosphorylation, suggesting that C kinase increases Ca²⁺ sensitivity through the inhibition of MLC phosphatase (Itoh et al., 1993; Masuo et al., 1994). There are six phosphorylation sites for C kinase in the myosin-binding subunit although the effects of phosphorylation are not known (Chen et al., 1995; Shimizu et al., 1994).

A third factor which may affect Ca²⁺ sensitivity is the availability of calmodulin. It is well known that concentration of the Ca²⁺-calmodulin complex can regulate the MLC kinase activity, and that Ca²⁺ concentration is regulated. However, it was postulated that the large intracellular pool of calmodulin is freely diffusible and saturating for kinase activity in living cells. From experiments of fluorescent recovery after photobleaching, however, it was found that only 5% of total calmodulin is freely diffusible in resting cells (Tansey et al., 1994; Luby-Phelps et al., 1995). Zimmermann et al. (1995) also estimated from flash photolysis studies with caged Ca²⁺ and caged ATP that the endogenous calmodulin concentration available in the resting state was of less than micromolar. Furthermore, Luby-Phelps et al. (1995) found that the diffusion coefficient and the percent mobile fraction of calmodulin were increased when [Ca²⁺]_i was elevated. These results suggest that endogenous calmodulin is compartmentalized into several intracellular pools with different affinities and is mobilized in a Ca²⁺-dependent manner. In neuroblastoma cells, it has been reported that carbachol stimulated a translocation of calmodulin from membrane to cytosol (Mangels and Gnegy, 1992). Thus, it is possible that not only Ca²⁺ concentration but also calmodulin concentration is regulated, and that changes in calmodulin concentration determine the Ca²⁺ sensitivity of MLC phosphorylation.

The agonist-induced increase in Ca²⁺ sensitivity may also result from activation of an actin-linked regulatory mechanism (Tansey et al., 1990; Stull et al., 1991; Sato et al., 1992; Hori et al., 1992; Karaki, 1995a, b, c; Kamm and Grange, 1996). In the absence of external Ca²⁺, prostaglandin F₂, endothelin-1, and phorbol esters induced sustained contractions in muscles in which Ca²⁺ stores had been depleted (Sato et al., 1992; Hori et al., 1992; Katsuyama and Morgan, 1993). These contractions were accompanied by increases in the rate of cross-bridge cycling of actomyosin although MLC phosphorylation stayed at a resting level (Sato et al., 1992; Hori et al., 1992). Wortmannin, an inhibitor of MLC kinase, inhibited the MLC phosphorylation with only partial inhibition of contractions induced by prostaglandin F₂ in the presence of external Ca²⁺ (Takayama et al., 1996). Takayanagi and coworkers also showed that the contractions mediated by the ₁-adrenoceptor were not inhibited by a MLC kinase inhibitor, KT5926 [(8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tetrahydro-8,11-epoxy,1H-8H-11H-2,7b,11a-triazadibenzo[a,g]cycloocta[cde]trinden-1-one] (Satoh et al., 1995; Takayanagi et al., 1997). These results suggest that smooth muscle contraction is regulated not only by MLC phosphorylation but also by a phosphorylation-independent mechanism, possibly a mechanism linked to actin. The actin-linked mechanism may be more sensitive to Ca²⁺ than MLC kinase and activated by agonists in the presence of resting level of [Ca²⁺]_i (Karaki, 1995a, b, c). Actin-binding proteins such as caldesmon (Sobue et al., 1981, 1982, 1991; Walsh, 1987, 1990), calponin (Takahashi et al., 1986, 1988; Nakamura et al., 1993; Ichikawa et al., 1993; Mino et al., 1995) and MLC kinase (Ebashi, 1990, 1991; Kohama et al., 1996) may be responsible for this regulatory mechanism (for review, see Kamm and Grange, 1996). Phosphorylation of caldesmon induced by mitogen-activated protein kinase was suggested to be one of the mechanisms of Ca²⁺ sensitization because the phosphorylation of caldesmon decreased its ability to inhibit actomyosin ATPase in vitro (Adam et al., 1989, 1992; Adam, 1996; Gerthoffer and Pohl, 1994). Khalil and Morgan (1993) also reported that the translocation of C kinase induced by phenylephrine was associated with transient translocation of cytosolic mitogen-activated protein kinase to the membrane before contraction and redistribution away to cytoplasm during contraction. They suggested a role for mitogen-activated protein kinase in the signal transduction cascade linking C kinase activation to smooth muscle contractility. In contrast to these reports, Nixon et al. (1995) reported that phosphorylation of caldesmon by recombinant mitogen-activated protein kinase (p42mapk) had no effect on resting tone or Ca²⁺ sensitivity of contraction in permeabilized smooth muscle.

Itoh et al. (1994b,d) showed that calponin inhibited actin-activated Mg²⁺-ATPase activity with a proportional increase in its binding to actomyosin and also attenuated Ca²⁺-induced contractions in permeabilized arterial strips in the presence or absence of calmodulin. Calponin, when phosphorylated by C kinase, reduced both its ability to bind to actomyosin and its inhibitory action on actomyosin Mg²⁺-ATPase. The phosphorylated calponin also had no effect on the maximum Ca²⁺-induced contraction in permeabilized smooth muscle, suggesting that these actions of calponin are specific. Calponin attenuated the Ca²⁺-independent contraction observed in MLC thiophosphorylated strips, or on application of trypsin-treated MLC kinase. A calponin peptide (calponin Phe-173-Arg-185), which inhibits the binding of calponin to actin, inhibited the action of calponin and enhanced the contraction induced by submaximal concentrations of Ca²⁺ in permeabilized vascular smooth muscle. Unlike calmodulin, this peptide enhanced the Ca²⁺-induced contraction without a corresponding increase in the level of MLC phosphorylation. These results suggest that calponin decreases the Ca²⁺ sensitivity of smooth muscle at a given level of MLC phosphorylation. However, Adam et al. (1995) showed that caldesmon but not calponin was phosphorylated during contractions of swine carotid arteries stimulated with histamine, high K⁺ or phorbol ester.

Agonists increase Ca²⁺ sensitivity of contractile elements in vascular (De Feo and Morgan, 1985; Sato et al., 1988a; Karaki et al., 1988a; Sakata et al., 1989; Takayanagi and Onozuka, 1989; Rembold, 1990), tracheal (Gerthoffer et al., 1989; Ozaki et al., 1990b) and gastric smooth muscle (Ozaki et al., 1991b, 1992a, 1993; Oishi et al., 1992). However, Ca²⁺ sensitivity is not increased in uterine (Sakata and Karaki, 1992; Szal et al., 1994; Kim et al., 1995a) and chicken gizzard smooth muscle (Anabuki et al., 1994). In rat anococcygeus muscle (Shimizu et al., 1995) and guinea pig taenia coli (Mitsui and Karaki, 1990, 1993), the increase in Ca²⁺ sensitivity was observed in permeabilized muscle but not in intact muscle. Because the mechanism of Ca²⁺ sensitization is not yet understood, the reasons for these tissue differences are not clear.

The increases in cyclic AMP due to -adrenergic stimulation and in cyclic GMP due to nitric oxide, atrial natriuretic peptides and nitro-vasodilators result in inhibition of contraction in intact smooth muscle (see Bulbring and Tomita, 1987; Kamm and Stull, 1989; Ignarro and Kadowitz, 1985; Ignarro, 1989). One of the mechanisms for the relaxation induced by these cyclic nucleotides was considered to be a decrease in [Ca²⁺]_i (see McDaniel et al., 1994; Kotlikoff and Kamm, 1996). Simultaneous measurements of [Ca²⁺]_i and muscle force, however, showed that these cyclic nucleotides more strongly inhibited contraction than [Ca²⁺]_i, suggesting that cyclic nucleotides caused muscle relaxation by desensitization of contractile elements to Ca²⁺ (Karaki et al., 1988b; Abe and Karaki, 1989, 1992b; Gunst and Bandyopadhyay, 1989; Tajimi et al., 1991; Chen and Rembold, 1992; McDaniel et al., 1992; Ozaki et al., 1992b, 1993; Kwon et al., 1993; Yamagishi et al., 1994). Furthermore, cyclic AMP and cyclic GMP inhibited Ca²⁺-induced contraction and agonist-induced augmentation of Ca²⁺-induced contraction in permeabilized smooth muscle (Nishimura and Van Breemen, 1989; Ozaki et al., 1992a, b; Tajimi et al., 1995). Paglin et al. (1988) found that, in rabbit aorta, atrial natriuretic peptide uncoupled MLC phosphorylation from the increase in [Ca²⁺]_i elicited by angiotensin II or histamine. Suematsu et al. (1991a) reported that forskolin significantly shifted the Ca²⁺-force curve and the Ca²⁺-MLC-phosphorylation curve to the right without changing the phosphorylation-force curve. These results suggest that both cyclic AMP and cyclic GMP increase the Ca²⁺ requirement for MLC phosphorylation (Ca²⁺ desensitization of MLC phosphorylation) either by inhibiting MLC kinase or activating MLC phosphatase. Phosphorylation of MLC kinase induced by cyclic AMP-dependent protein kinase would decrease the affinity of MLC kinase for Ca²⁺, resulting in a decrease of MLC kinase activity at a given Ca²⁺ in vitro (Adelstein et al., 1978; de Lanerolle et al., 1984). Recent work, however, demonstrated that the cyclic AMP-induced phosphorylation of MLC kinase is not the physiological mechanism for cyclic AMP-induced smooth muscle relaxation (Miller et al., 1983; Stull et al., 1990; Tang et al., 1992; Van Riper et al., 1995). Itoh et al. (1993) reported that a water-soluble forskolin, NKH477, activated MLC phosphatase in rat aorta. There are four phosphorylation sites in the smooth muscle phosphatase by A kinase (Shimizu et al., 1994), although the effects of phosphorylation on the phosphatase activity have not been defined.

Activation of G kinase did not decrease MLC kinase activity by phosphorylating MLC kinase (Nishikawa et al., 1984). Recently, it has also been reported that cyclic GMP inhibited Ca²⁺-induced contraction accompanied by a decrease in MLC phosphorylation (Kitazawa et al., 1996; Wu et al., 1996). The rate of relaxation and dephosphorylation of MLC was accelerated by 8-bromo-cyclic GMP in permeabilized muscle, suggesting that cyclic GMP activates the MLC phosphatase via G kinase. However, it has also reported that cyclic AMP and cyclic GMP relaxed the contraction without a proportional change in MLC phosphorylation in intact (McDaniel et al., 1992) and permeabilized muscle preparations (Su et al., 1996). Furthermore, these cyclic nucleotides also inhibited the contractions that are dependent neither on Ca²⁺ nor on MLC phosphorylation elicited by receptor agonists and phorbol esters in the absence of external Ca²⁺ (Ozaki et al., 1990c; Tajimi et al., 1995). These results suggest that cyclic nucleotides inhibit not only MLC phosphorylation-dependent pathway but also -independent pathway regulating contractile elements, although the details of the inhibitory mechanisms are not yet understood.

	IV. Effects of Pharmacological Agents

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A. Activators and Inhibitors of Protein Kinases and Phosphatases

1. Myosin light chain kinase. Wortmannin is a potent inhibitor of smooth muscle MLC kinase produced by a fungal strain, Talaromyces wortmannin (Nakanishi et al., 1992). It inhibits MLC kinase at 10 nM to 1 µM concentrations without affecting A kinase, G kinase, C kinase and Ca²⁺/calmodulin-dependent protein kinase II. However, it also inhibits phosphatidylinositol 3-kinase at concentrations lower than 10 nM (Okada et al., 1994). In rabbit aorta (Asano et al., 1995a), wortmannin inhibited high K⁺-induced contraction without changing [Ca²⁺]_i. In rat aorta, Takayama et al. (1996) showed that wortmannin decreased MLC phosphorylation to resting level and inhibited contractions induced by high K⁺. However, wortmannin did not change the high K⁺-induced increase in [Ca²⁺]_i. Wortmannin also decreased MLC phosphorylation to resting level in the presence of phenylephrine or prostaglandin F₂ without changing [Ca²⁺]_i. In canine gastric antrum, wortmannin changed neither resting membrane potential nor spontaneous slow waves (Burke et al., 1996). These results suggest that wortmannin inhibited MLC kinase without changing Ca²⁺ mobilization. However, a part of the contraction induced by prostaglandin F₂ was not inhibited by wortmannin (Takayama et al., 1996), suggesting that although contractions in rat aorta are due mainly to phosphorylation of MLC, another contractile mechanism exists which is not dependent on MLC phosphorylation or dependent only on resting level of MLC phosphorylation (Hori et al., 1992; Sato et al., 1992; Karaki, 1995a, b, c). Wortmannin also inhibited the release of human immunodeficiency virus type 1 from host cells by inhibiting myosin-actin interaction (Sasaki et al., 1995).

2. A kinase. The role of A kinase on smooth muscle contraction has been reviewed by Bulbring and Tomita (1987), Kamm and Stull (1989) and Kotlikoff and Kamm (1996). A kinase is activated by cyclic AMP produced by activation of adenylate cyclase. The -adrenoceptor agonists and forskolin are widely used to activate this enzyme. Inhibitors of phosphodiesterase also increase cyclic AMP. The effects of these agents on Ca²⁺ movements in smooth muscle are variable.

3. G kinase. Role of G kinase on smooth muscle contraction has been reviewed by Ignarro and Kadowitz (1985) and Kamm and Stull (1989). G kinase is activated by cyclic GMP produced by stimulation of guanylate cyclase by nitric oxide, atrial natriuretic peptide, and nitro-vasodilators. Effects of nitric oxide on Ca²⁺ movements will be described in section IV.E. Effects of G kinase on SR functions have been described in section II.D.5. Similar to A kinase, effects of G kinase on Ca²⁺ movements are diverse.

4. C kinase. a. ISOFORMS OF C KINASE IN SMOOTH MUSCLE. There are several isoforms of C kinase (see Nishizuka, 1995; Singer, 1996) and smooth muscle cells have -, -, -, and -isoforms (Schworer and Singer, 1991; Inoguchi et al., 1992; Khalil et al., 1992; Assender et al., 1994; Ali et al., 1994; Dixon et al., 1994; Khalil and Morgan, 1993; Ohanian et al., 1996). In freshly isolated vascular smooth muscle cells loaded with fura-2, Khalil et al. (1994) reported that increasing [Ca²⁺]_i caused translocation of -isoform of C kinase and suggested that the [Ca²⁺]_i threshold of translocation of -isoform in situ is less than that reported in most in vitro assays and is consistent with an effect of agonist-induced generation of other second-messengers that cause cooperative interactions leading to translocation. Contractions induced by phorbol esters may be mediated by - and -isoforms (Ohanian et al., 1996), whereas phorbol ester-induced contractions induced in the absence of external Ca²⁺ may be mediated by - and -isoforms (Khalil et al., 1992).

5. Tyrosine kinase. Tyrosine kinases are functionally classified into three groups; tyrosine kinases associated with cell surface receptors (group 1), the focal adhesion kinase (group 2) and nucleus tyrosine kinases (group 3) (see Wang and McMhirter, 1994). Smooth muscle contraction may be modified by the receptor tyrosine kinases (group 1A) or receptor-coupled tyrosine kinases (group 1B). Tyrosine kinase inhibitors have been reviewed by Levitzki and Gazit (1995).

6. Phosphatases. Okadaic acid, isolated from marine sponges of the genus Halichondoria (Tachibana et al., 1981), is the first exogenous inhibitor of the serine/threonine protein phosphatase. Okadaic acid has a relatively high specificity for type 2A phosphatase rather than for type 1 phosphatase, with weak inhibitory effect on type 2B and no effect on type 2C in skeletal muscle protein phosphatases (Bialojan and Takai, 1988). In contrast, calyculin A, isolated from marine sponge genus Discodermia (Kato et al., 1986), nonselectively inhibits type 1 and type 2A phosphatase (Ishihara et al., 1989a). Microcystin-LR, isolated from the cyanobacterial genera Microcystis, has a similar inhibitory action on phosphatases with okadaic acid (Eriksson et al., 1990a, b; MacKintosh et al., 1990). Tautomycin, isolated from the bacterium Streptomyces verticillatus, in contrast, is a nonselective inhibitor of type 1 and type 2A protein phosphatases in a manner similar to calyculin A (MacKintosh and Klumpp, 1990; Hori et al., 1991).

B. Agents That Change Sarcoplasmic Reticulum Function

1. Caffeine. Caffeine is widely used as a pharmacological tool for studying excitation-contraction coupling in muscle physiology and pharmacology. The primary site of action has been assumed to be located on the SR. In the vascular smooth muscle of rabbit aorta (Karaki, 1987), caffeine induced a transient contraction which is attributable to the release of Ca²⁺ from internal stores. The caffeine-induced contraction was inhibited by external Mg²⁺ and by procaine and it was potentiated by low temperature. These results are compatible with the general characteristics of CICR, suggesting that the CICR plays an important role in the contraction induced by caffeine (see Karaki and Weiss, 1988).

2. Ryanodine. Ryanodine is a neutral alkaloid extracted from Ryania speciosa and has been demonstrated to alter specifically Ca²⁺ movements across SR membranes in cardiac and skeletal muscles (Sutko et al., 1979, 1997; Sutko and Willerson, 1980). Ito et al. (1986) first demonstrated that ryanodine suppressed the phasic contractions in smooth muscle elicited by caffeine and norepinephrine in Ca²⁺-free medium. This finding was confirmed in different smooth muscles using different stimulants; norepinephrine in rabbit aorta (Hwang and Van Breemen, 1987), caffeine and carbachol in canine tracheal muscle (Gerthoffer et al., 1988), caffeine and norepinephrine in rat aorta (Sato et al., 1988b; Hisayama et al., 1990), caffeine in guinea pig taenia coli (Hisayama and Takayanagi, 1988), norepinephrine in rabbit ear artery (Kanmura et al., 1988), caffeine in rat and guinea pig aorta (Ito et al., 1989), endothelin-1 in coronary arterial cells (Wagner-Mann and Sturek, 1991), acetylcholine and caffeine in porcine coronary artery (Katsuyama et al., 1991) and acetylcholine in canine colonic smooth muscle (Sato et al., 1994a). Ito et al. (1986) also reported that ryanodine prevented the stimulation of ⁴⁵Ca²⁺ efflux by norepinephrine and caffeine although it did not alter the high K⁺-induced contraction and accompanying increase in ⁴⁵Ca²⁺ influx. These data are consistent with the hypothesis that ryanodine inhibits SR Ca²⁺ release in vascular smooth muscle. Aoki and Ito (1988) further demonstrated that opening of the Ca²⁺ release channel enhanced the interaction of ryanodine with the channel and confirmed the previous finding (Sutko et al., 1985) that ryanodine irreversibly opens the Ca²⁺ channels in the SR.

3. Inhibitors of sarcoplasmic reticulum calcium pump. Three compounds have been identified to be selective inhibitors of the SR Ca²⁺ pump; thapsigargin isolated from the umberilliferous plant, a mycotoxin, cyclopiazonic acid, and 2,5-di-(tert-butyl)-1,4-benzohydroquinone. These inhibitors have been used to clarify the roles of SR Ca²⁺ pumps in the regulation of [Ca²⁺]_i in smooth muscle (see Goeger and Riley, 1989; Thastrup, 1990; Seidler et al., 1989; Ozaki et al., 1992c; Uyama et al., 1992; Luo et al., 1993; Darby et al., 1993; Kwan et al., 1994).

C. Stimulants

1. Membrane depolarization. Membrane depolarization opens the L-type Ca²⁺ channels, increases Ca²⁺ influx, increases [Ca²⁺]_i and induces contraction. Thus, contraction induced by high K⁺ is considered to be due to a relatively simple mechanism, an increase in [Ca²⁺]_i without changing other signal transduction systems including phosphatidylinositol turnover and Ca²⁺ sensitization.

2. Receptor agonists.

a. -ADRENOCEPTOR AGONISTS. In rat aorta (Hisayama et al., 1990