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).