INTRODUCTION

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

CALCIUM IONS DIRECTLY REGULATE the contraction of smooth muscle through the activation of Ca²⁺/calmodulin-dependent myosin light-chain kinase (MLCK). For this to occur, an elevation of Ca²⁺ must be broadly or globally distributed throughout the cytoplasm. The view that a spatially homogeneous elevation in cytoplasmic Ca²⁺ is necessary for effective Ca²⁺ signaling has been radically altered by the discovery of local Ca²⁺ transients ("Ca²⁺ sparks") in cardiac (37), skeletal (99,193), and smooth (140) muscle cells. Ca²⁺ sparks are caused by the opening of ryanodine-sensitive Ca²⁺-release (RyR) channels in the sarcoplasmic reticulum (SR). A single Ca²⁺ spark is capable of producing a very high (10-100 µM) local (~1% of the cell volume) increase in [Ca²⁺], while increasing the global intracellular concentration of Ca²⁺ ([Ca²⁺]_i) by <2 nM (85, 140). A Ca²⁺ spark, by virtue of its high local [Ca²⁺] elevation, has the potential to modulate Ca²⁺-dependent processes that are not responsive to global increases in [Ca²⁺]_i .

Early evidence for local Ca²⁺ release in cells came from measurements of spontaneous transient currents through Ca²⁺-sensitive K⁺ channels in frog sympathetic ganglia neurons (26, 125, 126). Subsequently, Benham & Bolton (13) measured transient outward currents through large-conductance Ca²⁺-sensitive K⁺ (BK_Ca) channels (referred to as "spontaneous transient outward currents" or STOCs) in isolated single cells of longitudinal smooth muscle from rabbit jejunum and ear artery (13). STOCs, which are caused by Ca²⁺ sparks (140), have since been described in a wide variety of types of smooth muscle (13, 19, 43, 75, 79, 82, 89, 95, 137, 140, 143, 145, 157, 163, 167, 217-219). The existence of STOCs suggested the possibility that subsarcolemmal [Ca²⁺] is different from global cytoplasmic [Ca²⁺] and that, under certain circumstances, subsarcolemmal [Ca²⁺] could be significantly higher than global [Ca²⁺]_i. Further support for this idea has come from studies that demonstrated a dissociation between global [Ca²⁺]_i and BK_Ca channel currents (56, 181). Also, Yamaguchi (215) provided direct evidence, using a modified confocal microscope, for an isoproterenol-induced increase in peripheral [Ca²⁺], which was ryanodine sensitive and distinct from [Ca²⁺] changes that occur in the cytosol.

The impact of a Ca²⁺ spark depends on a number of key factors: 1) the distance of the Ca²⁺ spark site from a Ca²⁺-sensitive target, 2) the number of target molecules affected by a Ca²⁺ spark, 3) the Ca²⁺ sensitivity of the target protein, and 4) the nature of the Ca²⁺-sensitive target. The physiological outcome of the Ca²⁺ spark depends on the [Ca²⁺] that reaches the target. Ca²⁺ reaches very high levels (10-100 µM) very close (e.g., 20 nm) to the release site (Ref. 153; see also Ref. 139). However, at several hundred nanometers from a Ca²⁺ spark release site, Ca²⁺ approaches equilibrium with mobile buffers (including fluorescent indicators), and the local elevation in [Ca²⁺] by a spark is less pronounced (< 500 nM; see Ref. 139). Molecular targets with low affinity for Ca²⁺, such as BK_Ca channels, are uniquely suited to respond to these very high local [Ca²⁺], since these channels require micromolar Ca²⁺ for significant levels of activity under physiological conditions (153). A single Ca²⁺ spark has a tremendous impact on cell membrane potential [up to 20 mV hyperpolarization of single cells (52)] through activation of BK_Ca channels (140). In contrast, some Ca²⁺/calmodulin (CaM)-dependent targets [e.g., MLCK, CaM kinase, small-conductance Ca²⁺-sensitive K⁺ (SK_Ca) channels, nitric oxide synthase (NOS), calcineurin, Ca²⁺-activated chloride (Cl_Ca) channels] are well suited to respond to changes in global Ca²⁺ (0.1-1 µM), since CaM is saturated by [Ca²⁺] below 1 µM.

The goal of this review is to provide up-to-date information on RyR channels and Ca²⁺ sparks in smooth muscle and to propose the following: 1) voltage-dependent Ca²⁺ channels, Ca²⁺ sparks (RyR channels), and BK_Ca channels act as a functional unit to regulate smooth muscle function (Figs. 1 and 2); 2) communication between voltage-dependent Ca²⁺ channels and RyR channels is a critical element in the control of global [Ca²⁺]_i (Figs. 1 and 2); 3) Ca²⁺ sparks are involved in both positive- and negative-feedback regulation of global [Ca²⁺]_i (Figs. 1 and 2); 4) frequency and amplitude modulation (FM and AM) of Ca²⁺ sparks by relaxant and contractile agents are mechanisms to regulate smooth muscle function. Unitary Ca²⁺ release may also occur due to inositol trisphosphate (IP₃) activation of IP₃ receptors in the SR [termed "blips" and "puffs" (22, 216)]. In this review, the term `Ca²⁺ sparks' refers to Ca²⁺ release through RyR channels.

View larger version (29K): [in this window] [in a new window]   Fig. 1.   Proposed functional roles of Ca2+ sparks in smooth muscle cells. Left: local control of Ca2+ sparks by voltage-dependent Ca2+ channels in cardiac and smooth muscle. In cardiac muscle, high local Ca2+ concentration ([Ca2+]) from L-type voltage-dependent Ca2+ channels regulates Ca2+ spark frequency. In smooth muscle, the role of local Ca2+ entry is not known. Right: Ca2+ spark activation of BKCa channels to complete a negative-feedback loop (green arrow) through membrane potential hyperpolarization to decrease Ca2+ entry. Also illustrated is a positive-feedback contribution (red arrow) of Ca2+ release from RyR channels to contraction. In most smooth muscle types, the negative-feedback pathway appears to dominate.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Possible relationships among voltage-dependent Ca2+ channels, ryanodine-sensitive Ca2+-release (RyR) channels, large-conductance Ca2+-sensitive K+ (BKCa) channels, and Ca2+-activated Cl (ClCa) channels to regulate smooth muscle contractility. Voltage-dependent Ca2+ channels (VDCC) are the primary Ca2+ entry pathway in most types of smooth muscle. For tonic smooth muscle, such as resistance arteries, steady Ca2+entry through VDCC determines intracellular [Ca2+] ([Ca2+]i; indicated by thick arrow). Ca2+ entry through VDCCs also stimulates RyR channels as Ca2+ spark events or as non-Ca2+ spark events. The type of Ca2+ communication from VDCCs to RyR channels (e.g., local Ca2+ control) in smooth muscle is not known. RyR channels can serve as negative- and positive-feedback transducers to VDCCs. Ca2+ release from RyR channels could inactivate VDCCs (negative-feedback element); this is not known for smooth muscle. Ca2+ release from RyR channels may also contribute to global [Ca2+]i for contraction [i.e., Ca2+-induced Ca2+ release (CICR); positive feedback element]. Contribution of Ca2+ sparks to global [Ca2+]i may be significant in phasic smooth muscle and less important in tonic smooth muscle. Ca2+ sparks activate BKCa channels in virtually all types of smooth muscle to cause a very substantial membrane potential (Vm) hyperpolarization (negative-feedback element). Sparks activate ClCa channels in some types of smooth muscle to cause membrane depolarization (positive-feedback element). Ca2+ release through RyR channels could also activate SKCa channels to cause membrane potential hyperpolarization in some types of smooth muscle (effect not illustrated). The final outcome of these various signaling elements depends on a number of factors, including proximity of the various elements, Ca2+ sensitivity, and phosphorylation state. MLCK, myosin light-chain kinase.

	RYANODINE RECEPTORS IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

Smooth muscle SR elements are very close (within 20 nm) to the plasma membrane (Fig. 3A) (45). RyR channels have been localized to both the peripheral (Fig. 3A) and central SR (45, 61, 118). Dihydropyridine receptors (voltage-dependent Ca²⁺ channels) and RyR channels colocalize in smooth muscle (33, 61), suggesting an intimate communication between these proteins in the plasma membrane and the SR. A strong spatial association has also been shown for the Na⁺-K⁺ pump and the Na⁺/Ca²⁺ exchanger, and the Na⁺/Ca²⁺ exchanger and Ca²⁺ released from the SR (18, 136). This close spatial localization between RyR channels and the plasma membrane suggests a special communication between the SR and sarcolemmal ion channel and ion transport systems.

View larger version (83K): [in this window] [in a new window]   Fig. 3.   A: surface coupling between junctional sarcoplasmic reticulum (SR) and plasma membrane: leaflets of SR and cell membranes are separated by an ~12- to 20-nm gap. Electron-opaque material present between the two opposing membranes has a "periodicity" of ~20-25 nm (arrows). Section is taken from rabbit portal anterior mesenteric vein. Scale bar = 0.1 µm. [From Devine et al. (45) by copyright permission of The Rockefeller University Press.] B: confocal image of an adult cerebral artery smooth muscle cell stained with a monoclonal anti-RyR2 antibody illustrating distinct staining along the cell membrane. [Modified from Gollasch et al. (61).]

RyR channels are found in all muscle types and many other cell types, including those from mammals, birds, amphibians, reptiles, fish, insects, crustaceans, and molluscs (for a review see Ref. 183). RyR channels were first identified in insect and invertebrate striated muscles, due to the action of the plant alkaloid, ryanodine, a potent channel antagonist (see Ref. 183). Presently, three molecularly distinct subtypes of RyR channels (RyR1, RyR2, RyR3) have been identified and cloned, with each thought to exist as a homotetramer in the SR membrane. RyR1 is found primarily in skeletal muscle, whereas RyR2 and RyR3 are predominantly found in cardiac tissue and brain, respectively. mRNA transcripts for RyR1, RyR2, and RyR3 have been identified in smooth muscle preparations (see Refs. 67, 90, 117, 142). However, these results should be interpreted with some caution, since the detection of RyR1-3 transcripts may reflect contamination from cells other than smooth muscle cells (e.g., endothelium, neurons) and does not necessarily reflect the level of functional protein.

A major breakthrough in the investigation of the functional properties of RyR channels involved the incorporation of RyR channels from cardiac and skeletal muscle SR microsomes into planar lipid bilayers (176). One difficulty with isolating RyR channels from smooth muscle is that smooth muscle SR membranes cannot be readily separated from other membranes. As an alternative approach, RyR channels have been partially purified from aortic smooth muscle (71) (Table 1) and from toad stomach smooth muscle (214) and incorporated into microsomes and then into planar lipid bilayers. The molecular and functional properties of the three cloned RyR channel subtypes and the smooth muscle RyR channel are summarized in Table 1. RyR channels from smooth muscle are activated by micromolar cytoplasmic [Ca²⁺] (Fig. 4) and by caffeine and are inhibited by Mg²⁺ and ruthenium red (71), similar to RyR2 and RyR3. The single-channel conductance of the smooth muscle RyR channels is also similar to the cardiac (RyR2) and skeletal muscle (RyR1) channels. However, the density of RyR channels in smooth muscle is considerably lower than in other tissues (Table 1).

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Dependence of smooth muscle RyR channel activity on cytoplasmic free [Ca2+]. Data represent single-channel recordings of a Ca2+-activated Ca2+-release channel from aortic smooth muscle incorporated into a planar lipid bilayer. [From Herrmann-Frank et al. (71).] For experimental protocol see Ref. 71. Left: single-channel recordings in symmetrical 200 mM NaCl, 20mM PIPES, pH 7, in the presence of indicated free Ca2+ concentrations. Free Ca2+ concentration was reduced by adding EGTA to the cis (cytoplasmic) side of the channel. Channel openings are illustrated as upward deflections from baseline. Right: cumulative open and closed time histograms for different free Ca2+ concentrations with fitted time constants. Open time histogram: 1 = 42 ms, 2 = 110 ms (100 µM Ca2+) compared with 1 = 8.2 ms, 2 = 33ms (0.5 µM Ca2+). Closed time histogram: 1 = 23 ms, 2 = 150 ms (100 µM Ca2+) compared with 1 = 115 ms, 2 = 210 ms (0.5 µM Ca2+).

It has been suggested that RyR3 may be the prominent ryanodine receptor isoform in smooth muscle (67). However, a possible role of RyR3 in smooth muscle is unclear, since mice lacking RyR3 do not have impaired smooth muscle function, and smooth muscle cells from these animals respond normally to caffeine and norepinephrine (187). Indeed, RyR3-deficient mice mature to adulthood, with the only discernable abnormality being increased motor activity (187).

A number of factors suggest that the predominant RyR channel subtype in smooth muscle is similar to that in cardiac muscle (RyR2). For example, the pharmacological and biophysical characteristics of RyR channels isolated from native smooth muscle cells are similar to those of RyR2 channels incorporated into lipid bilayers (71, 214) (Table 1). Ca²⁺ sparks that occur in cardiac cells and smooth muscle cells have similar biophysical and pharmacological characteristics (e.g., see Refs. 37 and 140; Table 2). Furthermore, the ryanodine-binding receptors purified from toad stomach, and from cerebral artery smooth muscle cells, cross-react with monoclonal antibodies for RyR2 (Fig. 3B) (61, 214). Therefore, the RyR channel responsible for Ca²⁺ release from the SR in smooth muscle appears to be similar to the RyR2 subtype, although an involvement of other RyR channel subtypes is also possible.

	CA2+ SPARK PROPERTIES IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

Basic Properties

Ca²⁺ sparks in smooth muscle cells were first described in myocytes from rat cerebral arteries [Figs. 5 and 6 (140), see also Refs. 19, 61, 85, 86, 153, 157]. Subsequently, Ca²⁺ sparks have been measured in smooth muscle cells from coronary arteries (86, 157), mesenteric artery (132), rat portal vein (9), guinea pig (218) and porcine (172) trachea, guinea pig ileum (63), guinea pig urinary bladder (83), guinea pig vas deferens (83), and toad stomach (219). The properties of Ca²⁺ sparks appear to be similar in these different smooth muscle preparations (Table 3). Figure 5 illustrates recordings of Ca²⁺ sparks in the smooth muscle cells of an intact pressurized (60 mmHg) cerebral artery. In arterial smooth muscle, Ca²⁺ sparks have a rise time of ~20 ms, a half time of decay of 50-60 ms (Figs. 5 and 6), and a spatial spread (full width at the half-maximum amplitude) of 2.4 µm (19, 85, 140, 153). These unitary events occur most frequently close to the cell membrane (140), although events away from the cell membrane have been reported in smooth muscle cells from portal vein (9) and ileum (63). Ca²⁺ sparks occur in intact, nonpressurized (61, 85) and pressurized cerebral (Fig. 5) and mesenteric arteries (132), with an apparent frequency of ~1 · s ¹ · cell ¹ at physiological membrane potentials (60 to 40 mV). These results support the idea that [Ca²⁺]_i is dynamically and heterogeneously distributed in intact tonic smooth muscle.

View larger version (57K): [in this window] [in a new window]   Fig. 5.   Ca2+ sparks in a pressurized cerebral artery. Arteries were loaded with 10 µM fluo 3 and cannulated at each end as previously described (25). A steady luminal pressure of 60 mmHg was applied and the artery imaged using a Noran laser scanning confocal microscope. For similar experimental procedure see Ref. 85. Areas (56 × 52 µm) were acquired at a rate of 60 images/s for 10 s. Ca2+ sparks were observed in the smooth muscle cells of the artery (top; average of 100 images) as transient localized changes in fluorescence. Bottom: fractional increase in fluorescence (F/F0) vs. time for analysis boxes a-d. Gray lines illustrate the smooth muscle cells (3 cells are clearly identifiable) (G. C. Wellman and M. T. Nelson, unpublished observations).

View larger version (42K): [in this window] [in a new window]   Fig. 6.   Ca2+ sparks activate BKCa channel currents in smooth muscle cells from cerebral arteries. A: original sequence of two-dimensional confocal images obtained every 8.33 ms of an entire smooth muscle cell (top), followed by subsequent images of region of interest (dotted box) illustrating time course of fractional increase in fluorescence (F/F0) and decay of a typical Ca2+ spark. Images are color coded as indicated by color bar. B: simultaneous BKCa channel current and Ca2+ spark (F/F0) measurements at 40 mV illustrating temporal association. Current (blue) is indicated above F/F0 average (red and green) of red and green boxes (2.2 µm/side) indicated in A, respectively. Purple bar, segment of trace illustrated in A. [Modified from Perez et al. (153).]

A Ca²⁺ Spark is due to the Simultaneous Activation of a Cluster of RyR Channels

Fate of Ca²⁺ From a Ca²⁺ Spark

The smooth muscle cell Ca²⁺ spark represents the spatially localized, unitary release of a significant amount of Ca²⁺ very close to the plasma membrane, with essentially no effect on global [Ca²⁺]_i or on contraction (140). The localized release of Ca²⁺ into a subsarcolemmal domain can thus represent a measurable element of the "superficial buffer barrier" (SBB) hypothesis (for review see Ref. 196). This hypothesis suggests that Ca²⁺ entering a smooth muscle cell is continuously buffered by the SR and is then discharged "vectorially" toward the plasma membrane without any effect on global [Ca²⁺]_i. Subsequently, this Ca²⁺ is extruded from the cell via the sarcolemmal Na⁺/Ca²⁺ exchanger and Ca²⁺-ATPase. The anatomic requirements for a Ca²⁺ spark to activate BK_Ca channels are similar to those proposed for the SBB hypothesis. Additional studies on the decay of Ca²⁺ sparks should provide new insights into local Ca²⁺ extrusion and uptake mechanisms as well as the SBB hypothesis.

	CA2+ SPARKS ARE INVOLVED IN BOTH POSITIVE AND NEGATIVE FEEDBACK REGULATION OF GLOBAL INTRACELLULAR [CA2+] IN SMOOTH MUSCLE

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

The role of local Ca²⁺ signaling is cell type dependent and should be viewed in the context of cell function. For example, cardiac muscle must contract forcefully and relax rapidly at high rates (1-7 Hz). In these cells, activation of voltage-dependent Ca²⁺ channels, which is synchronized by the action potential, leads to an immediate rise in Ca²⁺ influx, which activates, in a coordinated and rapid fashion, large numbers of Ca²⁺ sparks, which summate to cause the global [Ca²⁺]_i transient. Thus Ca²⁺-induced Ca²⁺-release (CICR) through RyR channels acts as an amplifier for Ca²⁺ influx, contributing 90% of the cellular Ca²⁺ needed for contraction of heart muscle (Fig. 1). Although cardiac and smooth muscle share many of the same key components for excitation-contraction coupling (e.g., L-type Ca²⁺ channels and RyR channels), these elements are arranged to regulate function in very different ways. Importantly, cardiac muscle lacks BK_Ca channels. In smooth muscle, the triumvirate of dihydropyridine-sensitive voltage-dependent Ca²⁺ channels, RyR channels, and BK_Ca channels represent a functional unit to control the levels of [Ca²⁺]_i (86).

Most types of smooth muscle are involved in maintaining tonic contraction. One important element in graded, tonically contracted smooth muscle is the negative-feedback control of membrane potential, and hence Ca²⁺ entry, through activation of BK_Ca channels (Figs. 1, 2, and 6; cf. Refs. 25 and 140). Some types of smooth muscle (e.g., urinary bladder) exhibit phasic contractions, suggesting that Ca²⁺ release through RyR channels may contribute directly to contraction, like cardiac muscle, and indirectly to relaxation through activation of BK_Ca channels (Fig. 6) (69). Furthermore, as discussed later, some types of smooth muscle have SK_Ca channels and Cl_Ca channels (see Fig. 2 and Table 4), which will also contribute to the regulation of smooth muscle excitability.

Communication Between Voltage-Dependent Ca²⁺ Channels and RyR Channels is a Critical Element in the Control of Global Intracellular [Ca²⁺]

The communication between Ca²⁺ channels and RyR channels/Ca²⁺ sparks is less clear in smooth muscle. In smooth muscle, Ca²⁺ sparks are also regulated by Ca²⁺ influx through voltage-dependent Ca²⁺ channels, since Ca²⁺ spark frequency and amplitude increase with activation of voltage-dependent Ca²⁺ channels in the smooth muscle cells of intact arteries (85). Membrane potential depolarization of intact cerebral arteries from about 60 to 40 mV, a membrane potential that would occur in pressurized arteries with tone, increases Ca²⁺ spark frequency about fivefold (85). The voltage dependence is similar to that observed in isolated cardiac myocytes over a similar voltage range (30, 164). This increase in Ca²⁺ spark frequency and amplitude depends on Ca²⁺ influx through voltage-dependent Ca²⁺ channels, since it is reversed by diltiazem, a selective blocker of L-type Ca²⁺ channels (85). Additionally, in voltage-clamped rat portal vein myocytes, Ca²⁺ sparks have been observed to occur immediately following a depolarizing voltage step that elicits a Ca²⁺ current (9). Although these studies indicate that Ca²⁺ spark frequency is modulated by Ca²⁺ influx through voltage-dependent Ca²⁺ channels, it is still unresolved whether local Ca²⁺ regulates Ca²⁺ spark probability in smooth muscle.

Steady membrane depolarization of arterial smooth muscle increases voltage-dependent Ca²⁺ channel open probability (e.g., see Ref. 162), global [Ca²⁺]_i, and presumably SR [Ca²⁺]. An elevation in local [Ca²⁺] from Ca²⁺ channels, global [Ca²⁺]_i, or SR [Ca²⁺] could each contribute to the observed depolarization-induced increase in Ca²⁺ spark frequency and amplitude (Figs. 1 and 7). Indeed, recent evidence indicates that Ca²⁺ spark and STOC frequency in smooth muscle cells depends steeply on the SR Ca²⁺ load (219). Figure 7 illustrates modulation of the Ca²⁺ dependence of Ca²⁺ spark frequency by SR Ca²⁺ load and other factors (see FM AND AM OF CA²⁺ sparks: physiological implications).

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Hypothetical modulation of cytoplasmic Ca2+ dependence (activator [Ca2+]) of Ca2+ spark frequency. Activators of RyR channels (e.g., caffeine), an increase in SR Ca2+ load [e.g., through cAMP-dependent protein kinase (PKA) phosphorylation of phospholamban], and PKA/cGMP-dependent protein kinase (PKG) may increase Ca2+ sensitivity of RyR channel and increase frequency of Ca2+ sparks at a given [Ca2+]i. PKA/PKG may in part increase Ca2+ sensitivity of Ca2+ sparks by increasing SR Ca2+ load (see text). In contrast, PKC may induce a rightward shift in Ca2+ sensitivity of the RyR channel, since activators of PKC decrease Ca2+ spark frequency at a given [Ca2+]i (19).

Ca²⁺ release from RyR channels can inactivate voltage-dependent Ca²⁺ channels in cardiac myocytes (23, 134, 170), which would serve as another negative-feedback mechanism to regulate Ca²⁺ entry. The possibility that a similar mechanism plays a role in smooth muscle has not been extensively examined (although see Ref. 97). Indirect evidence from measurements of arterial wall [Ca²⁺]_i and diameter in pressurized arteries suggests that this pathway is not significant (103), at least in the absence of receptor agonists.

Direct Contribution of Ca²⁺ Sparks to Cytoplasmic [Ca²⁺]

1

1

In smooth muscle types that exhibit phasic contractions (e.g., urinary bladder), Ca²⁺ release through RyR channels may contribute Ca²⁺ directly for contraction. Unlike tonic smooth muscle, phasic smooth muscle exhibits action potentials, which activate voltage-dependent Ca²⁺ channels to trigger Ca²⁺ release. Ca²⁺ release through RyR channels can contribute as much as 70% of the increment in global Ca²⁺ (30% to Ca²⁺ influx) in isolated voltage-clamped urinary bladder myocytes (54). The excitability and duration of action potentials in phasic smooth muscle are also regulated in a negative-feedback manner by BK_Ca and SK_Ca channels (69, 70). Therefore, blocking RyR channels affects both positive- and negative-feedback regulation of [Ca²⁺]_i in these types of smooth muscle. The precise contribution of Ca²⁺ release through RyR channels to global [Ca²⁺]_i in intact phasic smooth muscle remains to be determined.

The ability of voltage-dependent Ca²⁺ influx to stimulate Ca²⁺ release through RyR channels appears to vary considerably among different types of smooth muscle. All types of smooth muscle are capable of releasing significant amounts of Ca²⁺ through RyR channels, since caffeine, an activator of RyR channels (Table 1), can induce global ryanodine-sensitive Ca²⁺ transients in virtually all smooth muscle types (cf. Refs. 12, 39, 49, 54, 61). Yet, influx through voltage-dependent Ca²⁺ channels can induce ryanodine-sensitive elevations in global [Ca²⁺]_i in some (54, 94), but not all (49, 128), types of smooth muscle. The lack of effective CICR through RyR channels suggests that, in smooth muscle, many of the RyR channels are spatially distant from the high local [Ca²⁺] caused by the opening of voltage-dependent Ca²⁺ channels. However, even in cell types (e.g., urinary bladder) that exhibit significant ryanodine-sensitive global Ca²⁺ transients, the situation is very different from cardiac muscle. CICR, when it does occur in smooth muscle, is very sluggish, developing slowly over hundreds of milliseconds (93, 94). In contrast, the close proximity of cardiac Ca²⁺ channels and RyR channels enables rapid and effective communication, i.e., Ca²⁺ influx stimulates Ca²⁺ release (Ca²⁺ sparks) within milliseconds (30, 121). Therefore, the failure of voltage-dependent Ca²⁺ influx to induce substantial Ca²⁺ transients does not seem to reflect a lack of releasable Ca²⁺ through RyR channels, but may reflect a physical separation of voltage-dependent Ca²⁺ channels from a majority of the RyR channels. Communication between Ca²⁺ channels and RyR channels in smooth muscle may be analogous to that in atrial muscle where the t-tubular network is virtually absent. Atrial muscle has been found to contain "feet-like" structures similar in appearance to those in smooth muscle (45) that occur close to the plasmalemma (50). Electrical stimulation of atrial myocytes has been demonstrated to elicit Ca²⁺ sparks immediately beneath the plasmalemma, which then trigger Ca²⁺ sparks in more central cellular regions to elicit a global Ca²⁺ transient (15, 80).

In heart, the Ca²⁺ transient is composed of the simultaneous activation of many Ca²⁺ sparks. However, in smooth muscle, the contribution of Ca²⁺ sparks to ryanodine-sensitive global Ca²⁺ transients is not known. It is also conceivable that, in smooth muscle, Ca²⁺ spark sites communicate only with local targets (e.g., BK_Ca channels), and ryanodine-sensitive global Ca²⁺ transients are caused by Ca²⁺ release through RyR channels, which are not in Ca²⁺ spark sites (these channels may be located in the central SR rather than at peripheral subplasmalemmal locations). The precise regulation of Ca²⁺ sparks and RyR channels by local Ca²⁺ influx and the direct contribution of Ca²⁺ release to contraction remain to be elucidated.

Negative Feedback Regulation of Global [Ca²⁺]_i Through Ca²⁺ Spark Activation of BK_Ca Channels in Smooth Muscle

Ca²⁺ sparks cause up to 20 mV hyperpolarizations of isolated arterial myocytes through activation of BK_Ca channels (Fig. 8, A and B, and Table 4) (52). Figure 8A illustrates a simultaneous measurement of Ca²⁺ sparks and associated membrane potential hyperpolarizations in an isolated cerebral artery myocyte. Smooth muscle cells are electrically coupled in the intact tissue, such that asynchronized hyperpolarizations that are caused by Ca²⁺ sparks should summate and lead to a graded level of hyperpolarization (see Fig. 8, B and C) (48). Ca²⁺ spark-induced membrane potential hyperpolarizations contribute significantly to the membrane potential of isolated smooth muscle cells. For example, in Fig. 8B, Ca²⁺ spark-induced hyperpolarizations caused an average membrane potential change of ~15 mV. Ryanodine caused a 15-mV depolarization by inhibiting the transient hyperpolarizations (Fig. 8B). In support of this mechanism, inhibition of Ca²⁺ sparks or BK_Ca channels causes a membrane potential depolarization of ~10 mV in pressurized cerebral arteries (Fig. 8C) (103), which leads to an elevation in global [Ca²⁺]_i and vasoconstriction (61, 103, 140, 157). The effects of blocking Ca²⁺ sparks and BK_Ca channels on membrane potential, arterial wall [Ca²⁺]_i, and diameter are not additive, supporting the idea that Ca²⁺ sparks regulate BK_Ca channel activity (61, 103, 140, 157). These results support the concept that Ca²⁺ sparks, in part, regulate vascular tone, in a negative feedback manner, through activation of BK_Ca channels (19, 48, 61, 85, 86, 103, 140, 153, 157).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   Ca2+ sparks hyperpolarize the membrane potential of arterial smooth muscle. A: Ca2+ sparks elicit transient membrane hyperpolarization. Simultaneous recording of Ca2+ sparks (top) and membrane potential (bottom) in an isolated cerebral artery smooth muscle cell. Changes in subcellular Ca2+ fluorescence were recorded using a laser scanning confocal microscope and the Ca2+-sensitive dye fluo 3. Top: fluorescence ratio recording of 2 Ca2+ sparks that occur at a single Ca2+ spark site. Bottom: simultaneous patch-clamp recording from same cell under current clamp. Both Ca2+ sparks elicit spontaneous transient membrane potential hyperpolarizations (STHs) due to activation of BKCa channels (L. F. Santana and M. T. Nelson, unpublished observations). B: STHs contribute to membrane potential hyperpolarization in arterial smooth muscle cells and are inhibited by application of 10 µM ryanodine. STHs were observed in an isolated cerebral artery smooth muscle cell, under current clamp, using perforated-patch configuration of patch-clamp technique. Data were acquired using an Axopatch 200 amplifier (Axon Instruments) at 2 kHz and subsequently filtered at 500 Hz with a bessel filter. Mean contribution of STHs to membrane potential (red line; obtained by averaging 1,000 adjacent data points) was ~15 mV. Application of 10 µM ryanodine inhibited STHs and reduced mean STH contribution to membrane potential. Green and blue dotted lines illustrate the basal membrane potential and peak Ca2+ spark-induced transient membrane potentials, respectively. Ryanodine (10 µM) induced membrane potential depolarization due to a reduction of STHs and the impact of these events on mean membrane potential (red line). Depolarization was not due to a steady-state depolarization of basal membrane potential (green dotted line) (J. H. Jaggar and M. T. Nelson, unpublished observations). C: ryanodine depolarizes membrane potential of smooth muscle cells in pressurized (60 mmHg) cerebral arteries. Inhibition of Ca2+ sparks by application of 10 µM ryanodine depolarizes membrane potential of a pressurized (60 mmHg) cerebral artery by 9 mV (103). [Modified from Knot et al. (103).] STHs are not observed in this cell, due presumably to cell-cell contact between smooth muscle cells in the artery. i.e., hyperpolarization due to individual Ca2+ sparks is averaged throughout the artery.

Positive Feedback Regulation of Global [Ca²⁺]_i Through Ca²⁺ Spark Activation of Cl_Ca Channels in Smooth Muscle

View larger version (54K): [in this window] [in a new window]   Fig. 9.   A single Ca2+ spark activates biphasic currents (STOICs) in isolated voltage-clamped tracheal myocytes. [Modified from ZhuGe et al. (218).] For experimental protocol see Ref. 218. A: time course of a single Ca2+ spark acquired at times designated in B (top). Images in bottom row show entire cell. Contour plots in top row show spark at higher spatial resolution. Bar = 5 µm. B, top: time course of the change in [Ca2+] in the one pixel (333 nm × 333 nm) where peak [Ca2+] is reached during the course of the Ca2+ spark. Bottom: current simultaneously recorded at a holding potential (HP) of 30 mV. Transient outward current is due to Ca2+ spark activation of BKCa channels, whereas longer inward current represents ClCa channel activation.

There are some notable differences in the properties of STOCs and STICs. First, the apparent number of channels involved in STOCs and STICs may be very different. The amplitudes of STICs and STOCs are similar (with similar driving forces for Cl and K⁺; e.g., Fig. 9), even though the unitary conductance of the BK_Ca channel (80 pS) is ~30-fold greater than the Cl_Ca channel [2.6 pS (100); for reviews see Refs. 31, 116, 141]. Therefore, a STIC appears to represent the activation of at least 600 Cl_Ca channels by a Ca²⁺ spark. Cl_Ca channels may be clustered above Ca²⁺ spark sites, since tracheal myocytes have <10,000 Cl_Ca channels/cell (116), and a STIC represents the activation of at least 5% of the Cl_Ca channels in 0.25% of the membrane surface area in this preparation. Second, the decay of STICs is much slower than the decay of STOCs and mirrors the decay of the fluorescence transient (see Figs. 6 and 9) (218).

The precise effect of the simultaneous activation of Cl_Ca and BK_Ca channels by a Ca²⁺ spark on membrane potential would depend on a number of factors, including the intrinsic voltage dependence of Cl_Ca (none) and BK_Ca channels [open probability changes about e-fold for 12 -14 mV (14, 115)] and the voltage and Ca²⁺ dependence of other conductances in the cell membrane (Table 4). It is curious that Cl_Ca channels and STICs have only been reported in a handful of smooth muscle preparations [rabbit portal vein (77, 78, 203), rabbit pulmonary artery (76), and canine and guinea pig tracheal myocytes (87, 218, and see Ref. 116)], suggesting that these channels subserve a functional role unique to these tissues. The coexistence of Cl_Ca and BK_Ca channels in the sarcolemmal membrane adjacent to Ca²⁺ spark sites suggests another level of fine control of membrane excitability through Ca²⁺ sparks (see Figs. 2 and 9). Indeed, differential modulation of Cl_Ca and BK_Ca channels by vasoactive substances would provide a means to alter the control of membrane potential by Ca²⁺ sparks. For example, vasoconstrictor-induced activation of Cl_Ca channels and inhibition of BK_Ca channels (see Decrease in Ca²⁺ Spark Frequency by PKC) could be a mechanism to enhance membrane excitability.

The decay of STICs [t_1/2 = 65-182 ms (89, 218)] is much slower than the decay of STOCs [t_1/2 = 9-43 ms (153, 218)] and is similar to the decay of Ca²⁺ sparks as measured with the fluorescent indicator fluo 3 (see Figs. 6 and 9) (218). This observation is consistent with the idea that the BK_Ca channels (STOCs) are activated by high (10-100 µM) local Ca²⁺ (Fig. 6). The rapid decay of the STOC is determined by dissipation of this local Ca²⁺ following closure of the RyR channels. In contrast, Cl_Ca channels are saturated with Ca²⁺ above 1 µM, and these currents do not decay until [Ca²⁺]_i falls below 1 µM, which are concentrations measured accurately by fluo 3. Interestingly, whole cell Cl_Ca currents decay much faster than whole cell Ca²⁺ transients, as the decay of the current is determined by CAM kinase-induced inactivation of Cl_Ca channels (207). Therefore, the decay of a STIC appears to be determined by the decline in [Ca²⁺], whereas the decay of the whole cell Cl_Ca currents is regulated by [Ca²⁺] and CAM kinase.

	FM AND AM OF CA2+ SPARKS: PHYSIOLOGICAL IMPLICATIONS

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

A number of factors regulate the apparent cytoplasmic Ca²⁺ sensitivity of RyR channels, including phosphorylation state, pharmacological agents, and the SR Ca²⁺ load (Fig. 7). Modulation of RyR channels is manifest as a change in Ca²⁺ spark frequency and/or amplitude. Ca²⁺ spark amplitude should depend on the open time of the RyR channels and the Ca²⁺ load of the SR, i.e., the driving force for Ca²⁺ (165). Increasing SR Ca²⁺ load (219) or elevating cAMP or cGMP (157) leads to an increase in Ca²⁺ spark frequency in smooth muscle, without elevating global [Ca²⁺]_i. Activators of protein kinase C (PKC) have the opposite effect (19).

FM and AM of Ca²⁺ sparks by SR Ca²⁺ load

In stomach smooth muscle cells isolated from the toad Bufo marinus, simultaneous measurements of SR [Ca²⁺], cytoplasmic [Ca²⁺], and Ca²⁺ sparks (or STOCs) have provided new insights into the relationship between SR Ca²⁺ load and Ca²⁺ spark properties (219). After emptying the SR with a bolus of caffeine, the frequency of Ca²⁺ sparks and STOCs increased steeply with the SR [Ca²⁺] (219), as has been reported in heart (30, 36, 164). Therefore, SR Ca²⁺ load appears to be an important regulator of Ca²⁺ spark frequency and amplitude in smooth muscle. However, the mechanism by which SR Ca²⁺ regulates Ca²⁺ spark properties in smooth muscle remains to be elucidated.

Increase in Ca²⁺ Spark Frequency by cGMP and cAMP

Smooth muscle relaxants that increase cGMP and cAMP have been shown to activate BK_Ca channels through direct phosphorylation effects on the channel protein (108, 161, 191, 208) and through elevation of Ca²⁺ spark frequency (86, 157). Agents that elevate cGMP or cAMP increase Ca²⁺ spark frequency, and therefore BK_Ca channel activity, threefold (86, 157). The direct effect of PKG and PKA on BK_Ca channel activity in intact cells appears to be much smaller (1.3-fold) than the activation caused by increased Ca²⁺ spark frequency. These results suggest that cyclic nucleotide-mediated smooth muscle relaxation may occur partially through an increase in the frequency of Ca²⁺ sparks and a subsequent increase in the activity of BK_Ca channels.

An increase in Ca²⁺ spark frequency following elevation of cGMP and cAMP may be due to phosphorylation of RyR channels, phospholamban, or an unknown regulatory protein (Figs. 7 and 10). Phospholamban, when phosphorylated by PKG or PKA, dissociates from the SR Ca²⁺-ATPase, which leads to increased Ca²⁺ pumping and an elevated SR Ca²⁺ load, which increases Ca²⁺ spark frequency (165, 219). Recent results, using arteries from phospholamban-deficient mice, indicate that some of the frequency and amplitude modulation of Ca²⁺ sparks and STOCs by forskolin and sodium nitroprusside could occur through modulation of the SR Ca²⁺ load by phospholamban (47). RyR channels are also directly regulated by PKA (195), and this may contribute to the actions of cyclic nucleotides in smooth muscle. The emerging view of cyclic nucleotide action through BK_Ca channels is that the concerted action of PKG or PKA on phospholamban, RyR channels, and BK_Ca channels leads to an elevation of Ca²⁺ spark and STOC frequency and amplitude (Fig. 10).

View larger version (33K): [in this window] [in a new window]   Fig. 10.   Possible mechanisms of action of PKA/PKG and PKC on Ca2+ sparks, BKCa channels, and SR Ca2+-ATPase in arterial smooth muscle cells. Activation of PKA or PKG increases Ca2+ spark frequency and increases Ca2+ load of the SR (probably through activation of the SR Ca2+-ATPase via disinhibition of phospholamban). Increased Ca2+ spark frequency could occur due to a direct effect on the RyR channel and/or be a secondary effect from increased SR Ca2+ load. PKA/PKG activation also increases the activity of BKCa channels, which could manifest itself by an elevation in STOC amplitude and steady-state BKCa channel activity. Synergistic effect of increased Ca2+ spark frequency and a direct effect on BKCa channels will result in a significant increase of BKCa channel activity. Membrane potential hyperpolarization closes L-type Ca2+ channels, which reduces Ca2+ influx, lowering the cytoplasmic Ca2+ concentration, and leads to vasodilation (see Ref. 157). Activation of PKC reduces frequency of Ca2+ sparks and amplitude of STOCs, due to a direct inhibitory effect on RyR channels and BKCa channels, respectively. Additive effect results in decreased BKCa channel activity, a depolarization of the smooth muscle cell membrane potential, and activation of L-type Ca2+ channels. PKA, PKG, and PKC also modulate the voltage-dependent Ca2+ channel, which would contribute to modulation of the Ca2+ channel  RyR channel  BKCa channel pathways (not illustrated).

Decrease in Ca²⁺ Spark Frequency by PKC

PKC may also contribute to the action of vasoconstrictors on smooth muscle through a novel mechanism, by decreasing the frequency of Ca²⁺sparks, which would lead to membrane depolarization and activation of voltage-dependent Ca²⁺ channels (Fig. 10) (19). Activators of PKC, phorbol 12-myristate 13-acetate and sn-1,2-dioctanoyl-glycerol, decreased Ca²⁺ spark and STOC frequency by ~60% in isolated cells from rat cerebral arteries (19). Activators of PKC also decreased STOC amplitude by ~20%, which could be explained by a direct inhibition of BK_Ca channels (Fig. 10) (19). The inhibitory effect of PKC activators on Ca²⁺ spark frequency does not involve a reduction of SR Ca²⁺ load, suggesting that PKC directly inhibits RyR channels (Fig. 10) (19). The precise molecular mechanisms of PKC inhibition of Ca²⁺ sparks remain to be determined.

IP₃ induced Ca²⁺ release has been described in terms of a hierarchical nomenclature based on the spatiotemporal characteristics of the observed events. Fundamental Ca²⁺ release through a single IP₃ receptor has been termed a blip [amplitude, 20-30 nM; spread, 1-3 µm; decay (t_1/2) = 45 ms (22)], whereas intermediate release due the recruitment of blips are termed Ca²⁺ puffs (22, 216). Propagating Ca²⁺ waves occur due to the summation of these smaller events. Although these elementary and intermediate events have not been described in smooth muscle cells, application of an agonist that elevates cytoplasmic levels of IP₃ should activate fundamental Ca²⁺ release through IP₃ receptors.

Vasoconstrictors have been shown to increase Ca²⁺ wave frequency and amplitude in the smooth muscle cells of rat tail artery, an effect that has been attributed to the activation of IP₃ receptors by IP₃, and subsequent activation of RyR channels by Ca²⁺ released through IP₃ receptors (81). IP₃-induced Ca²⁺ release would increase the cytoplasmic [Ca²⁺] and would tend to increase Ca²⁺ spark frequency, particularly if RyR channels are situated close to IP₃ receptors and sense the high local [Ca²⁺]. However, the reduction in SR Ca²⁺ load would tend to decrease Ca²⁺ spark frequency (219). The extent to which IP₃-induced Ca²⁺ release increases or decreases Ca²⁺ spark activity (16, 52, 98, 106) would depend on a balance between an increase in Ca²⁺ near the cytoplasmic face of the RyR channels and the reduction in stored Ca²⁺ that would follow. It remains to be resolved how PKC and IP₃ affect the cytoplasmic and SR luminal communication between RyR channels and IP₃ receptors.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION RYANODINE RECEPTORS IN SMOOTH... CA2+ SPARK PROPERTIES IN... CA2+ SPARKS ARE INVOLVED... FM AND AM OF... CONCLUSIONS REFERENCES

Ca²⁺ sparks occur in a wide variety of smooth muscle types and are caused by the activation of a cluster of RyR channels. In smooth muscle, the majority of Ca²⁺ sparks occur very close to the cell membrane, consistent with a role in signaling ion channels in the plasma membrane. The close proximity of Ca²⁺ spark sites to the plasma membrane support earlier studies indicating that, under certain circumstances, subsarcolemmal [Ca²⁺] could be significantly higher than global [Ca²⁺]_i (215). Further support for this idea has come from a demonstrated dissociation between global [Ca²⁺]_i and BK_Ca channel currents, which should be a measure of subsarcolemmal [Ca²⁺] (56, 153, 181).

Voltage-dependent Ca²⁺ channels, Ca²⁺ sparks (RyR channels), and BK_Ca channels act as a functional unit to regulate smooth muscle function (Figs. 1 and 2). In some cases Cl_Ca channels may be regulated by Ca²⁺ sparks, adding another level of control to smooth muscle membrane potential (Fig. 2). Apamin-sensitive SK_Ca channels are also expressed in some types of smooth muscle (Table 4), and the effect of Ca²⁺ sparks on SK_Ca channels remains to be established.

Ca²⁺ entry through dihydropyridine-sensitive, voltage-dependent Ca²⁺ channels regulates Ca²⁺ spark properties. The speed and efficacy of comm