Jaggar, Jonathan H., Valerie A. Porter, W. Jonathan Lederer, and Mark T. Nelson.

Transcription

1 Am. J. Physiol. Cell Physiol. 278: C235 C256, Calcium sparks in smooth muscle invited review JONATHAN H. JAGGAR, 1 VALERIE A. PORTER, 1 W. JONATHAN LEDERER, 2 AND MARK T. NELSON 1 1 Department of Pharmacology, College of Medicine, The University of Vermont, Burlington, Vermont 05405; and 2 Medical Biotechnology Center, University of Maryland Biotechnology Institute, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland Jaggar, Jonathan H., Valerie A. Porter, W. Jonathan Lederer, and Mark T. Nelson. Calcium sparks in smooth muscle. Am. J. Physiol. Cell Physiol. 278: C235 C256, Local intracellular Ca 2 transients, termed Ca 2 sparks, are caused by the coordinated opening of a cluster of ryanodine-sensitive Ca 2 release channels in the sarcoplasmic reticulum of smooth muscle cells. Ca 2 sparks are activated by Ca 2 entry through dihydropyridine-sensitive voltage-dependent Ca 2 channels, although the precise mechanisms of communication of Ca 2 entry to Ca 2 spark activation are not clear in smooth muscle. Ca 2 sparks act as a positive-feedback element to increase smooth muscle contractility, directly by contributing to the global cytoplasmic Ca 2 concentration ([Ca 2 ]) and indirectly by increasing Ca 2 entry through membrane potential depolarization, caused by activation of Ca 2 spark-activated Cl channels. Ca 2 sparks also have a profound negative-feedback effect on contractility by decreasing Ca 2 entry through membrane potential hyperpolarization, caused by activation of large-conductance, Ca 2 - sensitive K channels. In this review, the roles of Ca 2 sparks in positiveand negative-feedback regulation of smooth muscle function are explored. We also propose that frequency and amplitude modulation of Ca 2 sparks by contractile and relaxant agents is an important mechanism to regulate smooth muscle function. ryanodine-sensitive calcium-release channel; voltage-dependent calcium channel; calcium-sensitive potassium channel; calcium-activated chloride channel; sarcoplasmic reticulum CALCIUM IONS DIRECTLY REGULATE the contraction of smooth muscle through the activation of Ca 2 /calmodulin-dependent myosin light-chain kinase (MLCK). For this to occur, an elevation of Ca 2 must be broadly or globally distributed throughout the cytoplasm. The view that a spatially homogeneous elevation in cytoplasmic Ca 2 is necessary for effective Ca 2 signaling has been radically altered by the discovery of local Ca 2 transients ( Ca 2 sparks ) in cardiac (37), skeletal (99,193), and smooth (140) muscle cells. Ca 2 sparks are caused by the opening of ryanodine-sensitive Ca 2 - release (RyR) channels in the sarcoplasmic reticulum (SR). A single Ca 2 spark is capable of producing a very high ( µm) local ( 1% of the cell volume) increase in [Ca 2 ], while increasing the global intracellular concentration of Ca 2 ([Ca 2 ] i )by 2 nm (85, 140). A Ca 2 spark, by virtue of its high local [Ca 2 ] elevation, has the potential to modulate Ca 2 -dependent processes that are not responsive to global increases in [Ca 2 ] i. Early evidence for local Ca 2 release in cells came from measurements of spontaneous transient currents through Ca 2 -sensitive K channels in frog sympathetic ganglia neurons (26, 125, 126). Subsequently, Benham & Bolton (13) measured transient outward currents through large-conductance Ca 2 -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 Ca 2 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, ). The existence of STOCs suggested the possibility that subsarcolemmal [Ca 2 ] is different from global cytoplasmic [Ca 2 ] and that, under certain circumstances, subsarcolemmal [Ca 2 ] could be significantly higher than global [Ca 2 ] i. Further support for this idea has come from studies that demonstrated a dissociation between glo /00 $5.00 Copyright 2000 the American Physiological Society C235

2 C236 INVITED REVIEW Fig. 1. Proposed functional roles of Ca 2 sparks in smooth muscle cells. Left: local control of Ca 2 sparks by voltage-dependent Ca 2 channels in cardiac and smooth muscle. In cardiac muscle, high local Ca 2 concentration ([Ca 2 ]) from L-type voltagedependent Ca 2 channels regulates Ca 2 spark frequency. In smooth muscle, the role of local Ca 2 entry is not known. Right: Ca 2 spark activation of BK Ca channels to complete a negative-feedback loop (green arrow) through membrane potential hyperpolarization to decrease Ca 2 entry. Also illustrated is a positive-feedback contribution (red arrow) of Ca 2 release from RyR channels to contraction. In most smooth muscle types, the negative-feedback pathway appears to dominate. bal [Ca 2 ] 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 [Ca 2 ], which was ryanodine sensitive and distinct from [Ca 2 ] changes that occur in the cytosol. The impact of a Ca 2 spark depends on a number of key factors: 1) the distance of the Ca 2 spark site from a Ca 2 -sensitive target, 2) the number of target molecules affected by a Ca 2 spark, 3) the Ca 2 sensitivity of the target protein, and 4) the nature of the Ca 2 - sensitive target. The physiological outcome of the Ca 2 spark depends on the [Ca 2 ] that reaches the target. Ca 2 reaches very high levels ( µm) very close (e.g., 20 nm) to the release site (Ref. 153; see also Ref. 139). However, at several hundred nanometers from a Ca 2 spark release site, Ca 2 approaches equilibrium with mobile buffers (including fluorescent indicators), and the local elevation in [Ca 2 ] by a spark is less pronounced ( 500 nm; see Ref. 139). Molecular targets with low affinity for Ca 2, such as BK Ca channels, are uniquely suited to respond to these very high local [Ca 2 ], since these channels require micromolar Ca 2 for significant levels of activity under physiological conditions (153). A single Ca 2 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 Ca 2 /calmodulin (CaM)-dependent targets [e.g., MLCK, CaM kinase, small-conductance Ca 2 - sensitive K (SK Ca ) channels, nitric oxide synthase (NOS), calcineurin, Ca 2 -activated chloride (Cl Ca ) channels] are well suited to respond to changes in global Ca 2 (0.1 1 µm), since CaM is saturated by [Ca 2 ] below 1 µm. The goal of this review is to provide up-to-date information on RyR channels and Ca 2 sparks in smooth muscle and to propose the following: 1) voltagedependent Ca 2 channels, Ca 2 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 Ca 2 channels and RyR channels is a critical element in the control of global [Ca 2 ] i (Figs. 1 and 2); 3) Ca 2 sparks are involved in both positive- and negative-feedback regulation of global [Ca 2 ] i (Figs. 1 and 2); 4) frequency and amplitude modulation (FM and AM) of Ca 2 sparks by relaxant and contractile agents are mechanisms to regulate smooth muscle function. Unitary Ca 2 release may also occur due to inositol trisphosphate (IP 3 ) activation of IP 3 receptors in the SR [termed blips and puffs (22, 216)]. In this review, the term Ca 2 sparks refers to Ca 2 release through RyR channels. RYANODINE RECEPTORS IN SMOOTH MUSCLE 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 (voltagedependent Ca 2 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 /Ca 2 exchanger, and the Na /Ca 2 exchanger and Ca 2 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. 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

3 INVITED REVIEW C237 Fig. 2. Possible relationships among voltage-dependent Ca 2 channels, ryanodine-sensitive Ca 2 -release (RyR) channels, large-conductance Ca 2 -sensitive K (BK Ca ) channels, and Ca 2 -activated Cl (Cl Ca ) channels to regulate smooth muscle contractility. Voltage-dependent Ca 2 channels (VDCC) are the primary Ca 2 entry pathway in most types of smooth muscle. For tonic smooth muscle, such as resistance arteries, steady Ca 2 entry through VDCC determines intracellular [Ca 2 ] ([Ca 2 ] i ; indicated by thick arrow). Ca 2 entry through VDCCs also stimulates RyR channels as Ca 2 spark events or as non-ca 2 spark events. The type of Ca 2 communication from VDCCs to RyR channels (e.g., local Ca 2 control) in smooth muscle is not known. RyR channels can serve as negative- and positive-feedback transducers to VDCCs. Ca 2 release from RyR channels could inactivate VDCCs (negativefeedback element); this is not known for smooth muscle. Ca 2 release from RyR channels may also contribute to global [Ca 2 ] i for contraction [i.e., Ca 2 -induced Ca 2 release (CICR); positive feedback element]. Contribution of Ca 2 sparks to global [Ca 2 ] i may be significant in phasic smooth muscle and less important in tonic smooth muscle. Ca 2 sparks activate BK Ca channels in virtually all types of smooth muscle to cause a very substantial membrane potential (V m ) hyperpolarization (negative-feedback element). Sparks activate Cl Ca channels in some types of smooth muscle to cause membrane depolarization (positive-feedback element). Ca 2 release through RyR channels could also activate SK Ca 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, Ca 2 sensitivity, and phosphorylation state. MLCK, myosin light-chain kinase. 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 [Ca 2 ] (Fig. 4) and by caffeine and are inhibited by Mg 2 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). 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). Ca 2 sparks that occur

4 C238 INVITED REVIEW 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 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).] 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, Table 1. Characteristics of ryanodine receptor subtypes Mammalian Skeletal Muscle (ryr1) cross-react with monoclonal antibodies for RyR2 (Fig. 3B) (61, 214). Therefore, the RyR channel responsible for Ca 2 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. Mammalian Cardiac Muscle (ryr2) Mammalian Brain (ryr3) Mammalian Smooth Muscle (?) (71) Human chromosome 19q13.1 (124, 129) 1 a (148, 149) 15q14-q15 (180) Mouse chromosome 7A2-7A3 (34, 127) 13A1-13A2 (34, 127) 2E5-2F3 (34, 127) Molecular weight 564 kda (220), 565 kda 565 kda (138; 149) 500 kda (130) (188) Number of residues 5,032 (220), 5,037 (188) 4,968 or 4,976 (138), 4,969 4,872 (67) (149) Morphology Quatrefoil (111) Quatrefoil (5) Quatrefoil (90, 110) Conductance ( ps (111, 112, 177) ps (5, 120, 160) 140 ps (110) 110 ps mm Ca 2 ) Mechanism of activation Direct coupling, CICR CICR Ca 2 Ca 2 Activation by µm Ca 2 Yes Yes Yes (90) Yes ATP Yes?? Yes (in the presence of µm Ca 2 ) Caffeine Yes Yes Yes (90) Yes Inhibition by mm Mg 2 b Yes Yes Yes (90) Yes Ruthenium red Yes Yes Yes (90) Yes Modulation by Protein kinase A Activation (59, 66, 73) c Activation (195) d?? Inactivation (202) Ryanodine binding pmol/mg protein 450 pmol/mg protein 460 pmol/mg protein 1 3 pmol/mg protein K d 4 8 nm e (111, 113) 2 nm e (5) 3 12 nm e (90, 110, 130) 5, 53, 555, 2,800 nm f (154) 2, 36, 681, 4,320 nm f (154) CICR, Ca 2 -induced Ca 2 release. a Located at the interval between 1q42.1 and 1q43. b Mg 2 has been suggested to competitively displace Ca 2 from its activation site and may also interact with the low-affinity Ca 2 inhibitory site (42). c Phosphorylation by PKA reduced Mg 2 inhibition. d Phosphorylation by protein kinase A increased the responsiveness of the channel to Ca 2. e Refers to high-affinity ryanodine binding site. f One high-affinity and 3 low-affinity binding sites. Nos. in parentheses are reference nos.

5 INVITED REVIEW C239 CA 2 SPARK PROPERTIES IN SMOOTH MUSCLE Basic Properties Ca 2 sparks have been observed in cardiac (37), skeletal (99, 193), and smooth (140) muscle cells. These measurements have been made using laser scanning confocal microscopy and the fluorescent Ca 2 indicator, fluo 3. A number of lines of evidence indicate that Ca 2 sparks are caused by the opening of RyR channels in the SR, including modulation by ryanodine, voltage, Table 2. Ca 2 sparks in cardiac, skeletal, and smooth muscle cells Fig. 4. Dependence of smooth muscle RyR channel activity on cytoplasmic free [Ca 2 ]. Data represent singlechannel recordings of a Ca 2 -activated Ca 2 -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 Ca 2 concentrations. Free Ca 2 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 Ca 2 concentrations with fitted time constants. Open time histogram: 1 42 ms, ms (100 µm Ca 2 ) compared with ms, 2 33ms (0.5 µm Ca 2 ). Closed time histogram: 1 23 ms, ms (100 µm Ca 2 ) compared with ms, ms (0.5 µm Ca 2 ). Ca 2, and caffeine, and location of events (30, 37, 99, 121, 140, 193). Table 2 summarizes the properties of Ca 2 sparks in cardiac, skeletal, and smooth muscle cells. Ca 2 sparks recorded in skeletal muscle are approximately one-third the size of those observed in cardiac and smooth muscle (Table 2), although the single-channel currents through skeletal and cardiac and smooth muscle RyR channels incorporated into bilayers are about the same (Table 1). It has been postulated that this difference in Ca 2 spark amplitude Muscle Type Cardiac Skeletal Smooth Change in Ca 2 * nm (37, 62, 123) nm (99, 193) nm (85, 133, 140, 172) Rise time 10 ms (37) 4.7 ms (109) ms (133, 140, 153, 172) Decay t 1/ ms (30, 37, 123) 21 ms (62) Duration ms (99) 8.5 ms (109) t 1/ ms (19, 63, 85, 133, 140, 153) biexponential decay: 1 32 ms, ms (153) Spread (FWHM) µm (30, 35, 37) (62, 123) 2.4 µm (99) µm (133, 140), 14 µm 2 (153) Unstimulated frequency 100 s 1 cell 1 (37) triad 1 s 1 (99) s 1 cell 1 (19, 85, 140, 157, 172) Intracellular location t-tubule (ventricular) (37, 169); t-tubule (99) Subplasmalemmal (140) subplasmalemmal and central (atrial) (15, 80) Stimulation Local Ca 2 (30, 37, 121) Coupled to DHPR/Ca 2 (99) Ca 2 (local?) Proposed effect on muscle Contraction (37) Contraction (99) Relaxation (140) and contraction Pharmacology 2 µm ryanodine Activation (37, 166) Activation (133, 172) µm ryanodine Inhibition (37) Inhibition (99) Inhibition (9, 19, 63, 85, 133, 140, 157) Effect of caffeine Activation (500 µm) (166) Activation (41, 99) Activation (50 µm 1 mm) (61, 133, 172) FWHM, full width of the Ca 2 spark spread at the half-maximal amplitude. *Ca 2 sparks were measured in unstimulated cells using Ca 2 -sensitive fluorescent dyes such as fluo 3. Assessed using rapid 2-dimensional (2D) planar imaging.

6 C240 INVITED REVIEW is due to the number of RyR channels contributing to the Ca 2 spark or to different RyR channel kinetics (99, 131). Ca 2 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, Ca 2 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 Ca 2 sparks appear to be similar in these different smooth muscle preparations (Table 3). Figure 5 illustrates recordings of Ca 2 sparks in the smooth muscle cells of an intact pressurized (60 mmhg) cerebral artery. In arterial smooth muscle, Ca 2 sparks have a rise time of 20 ms, a half time of decay of ms (Figs. 5 and 6), and a spatial spread (full width at the halfmaximum 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). Ca 2 sparks Fig. 5. Ca 2 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. Ca 2 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/F 0 ) 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). 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 [Ca 2 ] i is dynamically and heterogeneously distributed in intact tonic smooth muscle. ACa 2 Spark is due to the Simultaneous Activation of a Cluster of RyR Channels Ca 2 sparks appear to represent the near-simultaneous activation of a cluster, or plaque, of RyR channels. The flux of Ca 2 from a spark site is substantial and corresponds to a Ca 2 current of 4 pa for 10 ms (37). With physiological levels of SR luminal [Ca 2 ], the unitary current of a single cardiac RyR channel is 0.6 pa (131). Therefore, a Ca 2 spark would correspond to the coordinated opening of at least 10 RyR channels (131). In cardiac muscle, the elementary physiological event appears to be Ca 2 sparks, since the global Ca 2 transient is composed of many Ca 2 sparks (29). In a similar fashion, the miniature end plate potential represents the release of one presynaptic vesicle (the elementary physiological event), and not the release of one neurotransmitter molecule. Fate of Ca 2 From a Ca 2 Spark The decay of the Ca 2 spark may be determined by diffusion, uptake into the SR by the thapsigarginsensitive Ca 2 -ATPase, and extrusion from the cell through the plasmalemmal Ca 2 -ATPase and Na /Ca 2 exchanger. In cardiac muscle, Ca 2 spark decay reflects diffusion and SR Ca 2 uptake, with a small contribution from Ca 2 extrusion (165). As measured with fluorescent dyes, the decay of Ca 2 sparks in arterial smooth muscle is biexponential, with both rate constants ( 1 32 ms and ms) contributing equally to the decay (49 and 51%, respectively) (153). It is unclear why Ca 2 sparks demonstrate a biexponential decay pattern, but this may reflect limited space diffusion, fluorescent dye kinetics, or extrusion/uptake of Ca 2 by SR or sarcolemmal Ca 2 pumps. The faster of the two time constants for decay is similar to that which has been measured for Ca 2 sparks in heart muscle (62, 165), consistent with diffusion of Ca 2 in the cytoplasm. The contribution of Ca 2 extrusion/ sequestration mechanisms to the decay of Ca 2 sparks in smooth muscle remains to be addressed. However, the decay of a whole cell Ca 2 transient, which reflects Ca 2 extrusion and SR Ca 2 uptake, is much slower [decay constant ( ) s (49, 207); see also Refs , 57, 93, 94] than the decay of a Ca 2 spark. This observation suggests that Ca 2 spark decay is largely determined by diffusion (see Ref. 62). The smooth muscle cell Ca 2 spark represents the spatially localized, unitary release of a significant amount of Ca 2 very close to the plasma membrane, with essentially no effect on global [Ca 2 ] i or on contraction (140). The localized release of Ca 2 into a subsarcolemmal domain can thus represent a measurable element of the superficial buffer barrier (SBB) hypothesis

7 INVITED REVIEW C241 Fig. 6. Ca 2 sparks activate BK Ca 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/F 0 ) and decay of a typical Ca 2 spark. Images are color coded as indicated by color bar. B: simultaneous BK Ca channel current and Ca 2 spark (F/F 0 ) measurements at 40 mv illustrating temporal association. Current (blue) is indicated above F/F 0 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).] (for review see Ref. 196). This hypothesis suggests that Ca 2 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 [Ca 2 ] i. Subsequently, this Ca 2 is extruded from the cell via the sarcolemmal Na /Ca 2 exchanger and Ca 2 -ATPase. The anatomic requirements for a Ca 2 spark to activate BK Ca channels are similar to those proposed for the SBB hypothesis. Additional studies on the decay of Ca 2 sparks should provide new insights into local Ca 2 extrusion and uptake mechanisms as well as the SBB hypothesis. CA 2 SPARKS ARE INVOLVED IN BOTH POSITIVE AND NEGATIVE FEEDBACK REGULATION OF GLOBAL INTRACELLULAR [CA 2 ] IN SMOOTH MUSCLE The role of local Ca 2 signaling is cell type dependent and should be viewed in the context of cell function. For example, cardiac muscle must contract forcefully and

8 C242 INVITED REVIEW Table 3. Ca 2 sparks in smooth muscle cells from arteries, veins, and nonvascular tissues Smooth Muscle Source Arteries Veins Non-vascular Change in Ca 2 * nm (85, 140) nm (133) nm (172) Rise time 20 ms (140) 22 ms (133) ms (63, 172) Decay (t 1/2 ) ms (19, 85, 140, 153) 27 ms (133) ms (63) Spread (FWHM) 2.4 µm (140), 14 µm 2 (153) 1.5 µm (133) µm (63) Frequency Unstimulated 0.24 Hz (85) 40 mv Hz (61, 85), 0.7 Hz (153) Location Subplasmalemmal Stimulation Ca 2 (local?) Ca 2 (local?) Ca 2 (local?) Effect on muscle Relaxation (140; 157) Contraction (9) Relaxation and contraction Effect of caffeine Activation (0.3 mm) (61) Activation (0.5 1 mm) (133) Activation (50 µm) (172) All measurements are obtained using Ca 2 -sensitive fluorescent dyes such as fluo 3. *Determined in unstimulated cells. Peak of a Gaussian distribution (133). Assessed using rapid 2D planar imaging of isolated, voltage-clamped ( 40 mv) cerebral artery myocytes under conditions where approximately one-half of the cell volume is captured. Rapid 2D confocal imaging of myocytes in an intact cerebral artery. Frequency measurements were obtained from 56 µm 53 µm areas, or 8 smooth muscle cells. Table 4. Cellular targets for Ca 2 sparks relax rapidly at high rates (1 7 Hz). In these cells, activation of voltage-dependent Ca 2 channels, which is synchronized by the action potential, leads to an immediate rise in Ca 2 influx, which activates, in a coordinated and rapid fashion, large numbers of Ca 2 sparks, which summate to cause the global [Ca 2 ] i transient. Thus Ca 2 -induced Ca 2 -release (CICR) through RyR channels acts as an amplifier for Ca 2 influx, contributing 90% of the cellular Ca 2 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 Ca 2 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 dihydropyridinesensitive voltage-dependent Ca 2 channels, RyR channels, and BK Ca channels represent a functional unit to control the levels of [Ca 2 ] i (86). Most types of smooth muscle are involved in maintaining tonic contraction. One important element in graded, tonically contracted smooth muscle is the negativefeedback control of membrane potential, and hence Ca 2 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 Ca 2 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 Ca 2 Channels and RyR Channels is a Critical Element in the Control of Global Intracellular [Ca 2 ] The first step in the flow of information following electrical excitation in heart muscle is an increase in Ca 2 influx through voltage-dependent Ca 2 channels. The communication between voltage-dependent Ca 2 channels and RyR channels is intimate, rapid, and BK Ca Channels Cl Channels SK Ca Channels MLCK Mechanism of activation Direct channel activation Unknown (calmodulin?) Calmodulin (213) Calmodulin by Ca 2 Ca 2 dependence K d 1.8 µm at 11 mv and 10 µm at K d 0.37 µm (150) K d 0.74 µm at 80 mv (74) K d 1µM (see Ref. 24) 83 mv (31) %Maximal activity at % (at 40 mv) 1.5% 3.2% 2 3% nm Ca 2 Single-channel conductance 80 ps 2.6 ps (100) 10 ps (physiological) Density 1 4 Channels/µm Channels/µm 2 Unknown Presence in different types of smooth muscle Voltage dependence Functional impact of one Ca 2 spark (13, 173, 190) (see Ref. 116) All Some (see text) Described in murine colon (104) and murine ileum (200) e-fold per 12- to 14-mV depolarization (14, 115) Up to 20-mV hyperpolarization (52) None None 5-mV depolarization (198) Unknown Activation of 1% of MLCK BK Ca, large-conductance Ca 2 -sensitive; SK Ca, small-conductance Ca 2 -sensitive; MLCK, myosin light-chain kinase. All

9 INVITED REVIEW C243 bidirectional. RyR channels are positioned in junctional SR elements within short distances ( 20 nm) of voltagedependent Ca 2 channels in the t-tubules of ventricular heart cells (for review see Ref. 51). RyR channels are activated by local Ca 2 shortly after they enter the cell through voltage-dependent Ca 2 channels ( local control ) (30, 36, 121). Local Ca 2 in the subsarcolemmal junctional space is likely to reach relatively high concentrations ( 10 µm), corresponding to levels required for significant RyR channel activation. Thus local Ca 2 control of Ca 2 release can explain the graded and stable increases in Ca 2 release through RyR channels in response to Ca 2 influx (see Fig. 1) (30, 121, 178). The communication is bidirectional, since Ca 2 sparks can also induce inactivation of voltage-dependent Ca 2 channels (1, 65, 170). The communication between Ca 2 channels and RyR channels/ca 2 sparks is less clear in smooth muscle. In smooth muscle, Ca 2 sparks are also regulated by Ca 2 influx through voltage-dependent Ca 2 channels, since Ca 2 spark frequency and amplitude increase with activation of voltage-dependent Ca 2 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 Ca 2 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 Ca 2 spark frequency and amplitude depends on Ca 2 influx through voltage-dependent Ca 2 Fig. 7. Hypothetical modulation of cytoplasmic Ca 2 dependence (activator [Ca 2 ]) of Ca 2 spark frequency. Activators of RyR channels (e.g., caffeine), an increase in SR Ca 2 load [e.g., through campdependent protein kinase (PKA) phosphorylation of phospholamban], and PKA/cGMP-dependent protein kinase (PKG) may increase Ca 2 sensitivity of RyR channel and increase frequency of Ca 2 sparks at a given [Ca 2 ] i. PKA/PKG may in part increase Ca 2 sensitivity of Ca 2 sparks by increasing SR Ca 2 load (see text). In contrast, PKC may induce a rightward shift in Ca 2 sensitivity of the RyR channel, since activators of PKC decrease Ca 2 spark frequency at a given [Ca 2 ] i (19). channels, since it is reversed by diltiazem, a selective blocker of L-type Ca 2 channels (85). Additionally, in voltage-clamped rat portal vein myocytes, Ca 2 sparks have been observed to occur immediately following a depolarizing voltage step that elicits a Ca 2 current (9). Although these studies indicate that Ca 2 spark frequency is modulated by Ca 2 influx through voltage-dependent Ca 2 channels, it is still unresolved whether local Ca 2 regulates Ca 2 spark probability in smooth muscle. Steady membrane depolarization of arterial smooth muscle increases voltage-dependent Ca 2 channel open probability (e.g., see Ref. 162), global [Ca 2 ] i, and presumably SR [Ca 2 ]. An elevation in local [Ca 2 ] from Ca 2 channels, global [Ca 2 ] i,orsr[ca 2 ] could each contribute to the observed depolarization-induced increase in Ca 2 spark frequency and amplitude (Figs. 1 and 7). Indeed, recent evidence indicates that Ca 2 spark and STOC frequency in smooth muscle cells depends steeply on the SR Ca 2 load (219). Figure 7 illustrates modulation of the Ca 2 dependence of Ca 2 spark frequency by SR Ca 2 load and other factors (see FM AND AM OF CA 2 SPARKS: PHYSIOLOGICAL IMPLICATIONS). Ca 2 release from RyR channels can inactivate voltage-dependent Ca 2 channels in cardiac myocytes (23, 134, 170), which would serve as another negativefeedback mechanism to regulate Ca 2 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 [Ca 2 ] 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 Ca 2 Sparks to Cytoplasmic [Ca 2 ] In cardiac muscle, Ca 2 release through RyR channels (CICR) contributes most ( 90%) of the Ca 2 for contraction. In tonic smooth muscle (e.g., arteries), in the absence of smooth muscle constrictors, global [Ca 2 ] i is largely determined by steady-state Ca 2 entry through voltage-dependent Ca 2 channels, and Ca 2 release likely contributes little to global [Ca 2 ] i (102, 103, 140). Ca 2 spark frequency is low in intact arteries [ 1 Ca 2 spark s 1 cell 1 (Fig. 5)] (19, 61, 85, 140, 153, 157), and blocking Ca 2 sparks or RyR channels (in the presence of blockers of BK Ca channels) has little direct effect on global [Ca 2 ] i and arterial tone (102, 103, 140). However, it is conceivable that, under conditions of elevated arterial tone (e.g., in hypertension), Ca 2 sparks might have a significant impact on global [Ca 2 ] i. In smooth muscle types that exhibit phasic contractions (e.g., urinary bladder), Ca 2 release through RyR channels may contribute Ca 2 directly for contraction. Unlike tonic smooth muscle, phasic smooth muscle exhibits action potentials, which activate voltagedependent Ca 2 channels to trigger Ca 2 release. Ca 2 release through RyR channels can contribute as much as 70% of the increment in global Ca 2 (30% to Ca 2 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 [Ca 2 ] i in these types of smooth muscle. The precise contribution of Ca 2 release through RyR channels to global [Ca 2 ] i in intact phasic smooth muscle remains to be determined. The ability of voltage-dependent Ca 2 influx to stimulate Ca 2 release through RyR channels appears to

10 C244 INVITED REVIEW vary considerably among different types of smooth muscle. All types of smooth muscle are capable of releasing significant amounts of Ca 2 through RyR channels, since caffeine, an activator of RyR channels (Table 1), can induce global ryanodine-sensitive Ca 2 transients in virtually all smooth muscle types (cf. Refs. 12, 39, 49, 54, 61). Yet, influx through voltagedependent Ca 2 channels can induce ryanodine-sensitive elevations in global [Ca 2 ] 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 [Ca 2 ] caused by the opening of voltage-dependent Ca 2 channels. However, even in cell types (e.g., urinary bladder) that exhibit significant ryanodine-sensitive global Ca 2 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 Ca 2 channels and RyR channels enables rapid and effective communication, i.e., Ca 2 influx stimulates Ca 2 release (Ca 2 sparks) within milliseconds (30, 121). Therefore, the failure of voltage-dependent Ca 2 influx to induce substantial Ca 2 transients does not seem to reflect a lack of releasable Ca 2 through RyR channels, but may reflect a physical separation of voltagedependent Ca 2 channels from a majority of the RyR channels. Communication between Ca 2 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 Ca 2 sparks immediately beneath the plasmalemma, which then trigger Ca 2 sparks in more central cellular regions to elicit a global Ca 2 transient (15, 80). In heart, the Ca 2 transient is composed of the simultaneous activation of many Ca 2 sparks. However, in smooth muscle, the contribution of Ca 2 sparks to ryanodine-sensitive global Ca 2 transients is not known. It is also conceivable that, in smooth muscle, Ca 2 spark sites communicate only with local targets (e.g., BK Ca channels), and ryanodine-sensitive global Ca 2 transients are caused by Ca 2 release through RyR channels, which are not in Ca 2 spark sites (these channels may be located in the central SR rather than at peripheral subplasmalemmal locations). The precise regulation of Ca 2 sparks and RyR channels by local Ca 2 influx and the direct contribution of Ca 2 release to contraction remain to be elucidated. Negative Feedback Regulation of Global [Ca 2 ] i Through Ca 2 Spark Activation of BK Ca Channels in Smooth Muscle BK Ca channels have an estimated single-channel conductance of 80 ps at 40 mv with a physiological K gradient and occur at a density of 1 4 channels/ µm 2 (Table 4) (13, 173, 190). Nelson and colleagues (140) proposed that Ca 2 sparks activate a number of nearby BK Ca channels to cause a macroscopic BK Ca channel current, which had been previously described as a STOC (140). Simultaneous optical and electrical measurements in isolated smooth muscle cells support the idea that Ca 2 sparks activate STOCs (133, 153, 218). Quantitative analysis of such simultaneous measurements indicates that virtually all Ca 2 sparks are associated with STOCs in arterial myocytes (Fig. 6, A and B) (153). These measurements also indicate that Ca 2 sparks increase the open probability of BK Ca channels fold, which is consistent with subsarcolemmal activator [Ca 2 ] in the order of µm (153). STOCs rise and decay quite rapidly [time to peak, ms; half time of decay, 9 ms (140, 153)], and are consistent with the simultaneous activation of at least 15 BK Ca channels in a membrane surface area of 15 µm 2 ( 1% of the surface area of an arterial myocyte). In contrast, the Ca 2 spark, as measured with the fluorescent indicator fluo 3, rises from baseline to peak in 20 ms but decays more slowly [half time of decay ms (85, 140, 153)] (Tables 2 and 3). These results indicate that the RyR channels that cause Ca 2 sparks are located very close to the sarcolemmal BK Ca channels. Disparities in the peak [Ca 2 ]ofaca 2 spark measured with fluo 3 (up to 0.5 µm) and the Ca 2 requirement for an associated increase in BK Ca channel activity (local Ca 2 may actually reach levels of µm), as well as kinetic differences between Ca 2 sparks and STOCs, suggest that the fluorescent Ca 2 indicator is unable to accurately track Ca 2 in the subsarcolemmal space. This is consistent with the known chemical properties of fluo 3. Ca 2 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 Ca 2 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 Ca 2 sparks should summate and lead to a graded level of hyperpolarization (see Fig. 8, B and C) (48). Ca 2 spark-induced membrane potential hyperpolarizations contribute significantly to the membrane potential of isolated smooth muscle cells. For example, in Fig. 8B, Ca 2 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 Ca 2 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 [Ca 2 ] i and vasoconstriction (61, 103, 140, 157). The effects of blocking Ca 2 sparks and BK Ca channels on membrane potential, arterial wall [Ca 2 ] i, and diameter are not additive, supporting the idea that Ca 2 sparks regulate BK Ca channel activity (61, 103, 140, 157). These results support the concept that Ca 2 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).

11 INVITED REVIEW C245 Positive Feedback Regulation of Global [Ca 2 ] i Through Ca 2 Spark Activation of Cl Ca Channels in Smooth Muscle Some types of smooth muscle, but not all, express Cl Ca channels. To date, Cl Ca currents have been described in smooth muscle cells from rat (206) and rabbit (68, 205) pulmonary artery, rabbit ear artery (3, 4), rabbit (114) and porcine (101) coronary artery, rat renal artery (64), human mesenteric artery (100, 101), rat (A7r5) (156, 199) and pig (46) aorta, rabbit (28, 203, 204) and rat portal vein (151), guinea pig mesenteric vein (197), rat small intestine (84, 144), rabbit colon (182), canine (87, 89) and guinea pig (87 89) trachea, rabbit esophagus (2), guinea pig uterus (40), rat myometrium (8), and rat anococcygeus (27). Cl Ca channels are activated by increases in [Ca 2 ] i with a half-maximal activation at 365 nm and full activation around 600 nm (150) and have a single-channel conductance of 2.6 ps (Table 4) (100). The simultaneous activation of a number of Cl Ca channels underlies the spontaneous transient inward currents ( STICs ) first described in tracheal (87) and portal vein smooth muscle cells (203) (for a review see Ref. 116). STICs are activated by Ca 2 sparks (218) in a manner similar to that of STOCs. Interestingly, the same Ca 2 sparks that activate STICs also activate STOCs in these cells, indicating that Cl Ca channels and BK Ca channels are colocalized in this preparation (tracheal smooth muscle) (218). Figure 9 reproduced from ZhuGe et al. (218) illustrates the biphasic nature of the current activated by a Ca 2 spark, which initially evokes an outward current due to the activation of BK Ca channels, followed by a longlasting inward current due to the activation of Cl Ca channels (218). There are some notable differences in the properties of STOCs and STICs. First, the apparent number of Fig. 8. Ca 2 sparks hyperpolarize the membrane potential of arterial smooth muscle. A:Ca 2 sparks elicit transient membrane hyperpolarization. Simultaneous recording of Ca 2 sparks (top) and membrane potential (bottom) in an isolated cerebral artery smooth muscle cell. Changes in subcellular Ca 2 fluorescence were recorded using a laser scanning confocal microscope and the Ca 2 -sensitive dye fluo 3. Top: fluorescence ratio recording of 2 Ca 2 sparks that occur at a single Ca 2 spark site. Bottom: simultaneous patch-clamp recording from same cell under current clamp. Both Ca 2 sparks elicit spontaneous transient membrane potential hyperpolarizations (STHs) due to activation of BK Ca 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 Ca 2 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 Ca 2 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 Ca 2 sparks is averaged throughout the artery.

12 C246 INVITED REVIEW 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 Ca 2 spark. Cl Ca channels may be clustered above Ca 2 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 Ca 2 spark on membrane Fig. 9. A single Ca 2 spark activates biphasic currents (STOICs) in isolated voltage-clamped tracheal myocytes. [Modified from ZhuGe et al. (218).] For experimental protocol see Ref A: time course of a single Ca 2 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 [Ca 2 ] in the one pixel (333 nm 333 nm) where peak [Ca 2 ] is reached during the course of the Ca 2 spark. Bottom: current simultaneously recorded at a holding potential (HP) of 30 mv. Transient outward current is due to Ca 2 spark activation of BK Ca channels, whereas longer inward current represents Cl Ca channel activation.

13 INVITED REVIEW C247 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 mv (14, 115)] and the voltage and Ca 2 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 Ca 2 spark sites suggests another level of fine control of membrane excitability through Ca 2 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 Ca 2 sparks. For example, vasoconstrictorinduced activation of Cl Ca channels and inhibition of BK Ca channels (see Decrease in Ca 2 Spark Frequency by PKC) could be a mechanism to enhance membrane excitability. The decay of STICs [t 1/ ms (89, 218)] is much slower than the decay of STOCs [t 1/ ms (153, 218)] and is similar to the decay of Ca 2 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 ( µm) local Ca 2 (Fig. 6). The rapid decay of the STOC is determined by dissipation of this local Ca 2 following closure of the RyR channels. In contrast, Cl Ca channels are saturated with Ca 2 above 1 µm, and these currents do not decay until [Ca 2 ] 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 Ca 2 transients, as the decay of the current is determined by CAM kinaseinduced inactivation of Cl Ca channels (207). Therefore, the decay of a STIC appears to be determined by the decline in [Ca 2 ], whereas the decay of the whole cell Cl Ca currents is regulated by [Ca 2 ] and CAM kinase. FM AND AM OF CA 2 SPARKS: PHYSIOLOGICAL IMPLICATIONS A number of factors regulate the apparent cytoplasmic Ca 2 sensitivity of RyR channels, including phosphorylation state, pharmacological agents, and the SR Ca 2 load (Fig. 7). Modulation of RyR channels is manifest as a change in Ca 2 spark frequency and/or amplitude. Ca 2 spark amplitude should depend on the open time of the RyR channels and the Ca 2 load of the SR, i.e., the driving force for Ca 2 (165). Increasing SR Ca 2 load (219) or elevating camp or cgmp (157) leads to an increase in Ca 2 spark frequency in smooth muscle, without elevating global [Ca 2 ] i. Activators of protein kinase C (PKC) have the opposite effect (19). FM and AM of Ca 2 sparks by SR Ca 2 load Increases in the cytoplasmic [Ca 2 ] increase the open probability of smooth muscle RyR channels (Fig. 4 and 7) through a direct effect on cytosolic Ca 2 -activating sites on the RyR channel protein (71). SR luminal [Ca 2 ] also modulates RyR channel activity, producing profound effects on both the frequency and the amplitude of Ca 2 sparks in smooth muscle (219). Skeletal and cardiac muscle RyR channels, incorporated into planar lipid bilayers, are activated by increases in luminal [Ca 2 ] (for reviews see Refs. 42, 51, 184). It has been proposed that skeletal and cardiac muscle RyR channels have luminal Ca 2 binding sites that interact with regulatory sites located in the cytosol, since luminal Ca 2 will only activate RyR channels when cytosolic ATP is present (174, 175). Recent evidence indicates that luminal Ca 2 that passes through the RyR channel may activate by interaction with cytosolic binding sites (72, 192). Regardless of the mechanism, increasing SR luminal Ca 2 elevates RyR channel open probability in bilayers (42) and Ca 2 spark frequency in intact myocytes (219). In stomach smooth muscle cells isolated from the toad Bufo marinus, simultaneous measurements of SR [Ca 2 ], cytoplasmic [Ca 2 ], and Ca 2 sparks (or STOCs) have provided new insights into the relationship between SR Ca 2 load and Ca 2 spark properties (219). After emptying the SR with a bolus of caffeine, the frequency of Ca 2 sparks and STOCs increased steeply with the SR [Ca 2 ] (219), as has been reported in heart (30, 36, 164). Therefore, SR Ca 2 load appears to be an important regulator of Ca 2 spark frequency and amplitude in smooth muscle. However, the mechanism by which SR Ca 2 regulates Ca 2 spark properties in smooth muscle remains to be elucidated. Increase in Ca 2 Spark Frequency by cgmp and camp Nitric oxide, a potent vasodilator produced by both the vascular endothelium and by clinically used nitrovasodilators, activates guanylyl cyclase, leading to increased production of cgmp and stimulation of cgmpdependent protein kinase (PKG) (119). Other endogenous smooth muscle relaxants coupled to G s proteins (e.g., calcitonin gene-related peptide, adenosine, -adrenoceptor agonists) activate adenylyl cyclase and increase camp, resulting in the stimulation of camp-dependent protein kinase (PKA). Several mechanisms of action have been proposed for PKG and PKA, including increased Ca 2 uptake into the SR and activation of BK Ca channels (158, 161, 186, 212). Indeed, cgmp- and camp-mediated relaxations in many types of smooth muscle are, in part, through activation of BK Ca channels, causing membrane potential hyperpolarization, which closes voltage-dependent Ca 2 channels and leads to smooth muscle relaxation (see Table 5) (6, 7, 17, 32, 91, 92, 96, 107, 135, 146, 152, , 168, 186, 189, 201, 212). 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 Ca 2 spark frequency (86, 157). Agents that elevate cgmp or camp increase Ca 2 spark frequency, and therefore BK Ca channel activity, threefold (86, 157). The

14 C248 INVITED REVIEW Table 5. Inhibitors of BK Ca channels attenuate the relaxation to agents that increase camp or cgmp Preparation Relaxant Response Cerebral artery Forskolin SIN-1, SNP, 8-BrcGMP, zaprinast Adenosine, DBcAMP Forskolin, DBcAMP IbTX-sensitive relaxation (157) ChTX-sensitive relaxation (146) IbTX- and TEA (1 mm)-sensitive relaxation (152) IbTX- and ChTX-sensitive relaxation (186) Coronary artery Adenosine, isoproterenol, forskolin SNP, ACh, ANP, 8-BrcGMP SNP IbTX- and TEA (1 mm)-sensitive relaxation (158) IbTX- and TEA (1 mm)-sensitive relaxation (159) IbTX-sensitive relaxation and hyperpolarization (212) Mesenteric artery SNP, SNAP ANP, 8-BrcGMP Nitroglycerine, NO, ACh TEA (1 mm)-sensitive relaxation (32) IbTX-sensitive relaxation (189) IbTX- and ChTX-sensitive relaxation (96) Pulmonary artery NO, 8-BrcGMP, zaprinast SIN-1, SNAP, TEA ChTX- and TEA (10 mm)-sensitive relaxation (7) IbTX- and ChTX-sensitive relaxation (17) Aorta SIN-1, SNAP, TEA IbTX- and ChTX-sensitive relaxation (17) Carotid artery SIN-1, SNAP, TEA IbTX- and ChTX-sensitive relaxation (17) Tail artery Illoprost, Sp-cBIMPS IbTX- and TEA (50 µm 1 mm)-sensitive relaxation (168) Trachea Isoproterenol, salbutamol, aminophylline, AcMP, SNP Salbutamol, SNP SIN-1, SNAP, TEA ChTX-sensitive relaxation (91) IbTX-sensitive relaxation (92) IbTX- and ChTX-sensitive relaxation (17) Human airway Isoproterenol ChTX-sensitive relaxation (135) Mesenteric lymphatic vessel Isoproterenol, forskolin IbTX- and ChTX-sensitive hyperpolarization (201) SIN, 1,3-morpholinosydnonimine; SNP, sodium nitroprusside; 8-BrcGMP, 8-bromoguanosine 3,5 -cyclic monophosphate; DBcAMP, dibutyryl camp; TEA, tetraethylammonium; ANP, atrial natriuretic peptide; SNAP, S-nitroso-N-acetylpenicillamine; Sp-cBIMPS, (Sp)-5,6- dichloro-1- -D-ribofuranosylbenzimidazole-3,5 -cyclic monophosphorothioate; NO, authentic nitric oxide; AcMP, N 6-2 -O-adenosine 3,5 cyclic monophosphate; IbTX, iberiotoxin; ChTX, charybdotoxin. 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 Ca 2 spark frequency. These results suggest that cyclic nucleotidemediated smooth muscle relaxation may occur partially through an increase in the frequency of Ca 2 sparks and a subsequent increase in the activity of BK Ca channels. An increase in Ca 2 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 Ca 2 -ATPase, which leads to increased Ca 2 pumping and an elevated SR Ca 2 load, which increases Ca 2 spark frequency (165, 219). Recent results, using arteries from phospholamban-deficient mice, indicate that some of the frequency and amplitude modulation of Ca 2 sparks and STOCs by forskolin and sodium nitroprusside could occur through modulation of the SR Ca 2 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 Ca 2 spark and STOC frequency and amplitude (Fig. 10). Decrease in Ca 2 Spark Frequency by PKC Vasoconstrictors [e.g., serotonin (58) or thromboxane A 2 (194)] stimulate phospholipase C (PLC) through activation of an associated G protein (G q ). PLC activation induces the cleavage of membrane-associated phophatidylinositol 4,5-bisphosphate to diacylglycerol (DAG) and IP 3. Each product of this pathway has its own distinct effect in the cell. IP 3 induces the release of stored Ca 2 through binding to an IP 3 receptor located on the membrane of intracellular stores, whereas DAG activates PKC. Ca 2 release through IP 3 receptors plays an important role to increase smooth muscle contractility, in particular to endogenous smooth muscle contractile agents (e.g., norepinephrine). PKC may regulate the contractility of smooth muscle cells in a variety of ways, including the phosphorylation of actin binding proteins (179) and ion channels (20, 21; for a review see Ref. 171) and increased Ca 2 sensitivity of contractile proteins (60, 147). PKC may also contribute to the action of vasoconstrictors on smooth muscle through a novel mechanism, by decreasing the frequency of Ca 2 sparks, which would lead to membrane depolarization and activation of voltage-dependent Ca 2 channels (Fig. 10) (19). Activators of PKC, phorbol 12-myristate 13-acetate and sn-1,2- dioctanoyl-glycerol, decreased Ca 2 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 Ca 2 spark frequency does not involve a reduction of SR Ca 2 load, suggesting that PKC directly inhibits RyR channels (Fig. 10) (19). The precise molecular mechanisms of PKC inhibition of Ca 2 sparks remain to be determined. IP 3 induced Ca 2 release has been described in terms of a hierarchical nomenclature based on the spatiotemporal characteristics of the observed events. Fundamental Ca 2 release through a single IP 3 receptor has been termed a blip [amplitude, nm; spread, 1 3 µm; decay (t 1/2 ) 45 ms (22)], whereas intermediate release

15 INVITED REVIEW C249 Fig. 10. Possible mechanisms of action of PKA/PKG and PKC on Ca 2 sparks, BK Ca channels, and SR Ca 2 -ATPase in arterial smooth muscle cells. Activation of PKA or PKG increases Ca 2 spark frequency and increases Ca 2 load of the SR (probably through activation of the SR Ca 2 -ATPase via disinhibition of phospholamban). Increased Ca 2 spark frequency could occur due to a direct effect on the RyR channel and/or be a secondary effect from increased SR Ca 2 load. PKA/PKG activation also increases the activity of BK Ca channels, which could manifest itself by an elevation in STOC amplitude and steady-state BK Ca channel activity. Synergistic effect of increased Ca 2 spark frequency and a direct effect on BK Ca channels will result in a significant increase of BK Ca channel activity. Membrane potential hyperpolarization closes L-type Ca 2 channels, which reduces Ca 2 influx, lowering the cytoplasmic Ca 2 concentration, and leads to vasodilation (see Ref. 157). Activation of PKC reduces frequency of Ca 2 sparks and amplitude of STOCs, due to a direct inhibitory effect on RyR channels and BK Ca channels, respectively. Additive effect results in decreased BK Ca channel activity, a depolarization of the smooth muscle cell membrane potential, and activation of L-type Ca 2 channels. PKA, PKG, and PKC also modulate the voltagedependent Ca 2 channel, which would contribute to modulation of the Ca 2 channel = RyR channel = BK Ca channel pathways (not illustrated). due the recruitment of blips are termed Ca 2 puffs (22, 216). Propagating Ca 2 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 IP 3 should activate fundamental Ca 2 release through IP 3 receptors. Vasoconstrictors have been shown to increase Ca 2 wave frequency and amplitude in the smooth muscle cells of rat tail artery, an effect that has been attributed to the activation of IP 3 receptors by IP 3, and subsequent activation of RyR channels by Ca 2 released through IP 3 receptors (81). IP 3 -induced Ca 2 release would increase the cytoplasmic [Ca 2 ] and would tend to increase Ca 2 spark frequency, particularly if RyR channels are situated close to IP 3 receptors and sense the high local [Ca 2 ]. However, the reduction in SR Ca 2 load would tend to decrease Ca 2 spark frequency (219). The extent to which IP 3 -induced Ca 2 release increases or decreases Ca 2 spark activity (16, 52, 98, 106) would depend on a balance between an increase in Ca 2 near the cytoplasmic face of the RyR channels and the reduction in stored Ca 2 that would follow. It remains to be resolved how PKC and IP 3 affect the cytoplasmic and SR luminal communication between RyR channels and IP 3 receptors. CONCLUSIONS Ca 2 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 Ca 2 sparks occur very close to the cell membrane, consistent with a role in signaling ion channels in the plasma membrane. The close proximity of Ca 2 spark sites to the plasma membrane support earlier studies indicating that, under certain circumstances, subsarcolemmal [Ca 2 ] could be significantly higher than global [Ca 2 ] i (215). Further support for this idea has come from a demonstrated dissociation between global [Ca 2 ] i and BK Ca channel currents, which should be a measure of subsarcolemmal [Ca 2 ] (56, 153, 181). Voltage-dependent Ca 2 channels, Ca 2 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 Ca 2 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 Ca 2 sparks on SK Ca channels remains to be established. Ca 2 entry through dihydropyridine-sensitive, voltage-dependent Ca 2 channels regulates Ca 2 spark

16 C250 INVITED REVIEW properties. The speed and efficacy of communication of voltage-dependent Ca 2 channels to activate Ca 2 sparks depends on proximity, and this issue is not resolved for smooth muscle. In contrast, it is clear that Ca 2 sparks in cardiac muscle are activated by local Ca 2 entry through voltage-dependent Ca 2 channels (see Fig. 1). Ca 2 spark communication to BK Ca channels is local, and the activator [Ca 2 ] for BK Ca channels likely reaches µm (153) within the 20-nm gap between the SR and plasma membrane. Activation of BK Ca channels by Ca 2 sparks causes membrane potential hyperpolarization (e.g., Fig. 8), which closes voltagedependent Ca 2 channels, leading to a decrease in global intracellular [Ca 2 ] and smooth muscle relaxation (Figs. 1 and 2) (102, 103, 140, 157). This negativefeedback pathway may be of particular importance for tonic smooth muscle. The ability of Ca 2 entry through voltage-dependent Ca 2 channels to stimulate Ca 2 release through RyR channels and contribute to a global Ca 2 transient seems to be quite variable and is not consistent with local Ca 2 controlling Ca 2 release. The precise mechanisms and significance of Ca 2 -induced Ca 2 release to global Ca 2 transients in smooth muscle are still unclear. It is likely that CICR does contribute Ca 2 for contraction in some types of phasic smooth muscle. Nonetheless, blocking Ca 2 release with ryanodine contracts most types of smooth muscle (10, 11, 38, 39, 44, 103, 105, 122, 140, 155, 185, ), suggesting that RyR channel-mediated negative-feedback control of [Ca 2 ] i dominates in smooth muscle (Fig. 2). It is possible that local Ca 2 release through RyR channels (Ca 2 sparks) provides a means for Ca 2 extrusion, thus decreasing cytoplasmic Ca 2 (the SBB hypothesis). RyR channels could exert negative-feedback control of [Ca 2 ] i through membrane potential hyperpolarization caused by activation of BK Ca channels (Fig. 2) and SK Ca channels and through inactivation of voltage-dependent Ca 2 channels (Fig. 2). Modulation of Ca 2 spark frequency and amplitude by smooth muscle relaxants and constrictors appears to regulate smooth muscle membrane potential and hence contractility. PKA, PKG, and PKC have especially prominent roles in frequency and amplitude modulation of Ca 2 sparks. These kinases appear to exert coordinated and concerted actions on the key elements of Ca 2 signaling, including voltage-dependent Ca 2 channels, RyR channels, phospholamban, and BK Ca channels (see Fig. 10). The exact molecular targets that determine the frequency and amplitude modulation of Ca 2 sparks by different kinases are still unresolved. However, it is clear that modulation of Ca 2 sparks provides a powerful means to regulate smooth muscle function. We thank Adrian D. Bonev, Joseph Brayden, Laura Cartin, Del-Rae Eckman, Thomas J. Heppner, Gerald Herrera, David Hill- Eubanks, and George C. Wellman for helpful comments on the manuscript. We also thank the following persons who have contributed to research on smooth muscle Ca 2 sparks at the University of Vermont: Adrian D. Bonev, Heping Cheng, Maik Gollasch, Thomas J. Heppner, Gerald Herrera, Thomas Kleppisch, Harm J. Knot, J. B. Patlait, Guillermo J. Perez, Michael Rubart, L. Fernando Santana, Andrá S. Stevenson, and George C. Wellman. Unpublished observations have been kindly provided by Drs. George C. Wellman and L. Fernando Santana. This review was supported by National Institutes of Health Grants HL-44455, HL-51728, and DK (to M. T. Nelson) and HL-25675, HL-36974, and HL (to W. J. Lederer), by National Science Foundation Grants IBN and BIR (to M. T. Nelson), and by fellowships from the American Heart Association, New England (to J. H. Jaggar) and Vermont (to V. A. 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