Invited Review: Significance of spatial and temporal heterogeneity of ...

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Ca2+ sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle ...
J Appl Physiol 91: 488–496, 2001.

highlighted topics Signal Transduction in Smooth Muscle Invited Review: Significance of spatial and temporal heterogeneity of calcium transients in smooth muscle CHRISTINA M. PABELICK, GARY C. SIECK, AND Y. S. PRAKASH Departments of Anesthesiology, and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905 Pabelick, Christina M., Gary C. Sieck, and Y. S. Prakash. Invited Review: Significance of spatial and temporal heterogeneity of calcium transients in smooth muscle. J Appl Physiol 91: 488–496, 2001.—The multiplicity of mechanisms involved in regulation of intracellular Ca2⫹ concentration ([Ca2⫹]i) in smooth muscle results in both intra- and intercellular heterogeneities in [Ca2⫹]i. Heterogeneity in [Ca2⫹]i regulation is reflected by the presence of spontaneous, localized [Ca2⫹]i transients (Ca2⫹ sparks) representing Ca2⫹ release through ryanodine receptor (RyR) channels. Ca2⫹ sparks display variable spatial Ca2⫹ distributions with every occurrence within and across cellular regions. Individual sparks are often grouped, and fusion of sparks produces large local elevations in [Ca2⫹]i that occasionally trigger propagating [Ca2⫹]i waves. Ca2⫹ sparks may modulate membrane potential and thus smooth muscle contractility. Sparks may also be the target of other regulatory factors in smooth muscle. Agonists induce propagating [Ca2⫹]i oscillations that originate from foci with high spark incidence and also represent Ca2⫹ release through RyR channels. With increasing agonist concentration, the peak of regional [Ca2⫹]i oscillations remains relatively constant, whereas both frequency and propagation velocity increase. In contrast, the global cellular response appears as a concentration-dependent increase in peak as well as mean cellular [Ca2⫹]i, representing a spatial and temporal integration of the oscillations. The significance of agonist-induced [Ca2⫹]i oscillations lies in the establishment of a global [Ca2⫹]i level for slower Ca2⫹-dependent physiological processes. calcium sparks; calcium oscillations; acetylcholine; confocal microscopy; fluorescence; sarcoplasmic reticulum; ryanodine receptor

Ca2⫹ concentration ([Ca2⫹]i) in smooth muscle involves multiple mechanisms including Ca2⫹ influx and efflux across the plasma membrane, second messenger production, sarcoplasmic reticulum (SR) Ca2⫹ release, and reuptake (which may themselves be feedback regulated by [Ca2⫹]i levels). Agonists elevate [Ca2⫹]i by increasing Ca2⫹ influx and by eliciting SR Ca2⫹ release, mediated through both inositol 1,4,5-trisphosphate (IP3) receptor channels (5, 21, 96) and ryanodine receptor (RyR) channels (53, 100, 101, 107). REGULATION OF INTRACELLULAR

Address for reprint requests and other correspondence: G. C. Sieck, 4-184W Jo. SMH, Mayo Clinic, 200 First St. SW, Rochester, MN 55905 (E-mail: [email protected]). 488

Spatial and kinetic or temporal differences in [Ca2⫹]i regulatory processes exist across cells, leading to intercellular heterogeneity in [Ca2⫹]i regulation both under basal conditions and during agonist stimulation. Intracellular heterogeneity in [Ca2⫹]i regulation may also arise from variations in the distribution of membrane receptors, production and/or diffusion of second messengers, and SR and membrane Ca2⫹ channels. Such heterogeneities in [Ca2⫹]i regulation are evidenced by spontaneous localized [Ca2⫹]i transients, termed Ca2⫹ sparks, which have been reported in several cell types, including smooth (77), cardiac (20, 71, 81), and skeletal muscles (57, 62, 99). Furthermore, agonist-induced propagated [Ca2⫹]i oscillations, which have been reported in vascular (12, 37, 42), colonic (70),

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uterine (55), and airway (67, 84, 93) smooth muscles, also reflect the nonhomogenous nature of [Ca2⫹]i regulation. However, ultimately, it is the global [Ca2⫹]i that determines the Ca2⫹-dependent force response of tissues such as smooth muscle. Accordingly, the spatial and temporal integration of [Ca2⫹]i transients is a key issue. In this brief review, we summarize the current literature on the spatial and temporal aspects of Ca2⫹ sparks and agonist-induced [Ca2⫹]i oscillations. We further propose a model for the significance of spatial and temporal heterogeneity in the establishment of global [Ca2⫹]i. CA2ⴙ SPARKS IN SMOOTH MUSCLE

Since their first description in cardiac muscle (20), spontaneous, localized Ca2⫹ transients, termed Ca2⫹ sparks, have now been observed in skeletal muscle fibers (57, 62, 99) and arterial (48, 77, 83), venous (4), gastrointestinal (34, 109) and airway (80, 93, 108) smooth muscles (see Refs. 19, 47, and 90 for detailed reviews). Converging evidence using modulation of spark properties by ryanodine, membrane potential, [Ca2⫹]i itself, and agonists such as caffeine indicates that Ca2⫹ sparks represent unitary Ca2⫹ release through RyR channels (4, 48, 74, 77, 80, 93). Properties of Ca2⫹ sparks. It is estimated that, in the vicinity of the Ca2⫹ spark, [Ca2⫹]i can increase by several micromolars but with negligible effects on global [Ca2⫹]i (48, 77). In this regard, spark amplitude (thought to reflect RyR channel open probability and the gradient for SR Ca2⫹ release; Ref. 89) has been found to be significantly smaller in skeletal muscle (57), compared with cardiac (20) and smooth muscles (48, 77, 80, 93). Because the single-channel currents through RyR channels in these muscle types are comparable, several investigators have combined imaging of [Ca2⫹]i with mathematical calculations to determine the numbers of individual RyR channels that likely contribute to a spark. The Ca2⫹ flux has been estimated to be ⬃4 pA over 10 ms (20). On the basis of an estimated 0.6 pA of current through a single RyR channel, sparks are estimated to involve activation of ⬃10 channels (72). The relative spatial distribution of the channels of a sparking unit has been observed only recently (35, 47, 48, 80). Using rapid real-time twodimensional confocal imaging of Ca2⫹ sparks, while maintaining a temporal resolution comparable to that of line scans used in the original and fundamental studies on sparks (57, 62, 71, 77, 81, 99), we found that the area occupied by a spark, at least in tracheal smooth muscle, is ⬃1.2–1.5 ␮m (80). Other studies in vascular smooth muscle have estimated the width of the spark to be ⬃1.5 ␮m (74, 77, 82). However, the area of sparking appears to vary from event to event within a sparking region (80), suggesting that, although ⬃10 RyR channels may contribute to a single sparking event, additional RyR channels in various states of activation or inactivation may be part of a sparking unit and may contribute Ca2⫹ at various times. The

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spatial limitation of foci may be due either to Ca2⫹ diffusion away from the sparking area or to SR Ca2⫹ reuptake acting as a barrier to propagation from the region of sparking. At least in smooth muscle, based on the time constants for various processes involved in decreasing [Ca2⫹]i, it has been suggested that diffusion rather than active uptake of Ca2⫹ contributes to inhibition of a spark in space and time (47). The concept of Ca2⫹ sparks representing unitary Ca2⫹ release is also generally supported by the finding that the distribution of spark amplitudes displays statistical modes that are multiples of a basic amplitude (⬃50 nM in tracheal smooth muscle) (80), thus resembling quantal neurotransmitter release at neuromuscular junctions. Although [Ca2⫹]i dynamics vary considerably across smooth muscle types, matching their function as tonic or phasic muscles, the properties of Ca2⫹ sparks appear to be similar across muscle types. For example, in arterial smooth muscle, the rise time of Ca2⫹ sparks is ⬃20 ms, with a fall time ⬎100 ms (48, 77). In our studies in porcine tracheal smooth muscle cells, the rise time of Ca2⫹ sparks ranged from 30 to 90 ms, whereas the fall time ranged from 60 to 300 ms (80). Within an intracellular region, the rise time of individual Ca2⫹ sparks (normalized for spark amplitude) was found to be relatively constant. However, across different regions displaying Ca2⫹ sparks within the same cell, there was considerable variability in the rise time of these sparks. A similar heterogeneity in the rise time was also observed across cells. The fall time of individual Ca2⫹ sparks was relatively constant, but across different regions of Ca2⫹ sparking within a cell, or across cells, there was greater variability in fall time. The rates of rise and fall of [Ca2⫹]i likely reflect the rate of Ca2⫹ release through the RyR channels and the rate of diffusion of Ca2⫹ away from the site of the spark. Indeed, in tracheal smooth muscle, we observed groups of three to four individual Ca2⫹ sparks, separated by periods of quiescence (80, 93). These events may represent localized facilitation of sparking from different groups of RyR channels with differing activation histories and kinetics. The frequency of sparks then likely reflects channel kinetics, local [Ca2⫹]i, and the sensitivity for RyR channel activation. Within single cells, multiple foci for Ca2⫹ sparks have been observed essentially in every smooth muscle type. In tracheal smooth muscle, we found that adjacent regions of Ca2⫹ sparking are often coupled, whereas more distant regions display sparks independently (80, 93). These data suggest that localized SR Ca2⫹ release may induce Ca2⫹ release from surrounding regions, perhaps via Ca2⫹-induced Ca2⫹ release (CICR), a key mechanism for elevating [Ca2⫹]i at least in cardiac muscle. In tracheal smooth muscle, we frequently observed larger [Ca2⫹]i responses with individual sparks superimposed on both the rising and falling phases of the larger response (80, 93). Similar events have been observed previously in cardiac myocytes (20). In the heart, Ca2⫹ influx occurs through voltagegated influx channels in response to electrical stimulation. The physical and functional coupling between

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influx channels and RyR channels of the SR is intimate in that RyR channels are positioned within 25 nm of the influx channels within the T tubules (28). Local Ca2⫹ in the subsarcolemmal space likely reaches several micromolars and activates RyR channels, producing CICR. Whether such intimate communication occurs between influx channels and RyR channels in smooth muscle is not clear. For example, spark amplitude and frequency in arterial smooth muscle are increased by influx through voltage-dependent Ca2⫹ channels (48), whereas removal of Ca2⫹ decreases spark frequency in both arterial (48) and tracheal (80, 93) smooth muscles. Therefore, it is possible that, even if the extent of CICR is less in smooth muscle, compared with cardiac muscle, Ca2⫹ influx is an important modulator of spark activation. Significance of Ca2⫹ sparks. Given the proven existence of sparks in several smooth muscle types, the physiological role of sparks has also been a subject of intense investigation. Certainly, although local Ca2⫹ may increase by several micromolars within a sparking region, global [Ca2⫹]i is negligibly affected (48, 77). Therefore, it is likely that whatever effect local Ca2⫹ via sparks has on global [Ca2⫹]i is indirect. In this regard, several potential roles for sparks have been proposed and investigated (Fig. 1). For example, in cardiac muscle, Ca2⫹ influx more or less simultaneously triggers a multitude of sparks that elevate global Ca2⫹ and produce muscle contraction (16, 68). In turn, Ca2⫹ sparks inactivate voltage-dependent Ca2⫹ channels (1, 36, 91), providing negative feedback for muscle contraction. Initial studies suggest that, at least in nonstimulated vascular smooth muscle, such a negative feedback is not a significant player (59). On the other hand, under resting conditions, tonically contracted smooth muscles, such as arterial smooth muscle, are largely dependent on Ca2⫹ influx rather than SR Ca2⫹ for global [Ca2⫹]i (59, 77). Accordingly, Ca2⫹ spark frequency is low (48, 77, 82, 83), and inhibition of sparks has a minimal effect on global [Ca2⫹]i and muscle tone (59, 77). However, a role for negative feedback of Ca2⫹ influx by sparks is possible under conditions of elevated global [Ca2⫹]i or during agonist stimulation where the relative contribution of SR Ca2⫹ is increased. In smooth muscle exhibiting phasic contraction, such as urinary or gastrointestinal smooth muscle, SR Ca2⫹ release is more significant (13, 33). Whether Ca2⫹ sparks regulate phasic contractions in these muscle types is not known. Smooth muscle differs from cardiac muscle in that Ca2⫹-sensitive K⫹ channels (BKCa) play an important role in regulation of membrane potential (15, 47, 49, 77), and thus Ca2⫹ influx and release. Nelson and colleagues (47, 77) proposed that the role of Ca2⫹ sparks lies in the activation of nearby BKCa channels resulting in a macroscopic K⫹ current [called a spontaneous transient outward current (STOC) (6, 40, 41, 77, 79)]. Since then, several studies using both imaging and electrophysiological measurements have demonstrated spark activation of STOCs (74, 82, 108). Ca2⫹ sparks increase the open probability of BKCa and pro-

Fig. 1. Ca2⫹ sparks in smooth muscle. Sparks arise from Ca2⫹ release through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). Sparks may be influenced by Ca2⫹ influx through voltage-dependent Ca2⫹ channels and may, in turn, inhibit these channels. In addition, sparks may activate Ca2⫹-activated K⫹ channels (BKCa) and Cl⫺ channels, resulting in hyperpolarization or depolarization of the plasma membrane, respectively. Accordingly, sparks may significantly influence Ca2⫹ regulation at the plasma membrane. By increasing Ca2⫹ locally, sparks may also influence the sensitivity of RyR channels for further Ca2⫹ release, thus facilitating the effect of stimulatory agonists.

duce hyperpolarization in arterial smooth muscle of ⬃20 mV (33). It has been suggested that, due to the electrical coupling between smooth muscle cells, even local changes in membrane potential should lead to hyperpolarization of the entire muscle, thus changing arterial tone (27). In addition to BKCa, Ca2⫹ sparks have been shown to modulate membrane potential via Ca2⫹-activated Cl⫺ channels in guinea-pig tracheal smooth muscle (108). Such channels have been demonstrated in several vascular (58, 63, 104), gastrointestinal (2, 43), and tracheal (50, 51) smooth muscles and are activated by increased [Ca2⫹]i, resulting in membrane depolarization. Spontaneous transient inward currents via such channels have been demonstrated to be activated by sparks either in isolation or in conjunction with STOCs, at least in tracheal smooth muscle (108). Studies have also shown that the activation characteristics and Ca2⫹ sensitivities of BKCa and Ca2⫹-activated Cl⫺ channels are different (17, 78). Accordingly, at least in some muscle types, Ca2⫹ sparks regulate membrane potential by controlling the relative amount of depolarization and hyperpolarization by activating these channels. Furthermore, differential modulation of these channels by agonists and other substances may provide an additional means to alter the control of membrane potential by Ca2⫹ sparks. The functional significance of Ca2⫹ sparks may vary between airway and vascular smooth muscle. In a study on tracheal smooth muscle, we observed that

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regions of increased incidence of Ca2⫹ sparks corresponded with the site of initiation of propagating AChinduced [Ca2⫹]i oscillations, as well as spontaneous summation of individual sparks, leading to larger [Ca2⫹]i transients. The amplitudes of the spontaneous, summated responses were comparable, if not identical, to those of agonist-induced [Ca2⫹]i responses. Therefore, we propose that, in addition to regulation of membrane potential, Ca2⫹ sparks act as “primers” for agonist stimulation and arise from “trigger” sites that reflect areas of high RyR channel density, also suggested by studies in vascular smooth muscle (65). However, this does not rule out the possibility that Ca2⫹ sparks also regulate membrane potential in tracheal smooth muscle, as observed in vascular smooth muscle. Calcium sparks are further regulated by several intracellular substances such as cAMP, cGMP, and protein kinase C (PKC). For example, both cAMP and cGMP increase Ca2⫹ spark frequency in vascular smooth muscle (47, 49, 83), leading to increased BKCa activity, hyperpolarization, and relaxation. On the other hand, activation of PKC has been shown to decrease spark frequency (14) and produce contraction of smooth muscle. Thus Ca2⫹ sparks may serve as intermediate targets for regulation of smooth muscle tone by cyclic nucleotides and protein kinases. The exact mechanisms by which Ca2⫹ sparks are regulated remain to be determined, although direct modulation of RyR channel activity has been suggested (14). CA2ⴙ OSCILLATIONS AND WAVES IN SMOOTH MUSCLE

Mechanisms underlying [Ca2⫹]i oscillations. Agonist-induced [Ca2⫹]i oscillations have been reported in vascular (12, 37, 42), colonic (70), uterine (55), and airway (67, 80, 84, 86, 93, 95) smooth muscles. Several mechanisms may underlie [Ca2⫹]i oscillations. Studies in gastrointestinal smooth muscle have shown that [Ca2⫹]i oscillations are accompanied by fluctuations in membrane potential (60) and may thus result from repetitive [Ca2⫹]i influx, rather than SR Ca2⫹ release. However, in tracheal smooth muscle, we found that [Ca2⫹]i oscillations were induced by ACh in the absence of Ca2⫹ influx as well as in the presence of KCl, which clamped membrane potential (84). A number of other studies have demonstrated that agonist-induced [Ca2⫹]i oscillations arise from Ca2⫹ release from intracellular stores (for a review, see Ref. 3). In smooth muscle, SR Ca2⫹ release involves both IP3 receptor channels (5, 21, 96) and RyR channels (53, 100, 101, 107). In this regard, it has been proposed that [Ca2⫹]i oscillations involve periodic variations in IP3 levels (73). Elevated [Ca2⫹]i may then activate PKC, which inhibits the G protein or the receptor for a specific agonist, thus establishing a negative-feedback oscillatory network (23). However, sustained IP3 oscillations have not been observed over longer time periods over which agonist-induced oscillations have been observed in tracheal and vascular smooth muscles, for example (38). Furthermore, the fact that [Ca2⫹]i oscillations can be elicited by a nonmetabolizable form of IP3 suggests

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that variations in IP3 do not necessarily underlie [Ca2⫹]i oscillations (102). It has also been proposed that [Ca2⫹]i oscillations arise from CICR via an IP3-independent SR Ca2⫹ pool (7–10, 26) (Fig. 2). In this scenario, agonist-induced IP3 stimulates Ca2⫹ release from an IP3-dependent pool, which then triggers Ca2⫹ release from an IP3-independent pool, i.e., CICR via RyR channels. After depletion of SR Ca2⫹, the release channels are inhibited and SR Ca2⫹ stores are replenished by Ca2⫹ reuptake, allowing a retriggering of CICR. Thus an oscillatory pattern of SR Ca2⫹ release and reuptake is established. For example, in our studies in porcine tracheal smooth muscle cells, we demonstrated that ACh-induced propagating [Ca2⫹]i oscillations reflect an all-or-none SR Ca2⫹ release via RyR channels but require SR Ca2⫹ release through IP3 receptor channels for initiation (54, 80, 84, 93). The initiation of [Ca2⫹]i oscillations was found to be independent of Ca2⫹ influx; however, the maintenance of [Ca2⫹]i oscillations was dependent on the balance between Ca2⫹ influx and efflux (84). Furthermore, [Ca2⫹]i oscillations were also observed in ␤-escin-permeabilized smooth muscle cells, where the influence of Ca2⫹ influx and efflux were eliminated, thus demonstrating the central role of SR Ca2⫹ release in [Ca2⫹]i oscillations (54, 93). Studies in vascular (12, 105) and uterine (30) smooth muscles have also demonstrated that [Ca2⫹]i oscillations are dependent on Ca2⫹ influx only for maintenance, most likely via continued replenishment of SR Ca2⫹ stores. In cells where oscillations arise via RyR channels, Ca2⫹ alone need not be the only trigger for further Ca2⫹ release through RyR channels. There has been

Fig. 2. Intracellular Ca2⫹ concentration ([Ca2⫹]i) oscillations in smooth muscle. Agonist-induced oscillations may arise from repetitive Ca2⫹ influx through voltage-gated or receptor-gated channels. In several smooth muscle types, [Ca2⫹]i oscillations involve repetitive SR Ca2⫹ release through inositol trisphosphate (IP3) receptor (IP3R) channels or via RyR channels. RyR channels may be influenced by Ca2⫹ itself or by the novel second messenger cADP ribose (cADPR). Overall, [Ca2⫹]i is determined by the balance between various regulatory processes.

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considerable recent evidence for the role of cADP ribose (cADPR), a product of ␤-NAD, as a novel second messenger involved in the regulation of RyR function (31, 32, 64, 85), akin to IP3 for its receptor channel. However, in contrast to the direct activation of IP3 receptor channels by its second messenger, cADPR acts indirectly via a binding site on the SR and intermediate proteins including calmodulin. In a recent study, we demonstrated that ACh-induced [Ca2⫹]i oscillations are modulated by cADPR (85). Whether cADPR is involved in oscillations in other smooth muscle types remains to be determined. Propagation of the [Ca2⫹]i wave likely involves diffusion of a trigger for Ca2⫹ release. In cells in which [Ca2⫹]i oscillations arise from SR Ca2⫹ release, both IP3 and Ca2⫹ itself may diffuse along a concentration gradient along the length of the cell, resulting in a cascade of release events across the cell, each of which appears as a local [Ca2⫹]i oscillation. The origin of the wave would then be the SR site within the cell with the lowest threshold for release. Alternatively, an intra-SR signal may allow for Ca2⫹ release from adjacent areas. Furthermore, mitochondria may buffer Ca2⫹ locally, further modulating the propagation of the [Ca2⫹]i wave. The precise mechanisms underlying propagation of [Ca2⫹]i waves in smooth muscle are still being investigated. Spatial and temporal aspects of [Ca2⫹]i oscillations. With regard to the spatial aspect of [Ca2⫹]i oscillations, a general finding across smooth muscle types is that these oscillations tend to originate from one end along the long axis of the cell and propagate in a wave-like fashion toward the other end. With continued agonist exposure, the oscillations tend to maintain their direction of propagation. On occasion, oscillations have been noted to reverse in direction, starting at the opposite end of the cell, or even to initiate from the central areas of the cell and propagate in two directions (e.g., see Ref. 84). Such heterogeneity appears to exist across cells within a smooth muscle type as well as across muscle types. The site of initiation of an oscillation may be dependent on the relative distribution of agonist receptors on the cell surface and/or other involved mechanisms such as Ca2⫹ influx channels or SR Ca2⫹ release channels. In this regard, we recently observed that ACh-induced [Ca2⫹]i oscillations in porcine tracheal smooth muscle initiate from areas of the cell displaying the highest frequency of Ca2⫹ sparks (80). The incidence of Ca2⫹ sparks and the initiation of [Ca2⫹]i oscillations may be interrelated by a heterogeneous distribution of RyR channels, such that, in a localized region, [Ca2⫹]i oscillations are initiated when the incidence of Ca2⫹ sparks, especially if they occur frequently enough to fuse, reaches a critical threshold triggering CICR in adjacent regions. The sensitivity of RyR to CICR may be further affected by changes in factors such as second messengers (e.g., cADPR) and/or cyclic nucleotides. In terms of temporal aspects, the characteristics of oscillations have also been found to vary between cells and cell types. For example, in porcine tracheal smooth

muscle cells, we have found that ACh induces [Ca2⫹]i oscillations that initiate with higher frequency, smaller amplitude, and higher propagation velocity and then slowly settle down to a slower frequency and propagation velocity but larger amplitude for the remainder of agonist exposure (84, 86). Similar timedependent variations in oscillation frequency and amplitude have also been noted in vascular smooth muscle exposed to phenylephrine and norepinephrine (42, 56, 88). However, unlike tracheal smooth muscle, in human vascular smooth muscle (12) and guinea pig airway smooth muscle (95), the [Ca2⫹]i oscillations have been found to subside with continued agonist exposure suggesting differences in the underlying mechanisms. The frequency of the [Ca2⫹]i oscillations also varies across cell types, ranging from ⬍5 to 30 per min and amplitudes from 100 to ⬎500 nM of Ca2⫹ at steady state. It is likely that the spatial and temporal characteristics of [Ca2⫹]i oscillations reflect differences in the underlying mechanisms. However, the precise kinetics and dynamics of the various [Ca2⫹]i regulatory processes have not been examined vis-a`-vis their effects on oscillation parameters, except in mathematical models simulating agonist-induced [Ca2⫹]i oscillations in certain nonsmooth muscle cells (e.g., see Refs. 18, 22, 61). Studies in different smooth muscle types have demonstrated that [Ca2⫹]i oscillations display a dose-dependent modulation of amplitude, frequency, and/or propagation velocity. For example, in tracheal smooth muscle, increasing ACh concentration results in higher oscillation frequency but smaller amplitude (86). An interesting observation has been that the peak or maximum [Ca2⫹]i level reached (relative to zero) remains relatively constant across agonist concentrations. On the basis of our work in tracheal smooth muscle, we propose a conceptual framework for [Ca2⫹]i oscillations where 1) basal [Ca2⫹]i level represents an overall balance between Ca2⫹ influx and efflux across the cell membrane and SR Ca2⫹ release and reuptake, 2) oscillation amplitude represents SR Ca2⫹ content, and 3) oscillation frequency and propagation velocity reflect the sensitivity for SR Ca2⫹ release through RyR channels (CICR sensitivity). In our model, SR Ca2⫹ release is all or none. Accordingly, oscillation amplitude will depend on the state of repletion of the Ca2⫹ pool and the basal [Ca2⫹]i level that determines the Ca2⫹ gradient for SR Ca2⫹ release. As basal [Ca2⫹]i levels increase, oscillation amplitude would be expected to decrease. However, unlike a tetany response in skeletal muscle for example, the peak value of the [Ca2⫹]i response (relative to zero) does not increase with subsequent oscillations. These data have led us to conclude that [Ca2⫹]i oscillations involve repetitive release and reuptake via a limited SR Ca2⫹ pool. Modulation of oscillation frequency by agonist concentration, observed in a number of different cell types (see Ref. 3 for a review), could be related to agonist modulation of the sensitivity for CICR through RyR channels. Previous studies have demonstrated a relationship between basal Ca2⫹ and CICR, in which the extent of CICR

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increases with increasing basal Ca2⫹ (11, 69). Accordingly, with increasing agonist concentration, the rise in basal Ca2⫹ due to several mechanisms would lead to an increase in oscillation frequency. Furthermore, with increasing basal Ca2⫹, the rate of SR Ca2⫹ reuptake increases (39), leading to faster SR refilling and an increase in oscillation frequency. Increase in propagation velocity with agonist concentration, observed in some smooth muscle types (86, 87, 98), is consistent with a model for diffusion of trigger IP3 or Ca2⫹ down a concentration gradient (45, 46). Significance of [Ca2⫹]i oscillations. Global elevation of [Ca2⫹]i is an important determinant of agonist-induced elevation of force in smooth muscle cells. In both individual cells and multicellular preparations, studies have reported a biphasic pattern in the global cellular [Ca2⫹]i response to agonist stimulation, with an initially higher [Ca2⫹]i level followed by a lower steadystate level (52, 75, 76, 92, 95). It has been further reported that the steady-state [Ca2⫹]i response is dependent on agonist concentration. In our studies in tracheal smooth muscle, we found that, although the peak of the [Ca2⫹]i oscillations within an intracellular region was unchanged by agonist concentration, the global [Ca2⫹]i response for a cell was increased with increasing agonist concentration (86). Therefore, we conclude that the previously observed biphasic global [Ca2⫹]i response represents the spatial and temporal integration of local [Ca2⫹]i oscillations. Accordingly, we hypothesize that the major physiological significance of propagating [Ca2⫹]i oscillations lies in providing an effective mode of regulating global [Ca2⫹]i level using only a limited pool of SR Ca2⫹. Within a localized region, amplitude modulation is limited by the capacity of the SR Ca2⫹ stores and the gradient for Ca2⫹ release, whereas frequency modulation is limited only by the kinetics of Ca2⫹ release and reuptake. Accordingly, amplitude modulation would not be an efficient way of controlling global [Ca2⫹]i level. Indeed, studies in hormone-stimulated hepatocytes (87, 98, 106) and histamine-stimulated endothelial cells (44) have also found a lack of amplitude modulation of oscillations by agonist concentration. On the other hand, frequency modulation allows for more rapid adjustments in global [Ca2⫹]i levels using a limited amount of Ca2⫹. Most Ca2⫹-dependent physiological processes in smooth muscle have slower kinetics than individual [Ca2⫹]i oscillations. For example, in both vascular (110) and airway (94) smooth muscles, there is an ⬃250- to 500-ms delay between the elevation in [Ca2⫹]i and the development of force. This delay has been found to be predominantly due to the slow recruitment of calmodulin from intracellular pools and the isomerization of the Ca2⫹-calmodulin-myosin light chain kinase complex, with time constants in the hundreds of milliseconds (24). Because the duration of individual [Ca2⫹]i oscillations tends to be considerably less than the time constants, these intracellular processes effectively integrate the more rapid changes in [Ca2⫹]i. Furthermore, the slower rate of relaxation of smooth muscle cells introduces an additional delay in respond-

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ing to fluctuating levels in [Ca2⫹]i. Accordingly, recurring [Ca2⫹]i oscillations would be integrated, and the resultant force would be a sustained response. Therefore, it is unlikely that Ca2⫹-dependent processes such as contraction will reflect the faster local [Ca2⫹]i oscillations. In addition regulating the dynamic availability of Ca2⫹ for contraction, [Ca2⫹]i oscillations may also modulate cross-bridge cycling in smooth muscle. Studies have suggested that agonist stimulation leads to myosin cross bridges going through a rapid cycling phase followed by a low-energy “latch” state in which they maintain the force of contraction (97, 103). [Ca2⫹]i oscillations may be one way of preventing the formation of a latch and maintaining a rapid cross-bridge cycling phase. Finally, [Ca2⫹]i oscillations may serve a purpose other than providing Ca2⫹ for force production. Several intracellular processes, such as gene activation and regulation of protein expression, have also been found to be sensitive to cyclical changes in Ca2⫹. Using a calcium clamp technique, Dolmetsch et al. (25) examined the role of [Ca2⫹]i oscillation amplitude and frequency on regulation of gene expression by proinflammatory cytokines. They found that [Ca2⫹]i oscillations reduce the threshold for activation of transcription factors and that oscillation frequency can be used to selectively activate certain transcription factors. Li et al. (66) found that repetitive applications of IP3 result in more gene expression in activated T cells compared with steady application. Calcium waves have also been proposed to influence cardiac muscle growth and function (29). Although the role of [Ca2⫹]i oscillations has not been examined in terms of gene expression in smooth muscle, this represents an exciting area of future research. In this regard, if [Ca2⫹]i oscillations represent the summation of multiple sparks, with the latter being an elemental form of Ca2⫹ regulation, then the possibility exists that sparks themselves are involved in basic cell signaling for gene expression, cellular differentiation, and proliferation at a local level. In conclusion, spatial and temporal heterogeneity of [Ca2⫹]i is evident in several smooth muscle types. Such heterogeneity allows for both local and global control of cellular function. The mechanisms underlying the establishment of [Ca2⫹]i heterogeneity may be tailored toward the specific function of a cell type. Such heterogeneity may be important not only in regulation of force, a key function of smooth muscle, but also in other aspects of cell signaling. This work is supported by Grants GM-57816 and GM-56686 from the National Institutes of Health and the Mayo Foundation. REFERENCES 1. Adachi-Akahane S, Cleemann L, and Morad M. Crosssignaling between L-type Ca2⫹ channels and ryanodine receptors in rat ventricular myocytes. J Gen Physiol 108: 435–454, 1996. 2. Akbarali HI and Giles WR. Ca2⫹ and Ca2⫹-activated Cl⫺ currents in rabbit oesophageal smooth muscle. J Physiol (Lond) 460: 117–133, 1993.

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