HIGHLIGHTED TOPICS
Signal Transduction in Smooth Muscle
Invited Review: Significance of spatial and temporal heterogeneity of calcium transients in smooth muscle

Departments of Anesthesiology, and Physiology and Biophysics, Mayo Clinic and Foundation, Rochester, Minnesota 55905

	ABSTRACT

TOP ABSTRACT INTRODUCTION CA2+ SPARKS IN SMOOTH... CA2+ OSCILLATIONS AND WAVES... REFERENCES

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION CA2+ SPARKS IN SMOOTH... CA2+ OSCILLATIONS AND WAVES... REFERENCES

REGULATION OF INTRACELLULAR 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).

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), 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.

	CA2+ SPARKS IN SMOOTH MUSCLE

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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 two-dimensional 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 µm (80). Other studies in vascular smooth muscle have estimated the width of the spark to be ~1.5 µm (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 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 voltage-gated influx channels in response to electrical stimulation. The physical and functional coupling between 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.

View larger version (27K): [in this window] [in a new window]   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.

Smooth muscle differs from cardiac muscle in that Ca²⁺-sensitive K⁺ channels (BK_Ca) 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 BK_Ca 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 BK_Ca and produce 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 BK_Ca, 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 BK_Ca 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 regions of increased incidence of Ca²⁺ sparks corresponded with the site of initiation of propagating ACh-induced [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 BK_Ca 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).

	CA2+ OSCILLATIONS AND WAVES IN SMOOTH MUSCLE

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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 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.

View larger version (23K): [in this window] [in a new window]   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.

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 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 time-dependent 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-à-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 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 steady-state 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 responding 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.

	ACKNOWLEDGEMENTS

This work is supported by Grants GM-57816 and GM-56686 from the National Institutes of Health and the Mayo Foundation.

	FOOTNOTES

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: sieck.gary{at}mayo.edu).

	REFERENCES

TOP ABSTRACT INTRODUCTION CA2+ SPARKS IN SMOOTH... CA2+ OSCILLATIONS AND WAVES... REFERENCES

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