INTRODUCTION

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CALCIUM IS A FUNDAMENTAL second messenger in smooth muscle cells. Increasing cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i), and binding to calmodulin and activation of myosin light chain kinase, is the primary stimulus for contraction. To activate the contractile apparatus, Ca²⁺ must increase globally throughout the cytoplasm. The Ca²⁺ utilized for activation of the contractile apparatus enters the cytoplasmic compartment during periods of membrane depolarization, mechanical distortion, or stimulation by agonists. Release of Ca²⁺ from intracellular stores is a second means of increasing [Ca²⁺]_i. After an excitatory event, relaxation and Ca²⁺ homeostasis are achieved by reuptake of Ca²⁺ into stores and extrusion into the extracellular space. These events are accomplished by at least a dozen specialized Ca²⁺ transporters and ion channels, which are arranged in membranes separating at least five distinct compartments and capable of facilitating Ca²⁺ movements up and down significant electrochemical gradients. This brief review provides a general overview of Ca²⁺ entry mechanisms, factors that regulate uptake and release from intracellular stores, and extrusion mechanisms. Further discussion will be provided about integrated Ca²⁺ handling mechanisms such as localized Ca²⁺ transients, which can provide either positive or negative feedback in regulating the excitability of smooth muscle cells. Additional recent reviews on this general subject are also available from other authors (cf. Refs. 26, 57, 63, 75, 87).

	CA2+ ENTRY MECHANISMS

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Dihydropyridine-sensitive Ca²+ channels. Much of the Ca²⁺ that activates the contractile apparatus in smooth muscles enters cells during periods of depolarization via dihydropyridine (DHP)-sensitive Ca²⁺ channels (Fig. 1). These channels are composed of pore-forming -subunits and several accessory subunits that may regulate pore formation, gating, and kinetics of the channels. At least six genes encode Ca²⁺ channel -subunits, and a splice variant, _1C-b, forms channels in smooth muscles (46). The _1C-b-subunit carries Ca²⁺ current and provides the voltage and DHP sensitivity of these channels. The -subunits are large proteins with four repeating segments, each with six membrane-spanning domains (S₁-S₆). The pore selectivity for Ca²⁺ is thought to be due to the region between S₅ and S₆ (44).

View larger version (47K): [in this window] [in a new window]   Fig. 1.   Main essentials of Ca2+ handling. At least 5 compartments are relevant to Ca2+ signaling in smooth muscle: 1) extracellular solution, 2) subsarcolemmal region between sarcoplasmic reticulum (SR) and plasma membrane (PM), 3) SR, 4) mitochondria (M), and 5) general cytoplasm. As discussed in the text, many transport proteins are involved in Ca2+ handling. Depolarization activates dihydropyridine-sensitive Ca2+ channels (DHP Ca2+). Other Ca2+ entry mechanisms include agonist-activated nonselective cation channels (NSCC, activated by muscarinic stimulation featured in figure) and capacitative Ca2+ entry (CCE) channels. The amount of Ca2+ entry through NSCC is controversial, but these channels yield depolarization that activates DHP Ca2+ channels. Ca2+ entering cells can increase global cytoplasmic Ca2+ and cause contraction. Part of the Ca2+ entering cells may be taken up ("buffered") by superficial Ca2+ stores, such as the SR and mitochondria. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps provide the mechanism to sequester Ca2+ into the SR, and this requires energy to pump Ca2+ up a steep concentration gradient. Ca2+ is highly buffered within SR. The Ca2+ uniporter in the inner membrane of mitochondria (outer membrane depicted schematically by dotted line) provides an uptake mechanism, and this occurs down a large electrochemical gradient for Ca2+ (mitochondria inside very negative) generated by proton pumping by the electron transport chain. Ca2+ homeostasis in mitochondria is maintained by Na+/Ca2+ exchange (NCE). Many excitatory agonists bind to receptors coupled to G proteins (Gq/G11) and activate phospholipase C to generate inositol trisphosphate (IP3). IP3 binds to receptors in the SR membrane and causes Ca2+ release. This can sum with Ca2+ entry mechanisms and contribute to global Ca2+ transients. IP3-dependent Ca2+ release can also stimulate Ca2+ uptake into mitochondria and localized release through IP3 receptors (IP3R; Ca2+ puffs). Localized Ca2+ transients can also originate from ryanodine receptors (RyR; Ca2+ sparks). Local Ca2+ transients result in high concentrations of Ca2+ in the subsarcolemmal region and can stimulate Ca2+-activated conductances in the plasma membrane, such as small-conductance Ca2+-activated K+ channels (SK), large-conductance Ca2+-activated K+ channels (BK), and Ca2+-activated Cl channels (ClCa). The response to Ca2+ sparks and puffs depends on the spatial proximity of RyR and IP3R to specific types of Ca2+-activated conductances and may vary between smooth muscle cells. Cellular Ca2+ homeostasis is maintained by 2 transporters that extrude Ca2+ into the extracellular medium: plasma membrane Ca2+ pump (PMCA) and NCE proteins. Many forms of intracellular regulation exist that affect the performance of the transporters shown in the figure. See text for details regarding regulatory mechanisms.

Other voltage-dependent Ca²+ channels. Ca²⁺ channels insensitive to DHP have been found in some smooth muscles. A recent example is the DHP-insensitive, rapidly inactivating, voltage-dependent Ca²⁺ channels in the terminal branches of guinea pig mesenteric artery (80). The fraction of these channels increased in lower branches of mesenteric arterial tree, and the conductance contributed significantly to Ca²⁺ entry. The DHP-insensitive channels had unique biophysical and pharmacological properties, but the molecular entity responsible for this conductance has not been identified. Others have reported that T-type or low-voltage-activated channels are expressed and contribute to Ca²⁺ entry in smooth muscles (45, 102). Mibefradil (Ro-40-5967) has been suggested as an antagonist of T-type channels in vascular muscles, but the selectivity of this compound has been questioned (cf. Refs. 10, 69).

Nonselective cation channels. Endogenous agonists activate nonselective cation currents and Ca²⁺-dependent Cl currents in smooth muscles. Both inward currents can contribute to Ca²⁺ entry via depolarization and activation of voltage-dependent Ca²⁺ channels. Although Cl current is important in this process, this review will not discuss this family of conductances because it is not a direct source of Ca²⁺. The reader is directed to other reviews (such as Ref. 66).

P2X receptors. ATP is released as a neurotransmitter from autonomic neurons and affects the activity of many smooth muscles (18). ATP can function as either an excitatory or inhibitory neurotransmitter. As an excitatory transmitter, ATP typically activates P2X receptors (P2X₁, P2X₂, and P2X₄), which are receptor-operated cation channels expressed by smooth muscle cells (16, 83, 103). In a variety of native smooth muscle cells, ATP activates a cation current (9) that is similar in characteristics to heterologously expressed P2X receptors (99). Activation of P2X receptors, such as those of human saphenous vein myocytes, is associated with a transient, nonselective cation current and increased [Ca²⁺]_i (72). These authors concluded that the rise in [Ca²⁺]_i due to ATP was partly due to Ca²⁺ entry through P2X channels.

Stretch-sensitive nonselective cation channels. Mechanical stretch can also activate Ca²⁺-permeable ion channels in smooth muscles. For example, in voltage-clamped urinary bladder cells, longitudinal stretch activated an inward current due to a Gd³⁺-sensitive, nonselective cation conductance (115). In cells from mesenteric resistance arteries, cell inflation generated an inwardly rectifying, nonselective cation conductance (95) that was permeable to Ca²⁺ and blocked by Gd³⁺.

Capacitative Ca²+ entry. In many cells, depletion of internal Ca²⁺ stores is coupled to activation of a Ca²⁺ entry pathway (cf. Ref. 91). This is known as store-operated Ca²⁺ entry or capacitative Ca²⁺ entry (CCE). Drugs that deplete stores without activating G-protein-coupled receptors have been used in investigations of CCE because this technique makes it easier to distinguish CCE from receptor-operated Ca²⁺ influx. In the presence of L-type Ca²⁺ channel blockers, depletion of Ca²⁺ stores with thapsigargin activated a sustained Ca²⁺ influx independent of IP₃-dependent Ca²⁺ release (119). Other studies have shown that depletion of stores with sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) pump inhibitors caused DHP-insensitive enhanced tone or increased [Ca²⁺]_i, and these effects were due to Ca²⁺ influx. How store depletion activates a conductance in the plasma membrane is unclear, but this process may involve a diffusible factor or some direct interaction between proteins in the sarcoplasmic reticulum (SR) and plasma membranes.

	INTRACELLULAR CA2+ UPTAKE MECHANISMS

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Sarcoplasmic reticulum. Storage of Ca²⁺ in cellular organelles also provides important physiological regulation and the potential for release of Ca²⁺ during physiological signaling (Fig. 1). The main storage compartment is the SR, and this organelle has a major role in maintaining low [Ca²⁺]_i. The volume of SR appears to vary between smooth muscles, but, in general, the SR forms an extensive intracellular network that is capable of Ca²⁺ uptake, storage, and specialized release. SR volume is estimated to be 1.5-7.5% of smooth muscle cell volume. SR is typically more abundant in tonic (e.g., aorta) than phasic (e.g., portal vein) smooth muscle. Much of the surface of SR in smooth muscles is closely associated with the plasma membrane (27), such that release of Ca²⁺ can greatly influence the concentration of Ca²⁺ near the inner surface of the plasma membrane. This organization has profound consequences for Ca²⁺ signaling (see below in Ca²⁺ sparks).

Mitochondria in Ca²+ uptake. Evidence from a variety of cell types suggests that mitochondria play an important role in Ca²⁺ homeostasis. Mitochondria develop quite negative membrane potentials by extrusion of protons via the electron transport chain. This creates a strong electrochemical gradient for Ca²⁺ entry, and a Ca²⁺ conductance in the inner membrane of mitochondria, the Ca²⁺ uniporter, facilitates the uptake of Ca²⁺. In voltage-clamped gastric smooth muscle cells, the rate of Ca²⁺ extrusion after Ca²⁺ loading by voltage-dependent mechanisms was reduced by 50% after treatment with inhibitors of mitochondrial Ca²⁺ uptake (28). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore that collapses the electrochemical gradient for Ca²⁺ uptake, prolonged the decay of Ca²⁺-activated Cl currents in portal vein myocytes that were activated by Ca²⁺ entry through voltage-dependent Ca²⁺ channels (41). Decay of Ca²⁺ transients was also prolonged in rat femoral artery cells by CCCP (61). These authors suggested that mitochondrial Ca²⁺ uptake may be most important when [Ca²⁺]_i levels are high and SERCA pumps may be more important when [Ca²⁺]_i levels are in a low range. Thus the duration of Ca²⁺ transients initiated by voltage-dependent mechanisms in smooth muscles appears to be reduced by mitochondrial Ca²⁺ uptake. Part of the effects of protonophores may be mediated by cell acidification. When pH buffering was increased in guinea pig urinary bladder cells, the effects of CCCP on slowing the decay of Ca²⁺ transients was greatly reduced (35). Thus intracellular pH may also be an important factor in regulating Ca²⁺ handling in smooth muscle cells.

	INTRACELLULAR CA2+ RELEASE MECHANISMS

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Ryanodine receptors. One of the channels that release Ca²⁺ from the SR binds the plant alkaloid, ryanodine, and is most commonly referred to as the RyR. Cytoplasmic Ca²⁺ activates RyR channels, and thus they are also referred to in the literature as Ca²⁺-induced Ca²⁺ release (CICR) channels. This term is less specific because the second type of Ca²⁺ release channel, IP₃ receptors (see below), can also produce CICR, but only in the presence of IP₃. At least three isoforms of RyRs have been cloned (RyR1-RyR3). RyR2 and RyR3 are the primary isoforms in smooth muscle cells. RyR2 channels are formed by four monomers, each of nearly 5,000 amino acids and weighing ~565 kDa (84). These channels are activated by caffeine and locked into a subconductance state by ryanodine (47). This explains the effectiveness of these compounds in emptying Ca²⁺ stores. Ruthenium red blocks RyRs.

IP₃ receptors. Stimulation by a variety of agonists binding to G-protein-coupled receptors in smooth muscles results in activation of phospholipase C and metabolism of phosphatidylinositol phosphate to IP₃. IP₃ activates Ca²⁺ release via a second class of Ca²⁺ release channels, known as IP₃ receptors. Three genes encode IP₃ receptors, and each channel is made of up of four subunits of ~300 kDa that form homotetrameric or heterotetrameric channels (87).

	CA2+ EXTRUSION MECHANISMS

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Plasma membrane Ca²+-ATPase. To offset the influx of Ca²⁺ during excitable events, cells need mechanisms to remove Ca²⁺ to restore Ca²⁺ homeostasis. A major mechanism for Ca²⁺ extrusion is the plasma membrane Ca²⁺-ATPase (PMCA), which uses energy from ATP to pump Ca²⁺ up the steep electrochemical gradient from cytosol to extracellular space. This pump is thought to be electron neutral because the Ca²⁺ pumped to the extracellular space is exchanged for two protons. Thus Ca²⁺ extrusion results in uptake of H⁺, and this has to be compensated for by transporters such as Na⁺/H⁺ exchange. There are no known specific inhibitors of PMCA, but nonspecific P-type transporter inhibitors, such as lantanides and vanadate, can inhibit PMCAs (19).

Na+/Ca⁺ exchange. In addition to active Ca²⁺ extrusion, some smooth muscles may rely on Na⁺/Ca²⁺ exchange as a means of rapid Ca²⁺ extrusion. Ca²⁺ extrusion by this mechanism utilizes energy from the electrochemical gradient for Na²⁺ and transports three Na²⁺ into the cell while removing one Ca²⁺. There is some controversy about the relative contribution of Na⁺/Ca²⁺ exchange in smooth muscles (see Ref. 63). Generally, the test for Na⁺/Ca²⁺ exchange is to determine whether smooth muscle accumulates Ca²⁺ in the presence of a reduced Na⁺ gradient; however, there are problems with this approach, such as the ability of the SR to capture much of the accumulated Ca²⁺. Recovery from elevated [Ca²⁺]_i in voltage-clamped myocytes from the guinea pig ureter was not seriously affected when the Na⁺ gradient was decreased by 25-50%, and these authors concluded that Na⁺-independent Ca²⁺ extrusion is mainly responsible for regulating [Ca²⁺]_i under the conditions of their experiments (1). In contrast, when toad gastric muscles were voltage clamped with a protocol designed to cause Ca²⁺ accumulation, clear evidence was obtained for Na⁺-dependent extrusion of Ca²⁺ (78). This became the dominant means of extrusion when [Ca²⁺]_i exceeded 400 nM. Recently, mice deficient in Na⁺/Ca²⁺ exchanger (NCX1) were shown to have markedly impaired tension development in aortic muscles in response to Na⁺-free solutions, suggesting a role for Na⁺/Ca²⁺ exchangers in Ca²⁺ handling in the aorta (109). In reviewing the literature, it is reasonable to conclude that the relative contribution of Na⁺/Ca²⁺ exchange to Ca²⁺ extrusion varies between preparations, and very careful experiments may be necessary to observe the contributions from this mechanism in some smooth muscle cells.

	INTEGRATED CA2+ SIGNALING

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Superficial buffer barrier. The proximity of the SR to the plasma membrane and the existence of Ca²⁺ entry mechanisms in the plasma membrane and uptake mechanisms in the SR provide the structure for what has been termed the superficial buffer barrier. This concept suggests that a significant portion of the Ca²⁺ entering cells may be taken up into the SR to "buffer" transmembrane Ca²⁺ signals (Fig. 1). Unloading of Ca²⁺ may also be preferentially directed at the plasma membrane to ensure efficient extrusion. One of the initial observations suggesting a superficial buffer barrier was the finding that, in some smooth muscles, the rate, more than the magnitude, of Ca²⁺ entry was important in determining contractile force (104). In accordance with this idea, it was found that preloading the SR increased the transduction of Ca²⁺ entry to contraction, and unloading the SR delayed the development of force when Ca²⁺ entry was initiated. Recent studies of canine airway smooth muscle cells confirmed these observations and showed that contractions induced by KCl were enhanced when the SR store was inactivated with CPA or with ryanodine or by overfilling (59). When the filled state of the SR was reduced, KCl contractions were reduced. Another feature of the superficial buffer barrier is that agonists that tend to increase Ca²⁺ release from IP₃ receptors effectively short-circuit the uptake in the SR and enhance contraction in this manner (104).

Ca²+ sparks. Release of Ca²⁺ from RyR can result in highly localized, transient increases in Ca²⁺ concentration (Fig. 1). These events have been referred to as Ca²⁺ sparks (58, 82). Ca²⁺ sparks can result in very high local increases in Ca²⁺ (i.e., estimated to be at least 10 µM close to the site of release; see Ref. 88), and the proximity of RyR in the SR to the plasma membrane creates significant transient elevations of Ca²⁺ near the plasma membrane where numerous important Ca²⁺-dependent proteins, including Ca²⁺-dependent ion conductances, are located. Coupling of Ca²⁺ sparks to activation of Ca²⁺-dependent conductances leads to transient changes in transmembrane ionic currents, but, in an intact tissues, where cells are electrically coupled, periodic Ca²⁺ sparks and transient currents may sum to affect the global conductance of the tissue. If there is a predominance of coupling between Ca²⁺ sparks to K⁺ currents (e.g., via large-conductance Ca²⁺-activated K⁺ channels or "BK" channels), then the syncytial effect of Ca²⁺ sparks will be net outward current and hyperpolarization. With this design, mechanisms that enhance Ca²⁺ spark frequency or amplitude will tend to increase outward current and provide negative feedback to depolarization. It is also possible for Ca²⁺ sparks to couple to channels that generate inward currents (e.g., Ca²⁺-activated Cl channels) and produce depolarization (120). Different smooth muscles utilize these mechanisms in a variety of ways, and other papers within this highlighted topic series of short reviews will discuss these specific mechanisms in more detail.

Ca²+ puffs. Localized Ca²⁺ transients in some smooth muscle cells are not blocked by ryanodine. In a study of murine colonic myocytes, Ca²⁺ transients were reduced in magnitude and frequency by xestospongin C, a blocker of IP₃ receptors (4). Thus it is more appropriate to refer to these events as "Ca²⁺ puffs" (Fig. 1). Ca²⁺ release via IP₃ receptors may be an important means of coupling between G-protein-regulated receptors and Ca²⁺-dependent ionic conductances in the plasma membrane. In support of this idea, it was shown that stimulating cells with the P2Y receptor agonist 2-methylthio-ATP (2-MeS-ATP) increased the incidence of Ca²⁺ puffs in colonic myocytes. Secondary support of the idea that Ca²⁺ transients were due to IP₃-dependent release came from the observation that spontaneous Ca²⁺ transients and the effects of 2-MeS-ATP were blocked by U-73122, an inhibitor of PLC.

	SUMMARY AND CONCLUSIONS

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In summary, Ca²⁺ homeostasis in smooth muscles is complicated and dependent on many cellular proteins and specialized compartments (Fig. 1). So important are these mechanisms in regulating [Ca²⁺]_i and the contractile state of muscles that minor defects in function can greatly affect the mechanical activity of smooth muscle organs. With what is already known about basic mechanisms that regulate Ca²⁺ transport proteins, we are beginning to understand how defects in these mechanisms contribute to pathophysiological conditions. In the near future with genetic analyses and experiments on transgenic animals, it should be possible to determine the defects in Ca²⁺ homeostasis mechanisms in a wider variety of pathphysiological conditions.