HIGHLIGHTED TOPICS
Signal Transduction in Smooth Muscle
Invited Review: Mechanisms of calcium handling in smooth muscles

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557

	ABSTRACT

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The concentration of cytoplasmic Ca²⁺ regulates the contractile state of smooth muscle cells and tissues. Elevations in global cytoplasmic Ca²⁺ resulting in contraction are accomplished by Ca²⁺ entry and release from intracellular stores. Pathways for Ca²⁺ entry include dihydropyridine-sensitive and -insensitive Ca²⁺ channels and receptor and store-operated nonselective channels permeable to Ca²⁺. Intracellular release from the sarcoplasmic reticulum (SR) is accomplished by ryanodine and inositol trisphosphate receptors. The impact of Ca²⁺ entry and release on cytoplasmic concentration is modulated by Ca²⁺ reuptake into the SR, uptake into mitochondria, and extrusion into the extracellular solution. Highly localized Ca²⁺ transients (i.e., sparks and puffs) regulate ionic conductances in the plasma membrane, which can provide feedback to cell excitability and affect Ca²⁺ entry. This short review describes the major transport mechanisms and compartments that are utilized for Ca²⁺ handling in smooth muscles.

calcium channel; ryanodine receptor; inositol trisphosphate receptor; calcium sparks; capacitative calcium entry

	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.

DHP-sensitive Ca²⁺ channels are activated by depolarization of the plasma membrane, and there is a presumed voltage sensor in the S₄ domain of the -subunit, as in other voltage-gated channels (21). In some smooth muscles, depolarization from extracellular stimuli, such as neurotransmitters, activates DHP-sensitive Ca²⁺ channels; if threshold is reached, a Ca²⁺ action potential is generated. An action potential brings substantial Ca²⁺ into cells and elicits strong contractions. In many cases, however, activation of delayed rectifier K⁺ channels, which have activation kinetics similar to the Ca²⁺ channels, impedes the generation of action potentials, and Ca²⁺ channels are activated in a more sustained manner (i.e., the channels maintain a low, but significant, open probability as long as the depolarization is maintained). Depolarization also results in inactivation that slightly lags the activation phase. Inactivation is both voltage and Ca²⁺ dependent (37, 107). The latter is conveyed by intracellular Ca²⁺ (probably to a large extent by the Ca²⁺ that enters cells through the channel). The voltage dependence of activation and inactivation is such that inactivation is incomplete through a range of potentials in which significant activation occurs (i.e., approximately 60 to 20 mV). Thus, at some voltages, DHP-sensitive Ca²⁺ channels are capable of sustained openings and sustained inward current. The voltage range in which this occurs is known as "window current" (24).

The magnitude of sustained Ca²⁺ current in the range of window current is small, but the amount of Ca²⁺ influx relative to cell volume is significant. DHP-sensitive Ca²⁺ channels have a high rate of Ca²⁺ permeation (38, 94). Integration of the inward current during step depolarization within the window current range showed that depolarization in the range of 40 to 20 mV increased cytoplasmic Ca²⁺ in colonic muscle cells by tens of micromolars (31, 106). With the assumption of 100-fold buffering (62), the increase in [Ca²⁺]_i is sufficient to elicit contraction (6, 106). Tonic smooth muscles with membrane potentials within the window current range have constant influx in Ca²⁺ by this pathway, and small voltage changes are capable of significantly altering [Ca²⁺]_i (31). Ca²⁺ influx through DHP-sensitive Ca²⁺ channels explains to a significant degree the coupling between changes in membrane potential and contraction and explains the steep relationship between voltage and force in smooth muscles (79, 85).

Regulating Ca²⁺ influx through DHP-sensitive Ca²⁺ channels is an important means of controlling the contractile state of smooth muscles. Some vasodilators, such as nitric oxide, working through cGMP and protein kinase G, directly regulate the open-probability DHP-sensitive Ca²⁺ channels (cf. Ref. 23). However, in many smooth muscles, these channels are not the primary target for regulation by agonists or second messengers. In many cases, alterations in Ca²⁺ influx are regulated by voltage, and changes are mediated by activation of subsidiary conductances. For example, K⁺ channels are activated to produce outward current, hyperpolarize membrane potential, and reduce Ca²⁺ influx or nonselective cation channels or Cl channels are opened to generate inward current, depolarize membrane potential, and increase Ca²⁺ influx.

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).

ACh, acting via muscarinic receptors, activates a nonselective cation current (I_ACh) in vascular and visceral smooth muscles (e.g., Refs. 8, 32, 51-54, 60, 73; Fig. 1). At the negative potentials of smooth muscle cells, most of the current through this conductance is carried by Na⁺, and the inward Na⁺ current is responsible for a significant part of the depolarization caused by muscarinic stimulation. I_ACh is voltage dependent in many cells, and the current reverses near 0 mV, demonstrating its nonselectivity. I_ACh is not directly activated but facilitated by intracellular Ca²⁺ (52, 86, 97, 110). Activation of I_ACh is blocked by pertussis toxin, and the current can be directly activated by dialysis of guanosine 5'-O-(3-thiotriphosphate) (53), demonstrating the role of a G-protein-dependent mechanism. Antibodies to the -subunit of G_i or G_o were also shown to block activation by ACh (110). I_ACh may be opened by ACh binding of M₂ receptors working through G_i/G_o and facilitated via M₃ receptors that are coupled to phospholipase C (PLC), D-myo-inositol 1,4,5-trisphosphate (IP₃) production, and Ca²⁺ release (14). The single-channel conductance of I_ACh appears to be 20-30 pS (55, 64, 108). Several ions and drugs block I_ACh (including Gd³⁺, Ni²⁺, Cd²⁺, quinine and fenamates), but specific blockers have not been identified.

I_ACh is permeable to Ca²⁺, but there is controversy over whether this conductance is a significant direct source for Ca²⁺ entry (see Ref. 86). I_ACh conducts Ca²⁺, and Inoue and Isenberg (51) showed that the current was of equal magnitude when external Na⁺ was replaced with Ca²⁺. The question remains, however, as to what extent the channels conduct Ca²⁺ in physiological ionic gradients. Some investigators argue that I_ACh provides enough Ca²⁺ influx to affect [Ca²⁺]_i, (cf. Refs. 32, 64). Such a conclusion is supported by the following observation: a rapid reduction in extracellular Ca²⁺ while I_ACh is activated immediately decreases [Ca²⁺]_i and a rapid increase in extracellular Ca²⁺ increases [Ca²⁺]_i. In addition, rapid application of a blocker of I_ACh, such as Ni²⁺, also immediately decreases [Ca²⁺]_i. Fleischmann and co-workers (32) calculated that up to 14% of I_ACh is carried by Ca²⁺ in airway smooth muscle cells.

Other agonists, such as adrenergic agents and peptides, also activate nonselective cation conductances in smooth muscle cells (cf. Refs. 70, 81, 111). These currents are similar but not identical to I_ACh. A major difference is that these conductances are not, in general, facilitated by intracellular Ca²⁺, suggesting they are due to species of ion channels different from I_ACh.

The molecular entities responsible for nonselective cation conductances in smooth muscles have not yet been identified; however, a recent study offers possible insights into the molecular nature of these channels. Inoue and co-workers (56) showed that expression of a transient receptor potential protein (TRP6) in HEK293 cells resulted in a current with biophysical and pharmacological properties similar to the nonselective cation current activated by adrenergic stimuli in portal vein cells. Treatment of cultured portal vein myocytes with TRP6 antisense oligonucleotides inhibited immunoreactivity to TRP6 antibodies and reduced the nonselective cation conductance activated by adrenergic stimulation.

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.

Most data suggesting the existence of CCE are from studies in which cells or tissues were loaded with fluorescent Ca²⁺ indicators to assay the end result of CCE-increased cytoplasmic Ca²⁺. If Ca²⁺ enters smooth muscle cells during this process, it should generate an inward current (Fig. 1). It has been far more difficult to measure this current; however, there are reports of inward currents resulting from pharmacological depletion of Ca²⁺ stores.

Freshly dispersed cells from the mouse anococcygeus were studied with the whole cell configuration of the patch-clamp technique, and membrane currents induced by cyclopiazonic acid (CPA) were characterized (113). After voltage-dependent Ca²⁺ currents and K⁺ currents were blocked, CPA activated two components of inward current. The first component, which was transient, was a Ca²⁺-activated Cl current. The second, sustained component had a nearly linear current-voltage relationship with a reversal potential of +31 mV. When extracellular Ca²⁺ was removed, the reversal potential shifted to +18 mV. The authors determined that this current was due to a nonselective cation conductance. Treating cells with caffeine generated a similar current. The CPA-induced nonselective cation was blocked by Cd²⁺ (100 µM) and SKF-96365 (10 µM) but not by La³⁺. In similar experiments, currents were measured while changes in cytosolic Ca²⁺ were monitored with fura 2 (114). The sustained current noted previously was associated with increased [Ca²⁺]_i. Both the current and the change in [Ca²⁺]_i were blocked by Cd²⁺ and SKF-96365, suggesting that the nonselective cation current was responsible for CCE in mouse anococcygeus cells.

Other studies have reported a conductance in vascular smooth muscle cells that is activated by a diffusible factor (Ca²⁺ influx factor) produced by yeast and human platelets (100). Application of thapsigargin activated 3-pS cation channels in cell-attached membrane patches (101). The same channels were activated when cells were loaded with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid to deplete stores without raising intracellular Ca²⁺. The 3-pS channels were shown to be cation channels and nonselective for Ca²⁺, Sr²⁺, Ba²⁺, Na⁺, K⁺, and Cs⁺. The authors concluded that this conductance might be responsible for CCE in vascular smooth muscle cells.

A recent report has proposed that the molecular entity responsible for CCE may be encoded by trp genes (118). Transcripts of trp1 were expressed in smooth muscle cells of resistance arterioles, arteries, and veins. Antibodies specific for TrpC1, a gene product of trp1, showed expression of TrpC1 protein in vascular smooth muscle cells and found the protein localized in the plasma membrane. Peptide-specific binding of the antibody blocked store-operated Ca²⁺ channel activity.

	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).

The SR is surrounded by a membrane that is not freely permeable to Ca²⁺. Specialized, active Ca²⁺-ATPases, known as SERCA pumps, exist in the SR membrane; these pumps generate and maintain about a 10,000-fold Ca²⁺ gradient between the SR lumen and the cytoplasm. Three genes encode SERCA pumps, and two subgroups of SERCA2 (SERCA2a and SERCA2b) have been identified. Most smooth muscles express SERCA2b (115 kDa) and SERCA 3 (105 kDa) (116). SERCA pumps utilize the energy from ATP hydrolysis to translocate Ca²⁺ from the cytoplasm to the lumen of the SR. After Ca²⁺ is pumped into the SR, it is buffered by proteins, such as calreticulin and calsequestrin. These proteins can bind large amounts of Ca²⁺. As a result of high-affinity Ca²⁺ uptake and intraluminal SR buffering, the actual Ca²⁺ store is estimated to reach Ca²⁺ concentrations of 10-15 mM (105).

SERCA pumps are regulated by phospholamban, a small transmembrane protein (52 amino acids) that assembles as a 6-kDa homopentamer (2). Regulation of SERCA pumps occurs through an inhibitory association between phospholamban and the Ca²⁺-ATPase that can be relieved by phosphorylation with either protein kinase A or G (92). Enhancing Ca²⁺ uptake tends to reduce basal levels of Ca²⁺ and shorten Ca²⁺ transients initiated by depolarization and/or agonist stimulation. Thus phosphorylation of phospholamban may be one of the ways in which agonists that enhance production of cAMP and cGMP produce net inhibitory effects.

Studies of the function of SERCA pumps have been strongly aided by specific SERCA pump inhibitors, such as thapsigargin and CPA (see review, Ref. 68). When SERCA pumps are inhibited, a major source of Ca²⁺ regulation is lost, Ca²⁺ leaks into the cytoplasm, and cells are unable to maintain typically low cytoplasmic concentrations. Uptake of Ca²⁺ after Ca²⁺ transients is also compromised, extending periods of contraction. For example, in guinea pig urinary bladder smooth muscle, CPA slowed recovery of basal Ca²⁺ levels after a depolarization-induced Ca²⁺ transient by a factor of four (35), thus demonstrating the importance of SERCA pumps in the process of relaxation.

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.

High-resolution imaging of HeLa cells with specifically targeted green fluorescent proteins have shown very close associations between endoplasmic reticulum and mitochondria (93). Thus, when IP₃-dependent release channels are opened, mitochondria are exposed to much higher local Ca²⁺ concentrations than reached during global Ca²⁺ transients. It is possible that a similar close relationship between SR and mitochondria exists in smooth muscle cells. Release of Ca²⁺ from the SR with caffeine also stimulated Ca²⁺ uptake into mitochondria, as shown by changes in rhod 2 fluorescence (a mitochondrial Ca²⁺ indicator) in toad gastric muscle cells (29). A close functional relationship between SR and mitochondria has also been suggested in experiments on aortic smooth muscle cells by showing that mitochondrial Ca²⁺ increased along with cytoplasmic Ca²⁺ when cells were stimulated with either phenylephrine (release from IP₃ receptors) or caffeine [release from ryanodine receptors (RyRs)] (43). However, mitochondrial Ca²⁺ transients were delayed and prolonged compared with cytoplasmic Ca²⁺ transients. Others have found that mitochondrial Ca²⁺ uptake affects Ca²⁺ transients initiated by IP₃-dependent (i.e., receptor-mediated) Ca²⁺ release (77). These authors suggested that, after IP₃-dependent release of Ca²⁺, mitochondrial Ca²⁺ uptake may regulate the Ca²⁺ concentration near IP₃ receptors and thus preserve the sensitivity of IP₃ receptors for subsequent Ca²⁺ release. A recent study has also suggested that mitochondrial Ca²⁺ uptake following Ca²⁺ release from IP₃ receptors is essential for pacemaker activity in interstitial cells of Cajal, the cells that provide electrical pacemaker activity in gastrointestinal muscles (112). More investigation is needed to fully appreciate the role of mitochondria in modulating Ca²⁺ transients 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.

Micromolar concentrations of cytoplasmic Ca²⁺ are the primary activator of RyR channels in smooth muscles (30, 48). The amount of Ca²⁺ necessary to initiate CICR in smooth muscles (>1 µM) may be much higher than experienced by smooth muscle cells during peak excitability. Therefore, the physiological significance of CICR was questioned. Voltage-clamp experiments on urinary bladder smooth muscle (36) and portal vein (42) demonstrated that Ca²⁺ entry can trigger Ca²⁺ release via RyRs. However, others have reached opposite conclusions about the importance of CICR in smooth muscles. For example, Kamishima and McCarron (62) were unable to demonstrate CICR in portal vein myocytes; similar findings were obtained in studies of tracheal myocytes (33). Recent studies have shown that Ca²⁺ entry through DHP-sensitive Ca²⁺ channels can activate CICR in smooth muscle cells of urinary bladder and couple to the occurrence of Ca²⁺ sparks and Ca²⁺ waves, but the coupling is loose (25). DHP-sensitive Ca²⁺ channels can open without initiating Ca²⁺ release, and Ca²⁺ sparks were observed after DHP-sensitive Ca²⁺ channels closed. Thus the amount of Ca²⁺ entering through DHP-sensitive Ca²⁺ channels was typically insufficient to initiate CICR, or the spatial organization between RyR and DHP-sensitive Ca²⁺ channels was such that Ca²⁺ entry did not necessarily achieve CICR. The physiological importance of this mechanism is likely to be limited to specific smooth muscles that have high-current densities through DHP-sensitive Ca²⁺ channels and appropriate spatial associations with RyR channels.

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).

Activation of IP₃ receptors by its ligand is regulated by cytoplasmic Ca²⁺, and there is a biphasic relationship between the open probability of IP₃ channels and Ca²⁺ release (11, 49, 74). A rise in [Ca²⁺]_i from basal levels to ~300 nM increases the potency of IP₃ in activating channel openings, but higher concentrations reduce the effectiveness of IP₃. Thus high levels of [Ca²⁺]_i provide negative feedback for the release of more Ca²⁺. The potentiating effects of <300 nM Ca²⁺ on open probability of IP₃ channels provides a mechanism for CICR via IP₃ receptors. Potentiation of openings of both IP₃ receptor channels and RyR channels provides the possibility of interactions between Ca²⁺ release mechanisms. If these channels are located close to each other in the SR membrane, then it is possible for release of Ca²⁺ from one to stimulate release from the other. This type of interaction tends to be amplified by agonists that enhance IP₃ levels, and under some conditions can lead to regenerative Ca²⁺ waves (see Ref. 50).

Studies of the role of IP₃ receptors in smooth muscle were complicated for many years by the lack of specific, cell-permeable antagonists. Heparin, a nonpermeable and relatively nonselective antagonist, was the main agent used, but it had to be introduced into cells with patch pipettes or through cell permeabilization. Others have used IP₃-receptor antibodies that specifically block channel activation (67, 98); however, these also proved to be impermeable. Membrane-permeable compounds, such as xestospongin C (34) and 2-aminoethoxydiphenyl borate (2-APB; Ref. 76), have been shown to block IP₃-dependent Ca²⁺ release. These agents are potentially useful for investigations of IP₃-receptor-dependent Ca²⁺ signaling. Xestospongins have some efficacy in blocking RyRs; however, these compounds are 30% less potent in this action than they are in blocking IP₃ receptors. 2-APB has no known effects on RyRs; however, at concentrations >90 µM, it causes Ca²⁺ release and elevation in [Ca²⁺]_i.

	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).

PMCAs are the products of at least four genes, and the isoforms 1 and 4 are widely expressed (20). PMCA1b is the most common and has a molecular mass of ~140 kDa. PMCAs are activated by binding of calmodulin to the COOH-terminal end. This removes autoinhibition and increases the affinity for Ca²⁺ and the transport rate (75). PMCAs are also regulated by protein kinases, and phosphorylation of sites near the calmodulin binding site by protein kinases A and G or by Ca²⁺/calmodulin kinase reduces autoinhibition and facilitates Ca²⁺ transport (117). For example, stimulation of cultured vascular smooth muscle cells with nitroglycerin caused enhanced Ca²⁺ extrusion (65).

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

TOP ABSTRACT INTRODUCTION CA2+ ENTRY MECHANISMS INTRACELLULAR CA2+ UPTAKE... INTRACELLULAR CA2+ RELEASE... CA2+ EXTRUSION MECHANISMS INTEGRATED CA2+ SIGNALING SUMMARY AND CONCLUSIONS REFERENCES

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.

The first evidence for the role of Ca²⁺ sparks in regulating plasma membrane ionic conductances came from the observation that voltage-clamped smooth muscle cells held at depolarized potentials (i.e., 40 to 10 mV) generated large spontaneous transients outward currents (STOCs; Refs. 7, 13). Benham and Bolton (7) found that STOCs were due to the periodic activation of many BK channels and found that, when Ca²⁺ stores were depleted by caffeine or agonists, STOCs ceased until the stores were reloaded. At the time, microfluorometry techniques were not sensitive enough to detect the localized Ca²⁺ transients that underlie STOCs. Application of confocal microscopy and the use of fluorescent Ca²⁺ binding molecules with high quantum yield (e.g., fluo 3) during the 1990s provided the resolution needed to detect Ca²⁺ sparks in smooth muscle. Utilization of these techniques have demonstrated Ca²⁺ sparks in a variety of smooth muscle cells (e.g., Refs. 40, 82, 88, 96, 120) and intact tissues (e.g., pressurized cerebral arteries; Ref. 58). Ca²⁺ sparks appear to be the result of a cluster of RyRs releasing Ca²⁺ at nearly the same time. Ryanodine (by blocking Ca²⁺ release) and thapsigargin (by unloading Ca²⁺ stores) inhibit Ca²⁺ sparks and the openings of BK channels that result from sparks. BK channels activated by Ca²⁺ sparks cause hyperpolarization and dilate pressurized arteries. Ryanodine and thapsigargin depolarize and constrict arteries, similar to blockers of BK channels. Thus, in vascular tissues that utilize this mechanism, Ca²⁺ sparks indirectly produce vasodilation via openings of BK channels.

The actual release of Ca²⁺ from a given spark site is significant and has been estimated to be due to a Ca²⁺ current of 4 pA of ~10-ms duration (22). This exceeds the amount of current due to a single RyR channel and suggests cooperativity between RyRs, possibly due to CICR. It is possible that Ca²⁺ from a single channel stimulates release from other closely clustered channels. In arterial muscle cells, Ca²⁺ sparks have a rise time of ~20 ms and a decay half-time of 50-60 ms. These events are highly localized and have a spatial spread of only ~2.4 µm at the point of half amplitude. Because RyRs are spatially close to the plasma membrane, relatively large changes in local Ca²⁺ result. The amplitude of sparks and the coupling between sparks and Ca²⁺-dependent proteins are of critical importance to the physiological consequence of this phenomenon. Regulation of the frequency and amplitude of Ca²⁺ sparks may be an important means of coupling receptor activation to electrical responses. Studies have shown that second-messenger-coupled mechanisms regulate Ca²⁺ sparks. For example, Ca²⁺ sparks recorded from rat coronary and cerebral arteriole myocytes were increased in frequency by cAMP-dependent mechanisms (90) and reduced by protein kinase C-dependent mechanisms (15). The changes in the frequency of sparks may have been modulated by altering Ca²⁺ uptake into the SR or by affecting the Ca²⁺ sensitivity of RyR. Ca²⁺ sparks in smooth muscles may regulate many cellular processes in addition to membrane conductance. Future studies will greatly expand our knowledge of this aspect of Ca²⁺ handling and additional cellular events, such as cell differentiation, proliferation, and gene expression (39).

Some authors have suggested that triggering of Ca²⁺ sparks is coupled to specific targeting of activator Ca²⁺ through DHP-sensitive Ca²⁺ channels to RyR (3, 71). This requires alignment of Ca²⁺ channels in the plasma membrane with RyR in the SR. Caveolae contain DHP-sensitive Ca²⁺ channels, and it was found that disruption of caveolae with methyl-ss-cyclodextrin (dextrin) reduced the amplitude, frequency, and spatial spread of Ca²⁺ sparks (71). These data suggest that Ca²⁺ sparks may be generated in a microdomain containing both caveolae and SR, and Ca²⁺ entry through the plasma membrane L-type Ca²⁺ channels may initiate Ca²⁺ release from a cluster of RyR.

The importance of coupling between Ca²⁺ sparks and BK channels has been demonstrated with transgenic mice. BK channels are composed of pore-forming - and regulatory ₁-subunits. The ₁-subunit increases the Ca²⁺ and voltage sensitivity of BK channels. Targeted deletion of ₁-subunits resulted in animals with elevated arterial blood pressure (17, 89). Studies on dispersed cells showed that the frequency and amplitude of Ca²⁺ sparks were unaffected in these animals; however, the coupling between Ca²⁺ sparks and BK channels was shown to be greatly diminished. Relative absence of STOCs resulting from the breakdown in coupling between Ca²⁺ sparks and BK coupling would tend to produce more depolarized cells and greater basal activation of voltage-dependent Ca²⁺ entry. Thus the defect in Ca²⁺ spark to BK coupling predisposed these animals to a greater degree of vasoconstriction and hypertension.

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.

It was also shown that, when Ca²⁺ release from IP₃ receptors was stimulated with 2-MeS-ATP, the localized Ca²⁺ puffs had a tendency to develop into Ca²⁺ waves, which spread locally or in some cases throughout the cells. The development of waves depended on recruitment of Ca²⁺ release from RyR, suggesting cooperation between these two release mechanisms for agonist responses. As discussed previously, because both IP₃ receptors and RyR are sensitive to cytoplasmic Ca²⁺, release of Ca²⁺ from one type of channel might increase the open probabilities of other channels nearby. Similar hypotheses have been put forward for stimulation of portal vein cells with norepinephrine (12) and of rat cerebral artery smooth muscle cells via UTP (57). Ca²⁺ waves in colonic myocytes may be restricted to a compartment near the plasma membrane because despite transcellular spread of waves contractions were not elicited. It was also found that IP₃-receptor-mediated puffs were coupled to both BK channels and small-conductance Ca²⁺-activated K⁺ channels (SK) in colonic myocytes. SK channels are known to be responsible for the hyperpolarization response due to release of ATP from enteric inhibitory motoneurons. Thus release of Ca²⁺ by G-protein-mediated activation of PLC can be linked to an inhibitory response in colonic cells via localized Ca²⁺ release and activation of Ca²⁺-activated K⁺ channels.

The finding that G-protein-dependent activation of PLC and subsequent activation of Ca²⁺ release is coupled to K⁺ channels seems contradictory to the well-described IP₃-dependent mechanism used by many excitatory agonists in smooth muscles. Thus the effects of ACh on Ca²⁺ transients were also examined because Ca²⁺ transients coupled to STOCs and hyperpolarization would tend to override the excitatory nature of cholinergic responses. In murine colonic smooth muscle cells, ACh reduced localized Ca²⁺ transients and STOCs (5). These effects were accompanied by a rise in [Ca²⁺]_i. The inhibitory effects of ACh on Ca²⁺ puffs were mimicked by nonreceptor-mediated increases in basal Ca²⁺ and blocked by inhibitors of nonselective cation conductances (e.g., Gd³⁺ and SKF-96365). When the rise in basal Ca²⁺ was blocked, ACh profoundly increased Ca²⁺ transients and promoted the generation of Ca²⁺ waves. These events were coupled to enhancement in STOCs. The results showed that the rise in [Ca²⁺]_i that accompanies muscarinic stimulation of colonic muscles inhibits localized Ca²⁺ transients that could undermine the excitatory effects of ACh by activating Ca²⁺-activated K⁺ channels. The inhibition of Ca²⁺ transients by increased [Ca²⁺]_i might be explained by the bell-shaped relationship between [Ca²⁺]_i and sensitivity of IP₃ receptors to IP₃ (Refs. 11, 49, 74; and see IP₃ receptors above).

	SUMMARY AND CONCLUSIONS

TOP ABSTRACT INTRODUCTION CA2+ ENTRY MECHANISMS INTRACELLULAR CA2+ UPTAKE... INTRACELLULAR CA2+ RELEASE... CA2+ EXTRUSION MECHANISMS INTEGRATED CA2+ SIGNALING SUMMARY AND CONCLUSIONS REFERENCES

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.

	ACKNOWLEDGEMENTS

Work on this review was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants PO1 DK-45569 and RO1 DK-40569.

	FOOTNOTES

Address for reprint requests and other correspondence: K. M. Sanders, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, NV 89511 (E-mail: kent{at}physio.unr.edu).

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TOP ABSTRACT INTRODUCTION CA2+ ENTRY MECHANISMS INTRACELLULAR CA2+ UPTAKE... INTRACELLULAR CA2+ RELEASE... CA2+ EXTRUSION MECHANISMS INTEGRATED CA2+ SIGNALING SUMMARY AND CONCLUSIONS REFERENCES

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