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INVITED REVIEW

Regulation of smooth muscle calcium sensitivity: KCl as a calcium-sensitizing stimulus

Departments of Biochemistry and Pediatrics, Virginia Commonwealth University, School of Medicine, Richmond, Virginia

	ABSTRACT

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

 
KCl has long been used as a convenient stimulus to bypass G protein-coupled receptors (GPCR) and activate smooth muscle by a highly reproducible and relatively "simple" mechanism involving activation of voltage-operated Ca²⁺ channels that leads to increases in cytosolic free Ca²⁺ ([Ca²⁺]_i), Ca²⁺-calmodulin-dependent myosin light chain (MLC) kinase activation, MLC phosphorylation and contraction. This KCl-induced stimulus-response coupling mechanism is a standard tool-set used in comparative studies to explore more complex mechanisms generated by activation of GPCRs. One area where this approach has been especially productive is in studies designed to understand Ca²⁺ sensitization, the relationship between [Ca²⁺]_i and force produced by GPCR agonists. Studies done in the late 1980s demonstrated that a unique relationship between stimulus-induced [Ca²⁺]_i and force does not exist: for a given increase in [Ca²⁺]_i, GPCR activation can produce greater force than KCl, and relaxant agents can produce the opposite effect to cause Ca²⁺ desensitization. Such changes in Ca²⁺ sensitivity are now known to involve multiple cell signaling strategies, including translocation of proteins from cytosol to plasma membrane, and activation of enzymes, including RhoA kinase and protein kinase C. However, recent studies show that KCl can also cause Ca²⁺ sensitization involving translocation and activation of RhoA kinase. Rather than complicating the Ca²⁺ sensitivity story, this surprising finding is already providing novel insights into mechanisms regulating Ca²⁺ sensitivity of smooth muscle contraction. KCl as a "simple" stimulus promises to remain a standard tool for smooth muscle cell physiologists, whose focus is to understand mechanisms regulating Ca²⁺ sensitivity.

K⁺ depolarization; cell signaling; signal transduction; contraction

Together, these and other studies provided the material to construct a model (84) showing that the degree of Ca²⁺ sensitivity is a regulated parameter; mechanisms shifting Ca²⁺ sensitivity to the left and right of a central curve generated in response to stimulation with KCl cause, respectively, Ca²⁺ sensitization and Ca²⁺ desensitization (Fig. 1A). What has emerged from research over the past 15 years is that Ca²⁺ sensitivity plays as important a role in regulation of smooth muscle contraction as does [Ca²⁺]_i (see Refs. 70, 91, 145, 170, 176, and 178 for reviews). Moreover, until recently, regulation of Ca²⁺ sensitivity has been attributed solely to Ca²⁺-independent cell signaling events. However, as with many cell signaling systems thought originally to reflect a direct cause-and-effect regulatory pathway and found later to involve cross-talk with other signaling systems, regulation of smooth muscle contraction by [Ca²⁺]_i and by Ca²⁺ sensitivity may not always be entirely separate systems, but may best be understood as part of an interacting spatiotemporal signaling network (150, 201, 202).

View larger version (26K): [in this window] [in a new window]   Fig. 1. A: the degrees of Ca2+ sensitization and Ca2+ desensitization produced by G protein coupled receptor (GPCR) agonists and relaxing agents, respectively, is often measured in relation to an assumed unique KCl-induced steady-state force cytosolic Ca2+ concentration ([Ca2+]i) curve. B: recent data indicate that KCl also can regulate the degree of Ca2+ sensitivity and that Ca2+ sensitivity produced by any stimulus is dependent on several factors, including stimulus concentration and the history of GPCR activation, suggesting a continuum of Ca2+ sensitivity curves.

	MECHANISMS REGULATING SMOOTH MUSCLE CA2+ SENSITIVITY

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

Studies done as early as 1984, with the use of intact smooth muscle tissues, showed that GPCR agonists can produce greater increases in force for a given increase in [Ca²⁺]_i than KCl (22, 32, 53, 66, 86, 127, 142, 158, 169). KCl-induced contraction has long been known to be due to membrane depolarization causing Ca²⁺ entry through voltage-operated Ca²⁺ channels (VOCCs) (52, 135, 205; see Refs. 20 and 24 for reviews), activation of Ca²⁺-dependent MLC kinase, and increases in MLC phosphorylation (38, 152). GPCR-induced contraction, on the other hand, is caused by activation of trimeric G proteins linked to G_q and G_12/13 (see Ref. 178 for review), leading to generation of multiple cell messengers, including inositol 1,4,5-trisphosphate, diacylglycerol, and the low molecular weight GTPase, RhoA (for a general review on RhoA and GPCRs, see Refs. 165, 171, and 208), activation of multiple Ca²⁺ channel types, and activation of at least two kinases, RhoA kinase (ROK) and protein kinase C (PKC), in addition to Ca²⁺-dependent MLC kinase (see Refs. 51, 120, 146, 168, and 176 for reviews). While the motor proteins responsible for generation of force reside in the cell interior, most of the upstream signals generated upon GPCR stimulation, including ROK and PKC, are regulated at the plasma membrane.

The smooth muscle plasma membrane can be divided into at least three domains: focal contacts, the dystroglycan complex, and caveolae. Caveolae are small 50–100 nm cavelike invaginations of the plasma membrane housing a unique lipid domain that contains high concentrations of cholesterol and sphingolipids (see Ref. 174 for review). The dystrophin complex and caveolae are complementary (i.e., spatially close), whereas caveolae and focal contacts are located in mutually exclusive areas that display an alternating, punctate pattern when viewed with the use of immunohistochemical fluorescence labeling of selected proteins (see Refs. 58, 138, 192, and Fig. 2). Several studies support the hypothesis that GPCR stimulation increases translocation of inactive cytosolic RhoA (bound to RhoA-GDI) to peripheral plasma membrane sites such as caveolae, where RhoA-GTP activates ROK (45, 56, 187), supporting a caveolar role in regulation of Ca²⁺ sensitization (for review, see Ref. 186). Moreover, the hypothesis that caveolae play an important role in GPCR-induced contraction is supported by data showing that disruption of the caveolar structure with the cholesterol-depleting agent, methyl--cyclodextrin, or by caveolin-1 knockout, reduces the ability of some GPCR stimuli, but not KCl, to produce contraction (36, 37, 78).

View larger version (57K): [in this window] [in a new window]   Fig. 2. Fluorescence confocal immunohistochemical three-dimensional image reconstruction (Volocity Software, Improvision, Lexington, MA) of a single vascular smooth muscle cell within an arterial tissue cut in cross-section and labeled with anti-talin (red) and anti-caveolin (green) antibodies to visualize, respectively, focal contacts and caveolin-containing domains (caveolae and dystroglycan complex) in the periphery of the cell. Note the alternating punctate staining with little to no colocalization (no yellow color).

The proposal that Ca²⁺ sensitization is caused primarily by inhibition of MLC phosphatase activity has gained widespread support (see Ref. 178 for a review, and for reviews on MLC phosphatase, see Refs. 59, 60, 75). The general model (Fig. 3; and reviewed by Ref. 177) is that GPCR stimulation reduces MLC phosphatase activity by phosphorylation of the MLC phosphatase regulatory subunit MYPT1 and the 17-kDa PKC-activated phosphatase inhibitor CPI-17 (39, 40, 43, 45, 59, 114, 116). Inhibitory activity of CPI-17 causing Ca²⁺ sensitization is activated by CPI-17-Thr38 phosphorylation (96). MLC phosphatase activity is also regulated independently of CPI-17 by MYPT1-Thr696 and MYPT1-Thr853 phosphorylation. MYPT1-Thr696 phosphorylation inhibits MLC phosphatase activity (42, 71) and MYPT1-Thr853 phosphorylation causes dissociation of MLC phosphatase from myosin, thereby effectively preventing MLC phosphatase from acting on its substrate, phospho-MLC (206). There is growing evidence that GPCR agonists that cause Ca²⁺ sensitization in smooth muscle act by increasing MYPT1-Thr853 phosphorylation more so than by increasing MYPT1-Thr696 phosphorylation (96,136). Several kinases, including ROK, can phosphorylate MYPT1 and CPI-17 (reviewed by Ref. 75, 178). Whereas GPCR activation can phosphorylate, KCl does not cause phosphorylation of CPI-17 in the tonic femoral artery or phasic vas deferens in the presence of -adrenergic receptor blockade (95, 96).

View larger version (76K): [in this window] [in a new window]   Fig. 3. Diagram of a general model depicting subcellular mechanisms by which contractile GPCR agonists, such as norepinephrine (NE), regulate [Ca2+]i and Ca2+ sensitivity, and thus myosin light chain (MLC) phosphorylation and force. Receptor activation elevates [Ca2+]i by causing Ca2+ entry through multiple plasma membrane (PM) channel types, and Ca2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors (activated by Ca2+) and inositol 1,4,5-trisphosphate (IP3) receptors. Ca2+ activates MLC kinase (MLCK) to increase MLC phosphorylation and contraction (shown as P on sidepolar thick filament moving actin cables). GPCR stimulation also generates other cell messengers in a (relatively) Ca2+-independent fashion, causing activation of kinases such as protein kinase C (PKC) and ROK (ROK*, activated Rho kinase) that inhibit MLC phosphatase (MLCP) activity. GPCR activation increases RhoA, ROK and PKC translocation to caveolin (blue hairpin-like structure within caveolar membrane invagination). Inhibition of MLCP activity by phosphorylation of the large molecular weight MLCP regulatory protein, MYPT1, or by phosphorylation of the MLCP inhibitory protein, CPI-17, produces a Ca2+-independent increase in the MLCK/MLCP activity ratio to further increase MLC phosphorylation and force (Ca2+ sensitization; note that MLCP signifies that MYPT1 phosphorylation by ROK reduces the ability of MLCP to dephosphorylate MLC-p). ROK may directly phosphorylate MYPT1 or may act through other kinases. PKC (and other kinases, including ROK) phosphorylate CPI-17 causing MLCP inhibition. Other modes of potential Ca2+ sensitization, including additional forms of regulation of MLCK activity and thin filament regulation, are not shown. VOCC, voltage-operated Ca2+ channel; SOCC, store-operated Ca2+ channel; ROCC, receptor-operated Ca2+ channel.

	ROK AND PKC PARTICIPATE IN ELEVATING CA2+ SENSITIVITY OF MANY SMOOTH MUSCLE TYPES IN RESPONSE TO GPCR STIMULATION

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

Y-27632 and a structurally unrelated ROK inhibitor, HA-1077, have proven invaluable in identification of the relative role that Ca²⁺ sensitization plays in regulation of contraction of smooth muscles of different organ systems. A recent study (31) documenting the specificity of 28 commercially available kinase inhibitors indicates that Y-27632 and HA-1077 are highly selective for inhibition of ROK when used at appropriate concentrations. Y-27632 and HA-1077 inhibit vascular (200), respiratory (27), ileal (185), stomach (156), bladder (79), and myometrial (106) smooth muscle contractions produced by GPCR stimulation. However, based on the degree to which these agents inhibit force, the extent to which ROK participates in contractions appears to differ in different smooth muscle types. For example, 10 µM Y-27632 modestly reduces spontaneous human myometrial contractions (106) yet abolishes tonic -adrenergic receptor-induced contraction of rabbit aorta (200). Muscarinic receptor stimulation induces a biphasic contraction of ileum, bladder, and chicken gizzard consisting of a phasic component (early peak response), followed by a tonic component that is weaker than the phasic component. In the ileum, ROK blockade by Y-27632 and HA-1077 leaves the phasic component nearly intact and abolishes the tonic component (185), whereas in the bladder, both components are greatly diminished (79). Although GTPS induces ROK activation and Ca²⁺ sensitization in chicken gizzard smooth muscle, muscarinic receptor stimulation does not cause ROK-dependent Ca²⁺ sensitization in this muscle type (6). However, Y-27632 reduces stomach antrum motility in conscious rats (197) and stomach fundus contraction induced by muscarinic receptor activation in vitro (156).

	EVIDENCE THAT KCL CAN ACTIVATE COMPLEX CELL SIGNALING SYSTEMS

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

 
KCl is often used as a tool to bypass GPCR stimulation and activate smooth muscle by changing the K⁺ equilibrium potential and clamping membrane potential at some value above the resting level (reviewed by Ref. 20). On the basis of the distinctive differences between muscle activation by membrane depolarization and receptor activation, originally termed electromechanical and pharmacomechanical coupling (180), quantitative comparisons between changes produced by stimulation with KCl and GPCR agonists in stimulus-response coupling mechanisms have provided invaluable information about how GPCRs cause contraction (see Ref. 85, 179). However, because membrane depolarization increases [Ca²⁺]_i, KCl can activate Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), which can cause MLC kinase site A phosphorylation, a reduction in MLC kinase affinity for Ca²⁺/calmodulin, and attenuated increases in MLC phosphorylation in airway smooth muscle (184, 194, 195). KCl can also increase ERK activity in both cultured aortic smooth muscle cells (2) and intact arteries, although the degree of activation may vary from sustained (89) to transient despite sustained increases in [Ca²⁺]_i and force (151). CaMKII has been proposed to activate ERK (92), and ERK can phosphorylate and activate MLC kinase (99). Thus a portion of KCl-induced force may be attributed to Ca²⁺ sensitization through enhanced activation of MLC kinase brought about by CaMKII-dependent activation of ERK (92). In short, KCl may cause both Ca²⁺-induced Ca²⁺ desensitization and Ca²⁺-induced Ca²⁺ sensitization through alterations in MLC kinase activity (for a review on MLC kinase, see Ref. 82), supporting the view that KCl can produce more than simply VOCC activation with resultant increases in [Ca²⁺]_i correlating directly (or simply) with increases in MLC kinase activity. That is, by increasing [Ca²⁺]_i, KCl has the potential to act as a stimulus to change the degree of Ca²⁺ sensitivity.

Prolonged membrane depolarization can maintain [Ca²⁺]_i and force above the basal level by continuous activation of Ca²⁺ influx through organic Ca²⁺ channel blocker-sensitive VOCCs (52, 65, 72, 100, 125, 131, 132, 191, 203). However, there is evidence that a portion of the early, phasic phase of a KCl-induced smooth muscle contraction is due also to release of Ca²⁺ from ryanodine- and caffeine-sensitive intracellular Ca²⁺ stores (101, 102). In coronary artery, membrane depolarization appears to modulate inositol 1,4,5-trisphosphate production (50). Moreover, selectivity for VOCCs of certain organic Ca²⁺ channel blockers, such as the dihydropyridine, nifedipine, has recently been challenged. Although nonselective actions at high concentrations of organic Ca²⁺ channel blockers are well known (83), recent studies (104, 109, 209) suggest that low nifedipine concentrations inhibit certain store-operated Ca²⁺ channels (SOCCs) with an IC₅₀ value equivalent to VOCC inhibition (28, 216). Although additional research is required, these data together support the hypothesis that smooth muscle membrane depolarization by KCl may stimulate more complex Ca²⁺ signaling systems than simply activation of VOCCs.

In 1990, the isoquinolinesulfonyl protein kinase inhibitor, H-7, was shown to more potently relax KCl-induced (IC₅₀ 4 µM) than phenylephrine-induced (IC₅₀ = 11–15 µM) tonic contractions of rabbit renal and femoral arteries (148). Reductions in force correlated with reductions in MLC phosphorylation and the maximum rate of muscle shortening (148). In the rat aorta, H-7 inhibits KCl-induced increases in force more strongly than increases in [Ca²⁺]_i (190). At the time of these studies, H-7 was considered a selective PKC inhibitor (63, 73, 90), and the data suggested involvement of PKC in KCl-induced tonic force maintenance. Another isoquinolinesulfonyl protein kinase inhibitor and potent vasorelaxant, HA-1077 when used at no more than 10 µM, was shown to also strongly inhibit KCl-induced contractions with a minimum inhibition of [Ca²⁺]_i (190). In canine basilar artery, 10 µM HA-1077 abolishes Ca²⁺ ionophore (ionomycin)-induced contraction, whereas organic Ca²⁺ channel blockers have no effect, suggesting that the inhibitory effect is due to intracellular antagonism of contraction (189). The K_i for MLC kinase is 36 µM (11), and at 20 µM, HA-1077 inhibits MLC kinase by <10% (31). These data, acquired before the discovery of ROK in 1995 (111), suggested that KCl stimulates a kinase other than MLC kinase that plays an important role downstream from Ca²⁺ mobilization in the regulation of smooth muscle contraction. It is now clear that H-7 (43) and HA-1077 inhibit ROK with nearly equal potency (200), that the IC₅₀ value is 3 µM for inhibition of GTPS-stimulated contraction of Ca²⁺-clamped (-escin) guinea pig ileum (185), and that HA-1077 is a poor inhibitor of conventional PKC isotypes (31, 185). KCl-induced tonic contraction of rabbit femoral artery is not inhibited by 1 µM of the bisindolymaleimide, GF-109203X (202), an effective and relatively selective inhibitor of conventional and novel PKC isotypes with no inhibitory activity toward ROK at concentrations effective against PKC (31, 40, 49). Thus these data together support the hypothesis that the kinase activated by KCl in smooth muscle is not PKC, but ROK. If KCl activates ROK, then KCl should cause Ca²⁺ sensitization.

	EVIDENCE THAT KCL CAN CAUSE INCREASES IN CA2+ SENSITIVITY OF SMOOTH MUSCLE CONTRACTION BY ACTIVATION OF ROK

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

 
In canine coronary artery, the degree of force produced for a given [Ca²⁺]_i is greater in tissues depolarized with 90 mM KCl and contracted by step increases in extracellular [Ca²⁺]_i from 0 to 2.5 mM than in tissues in 2.5 mM extracellular [Ca²⁺]_i and contracted by step increases in extracellular [KCl] from 5 to 90 mM (214). In 1994, these data led Yanagisawa et al. (214) to conclude that membrane depolarization by KCl can cause Ca²⁺ sensitization of arterial smooth muscle. Interestingly, the strength of KCl-induced tonic, but not phasic force of rabbit arteries is highly dependent on the history of contractile GPCR stimulation (149). More importantly, prior contractile GPCR stimulation can inhibit subsequent KCl-induced tonic force without inhibiting tonic increases in [Ca²⁺]_i (154, 155). This Ca²⁺ desensitization of KCl-induced tonic contraction by prior GPCR is reversed within a few hours (149), which is consistent with the hypothesis that the KCl-induced Ca²⁺ sensitivity curve is not unique (150, 214). Thus the degree of Ca²⁺ sensitivity produced by both GPCR and KCl stimulation is not fixed, but depends on the concentration of stimulus (47, 214), and for a given stimulus concentration, on the history of prior receptor activation (150), suggesting that Ca²⁺ sensitivity induced by both GPCR stimuli and KCl may fall within a range of values (Fig. 1B).

However, the argument that a stimulus increases Ca²⁺ sensitivity if it produces a [Ca²⁺]_i-force curve that is leftward shifted compared with that produced by KCl remains a valid argument, provided that consideration be made for the degree of Ca²⁺ sensitivity already induced by KCl. For example, the absence of a leftward shift in the [Ca²⁺]_i force curve produced by a novel GPCR agonist compared with the KCl-induced curve does not necessarily mean the absence of Ca²⁺ sensitization. However, the terminology remains loose, and an exact definition for the midpoint of the Ca²⁺ sensitivity spectrum representing neither Ca²⁺ sensitization nor Ca²⁺ desensitization should probably be eliminated. In its place, the two extremes of Ca²⁺ sensitivity should be defined and mechanistically equated with well-characterized signaling mechanisms.

Recent studies show that Y-27632 inhibits KCl-induced contraction of most smooth muscle types in many species, including the human myometrium (106), rat caudal artery (121), rat (6, 166) and rabbit aorta (166, 167), rat mesenteric artery (9, 166), rabbit femoral and renal arteries (202), mouse anococcygeus smooth muscle (13), pig and cow airway smooth muscle (77), and sheep ureter (113). KCl-induced contraction of chicken gizzard smooth muscle is not inhibited by 10 µM Y-27632, but this is consistent with the lack of inhibition by Y-27632 of GPCR stimulation in this smooth muscle despite the presence of RhoA and ROK and the ability to induce Ca²⁺ sensitization with GTPS in Ca²⁺-clamped tissues (6).

Y-27632 inhibits the tonic phase much more than the early phase of a KCl-induced contraction in rabbit femoral and renal arteries (202) and rat caudal artery (121), whereas 30 µM ML-9, a MLC kinase and ROK inhibitor (15, 200), attenuates both early and tonic force (121). Y-27632 inhibits KCl-induced force without inhibition of KCl-induced increases in [Ca²⁺]_i (77, 106, 121, 166, 202), but with concomitant inhibition of MLC phosphorylation (121, 166, 202). However, in the phasic rat ureter, 1 µM Y-27632 dramatically inhibits VOCC activity to reduce both [Ca²⁺]_i and force produced by electrical field stimulation, whereas action potential-generated force transients in guinea pig ureter are insensitive to Y-27632 (172). Y-27632 (10 µM) has also been shown to modestly inhibit muscarinic receptor-induced increases in [Ca²⁺]_i in guinea pig airway smooth muscle (76). Higher Y-27632 concentrations produce greater inhibition, and further reductions in [Ca²⁺]_i occur in the presence of 1 µM nifedipine, suggesting that non-VOCCs are inhibited. In rat arteries, Y-27632 inhibits nifedipine-insensitive Ca²⁺ entry induced by -adrenergic receptor activation, but does not inhibit thapsigargin-induced increases in Ca²⁺, suggesting that ROK can block nonselective cation channels distinct from VOCCs and SOCCs (54). Thus with the caveat that ROK may regulate Ca²⁺ channels in some smooth muscles (54, 76, 172), these data together indicate that KCl causes Ca²⁺ sensitization by activating ROK.

There are at least two reasons why these results do not necessarily contradict the original publication in 1997 stating that Y-27632 has little effect on KCl-induced force (200). First, a subsequent report by Sakamoto et al. (166) indicates that even 100 µM Y-27632 inhibits KCl-contracted rabbit aorta by only 45%, and with this minimum included in the calculation, the IC₅₀ value is 1.6 µM, a value comparable to that calculated for tissues activated by GPCR agonists. Thus, in this artery, the tonic phase of a KCl-induced contraction may consist of a Y-27632-resistant and Y-27632-sensitive component. A second reason is that the potency of inhibition of agonist-induced contraction by Y-27632 is dependent on agonist concentration. In rabbit femoral artery, the order of potency for inhibition by Y-27632 of contraction is 0.1 µM phenylephrine >KCl and 1 µM phenylephrine >100 µM phenylephrine (Fig. 4), and in bovine airway smooth muscle, although the IC₅₀ value for inhibition of 0.3 µM methacholine-induced contraction is 2.4 µM, this concentration of Y-27632 fails to inhibit contractions produced by 1 and 3 µM methacholine (130). Interestingly, in the rat aorta (166), 73 mM KCl-induced contraction is more potently (IC₅₀ 1 µM) inhibited by Y-27632 than are contractions produced by 1 µM phenylephrine (IC₅₀ 2 µM), 30 nM endothelin (IC₅₀ 4 µM), and 10 µM PGF₂ (IC₅₀ 5 µM), which is consistent with the earlier finding that the ROK inhibitor, H-7, produces a more potent inhibition of KCl-induced contraction than of phenylephrine-induced contraction (148).

View larger version (30K): [in this window] [in a new window]   Fig. 4. Steady-state force produced by KCl and 0.1, 1, and 100 µM phenylephrine (PE), an 1-adrenergic receptor agonist (A), and inhibitory potencies of Y-27632 (B), and GF-109203X (C) in rabbit femoral artery. The degree of ROK-dependent Ca2+ sensitization induced by 110 mM KCl (substituted isosmotically for Na+), as measured by the inhibitory potency of Y-27632, can be considered >, =, or < that induced by GPCR stimulation, depending on GPCR activation level (B). The potency of GF-109203X is also dependent on GPCR activation level, but KCl-induced contraction is not inhibited by 1 µM GF-109203X, a concentration that strongly inhibits conventional and novel PKC isotypes but has negligible inhibitory activity against ROK. Fo = maximum force produced by KCl at the optimum length for muscle contraction. Data are means ± SE, n = 3–4; *P < 0.05 compared with KCl.

	POTENTIAL MECHANISMS FOR KCL-INDUCED INCREASES IN CA2+ SENSITIVITY

TOP ABSTRACT MECHANISMS REGULATING SMOOTH... ROK AND PKC PARTICIPATE... EVIDENCE THAT KCl CAN... EVIDENCE THAT KCl CAN... POTENTIAL MECHANISMS FOR KCl... UNRESOLVED ISSUES SOME FUTURE DIRECTIONS IN... GRANTS REFERENCES

Before publication of Y-27632 as a ROK inhibitor (200), studies using cell-permeable RhoA inhibitors demonstrated attenuation of KCl-induced contractions. For example, Clostridium difficile toxin B reduces the peak of KCl-stimulated phasic contraction of guinea pig intestine by 30% (141), and DC3B (12, 21) significantly inhibits KCl-induced peak contraction of rabbit portal vein by 40% (45). The focus of these studies, however, was on inhibition of GPCR-induced contraction, and inhibition of KCl-induced force was not explored further. Nevertheless, RhoA seems to be involved in KCl-induced ROK activation. Although Ca²⁺ calmodulin-dependent ras GEFs exist, there are no reports of Ca²⁺- or depolarization-dependent RhoA GEFs. Thus there is not yet a viable model to explain how KCl might activate RhoA.

One function of caveolae is to serve as a signalosome (reviewed in Ref. 173), a subcellular compartment acting somewhat like an urban center to concentrate signaling molecules and systems, permitting efficient signal transduction. Caveolae are loci for ROK and RhoA interaction upon GPCR stimulation in smooth muscle (186), and a recent report indicates that caveolin-1 plays a crucial role in regulation of PKC- and ERK-based smooth muscle contraction (78). Whereas GPCR stimuli cause increased ROK and RhoA colocalization with caveolin (187, 188), KCl increases ROK, but not RhoA, colocalization with caveolin in intact rabbit femoral artery (201, 202). The fraction of RhoA colocalized basally with caveolin at the plasma membrane in rabbit artery appears to be substantial (201), and because membrane-bound RhoA is active (the inactive form is bound to Rho-GDI in the cytosol, see Ref. 178 for a review), any ROK that translocates to plasma membrane RhoA may become activated. Moreover, using a pull-down assay, Sakurada et al. (167) found that KCl increases the amount of cellular RhoA in the active (GTP bound) form, and that this stimulation is entirely Ca²⁺-dependent in rabbit aorta.

These data suggest that KCl may cause Ca²⁺ sensitization, in part, by increasing the fraction of active ROK at caveolae. However, intact caveolae are not required for KCl-induced contraction, because neither caveolin-1 knockout nor cholesterol depletion causing caveolae collapse alters the ability of KCl to cause contraction (36, 37, 78). Caveolin not only is found in caveolae but also is associated with the -dystroglycan (157) in a plasma membrane domain near caveolae (138, 192). Thus an alternative hypothesis consistent with the data that has not yet been tested is that RhoA and ROK do not require caveolin as a binding partner for activation and that KCl causes an increase in translocation of ROK to the dystroglycan complex located next to, but not within, caveolae. Although ₁-adrenergic receptor activation causes Ca²⁺ sensitization (66, 94) at agonist concentrations greater than the EC₅₀ value (48) and contraction that is sensitive to inhibition by Y-27632 (see Fig. 4), caveolin-1 knockout and caveolar collapse do not inhibit force produced by stimulation of this GPCR (29, 36). Thus, it appears that caveolae are necessary for some but not all stimuli that cause ROK activation and Ca²⁺ sensitization. A complicating factor in the interpretation of caveolin-1 knockout and caveolar collapse data is that many GPCR-linked signaling molecules other than RhoA and ROK, such as the receptors themselves, G proteins, and PLC that accumulate in caveolae would likewise be disrupted. For example, cholesterol depletion can reduce the affinity of receptors for their ligands (98). Caveolae also appear to play a dominant role in regulation of membrane potential and Ca²⁺ signaling (14, 30), especially activation of SOCCs (18, 198), and collapse of caveolae can reduce GPCR-induced Ca²⁺ entry (18, 37). Thus inhibition of GPCR stimulated contraction may reflect alterations in signaling other than those involved in ROK-induced Ca²⁺ sensitization. Although KCl doubles the amount of ROK found colocalized with caveolin in the cell periphery in rabbit femoral artery, the increase is from 18% basal colocalization to 35% (202), suggesting that a majority of ROK either is not translocated to peripheral sites or is translocated to other sites. These data together indicate that a major challenge will be to determine precisely what roles peripheral sites play in ROK activation leading to KCl (and GPCR)-induced Ca²⁺ sensitization.

How KCl causes ROK translocation to peripheral sites also remains to be determined. The directed movement from cytosol to specific plasma membrane locations suggests that ROK is cargo carried by molecular motors along intracellular cables (reviewed by Refs. 33, 80, 207). Microtubules that run along the long axis of the smooth muscle cell can be found near the caveolar membrane domain (Fig. 5 and Ref. 118), and not only do actin cables terminate nearby at focal contacts (192), but caveolae are largely immobilized by linkage to the cortical actin cytoskeleton, at least in certain cell types (196). Myosin II is known to transport vesicles along actin cables in clam oocyte extracts (210), but perhaps more importantly, myosin II colocalizes with cortical actin cables in chromaffin cells where MLC phosphorylation appears necessary for KCl-induced vesicle transport and secretion (133). Neuronal mitochondria are transported by molecular motors along microtubule and actin cables (128), and because of the propinquity of mitochondria, caveolae, microtubules and actin cables in smooth muscle (Fig. 5), it is tempting to speculate that KCl causes Ca²⁺ sensitization by activating a transport mechanism involving ROK attached to a molecular motor running along an actin or microtubule cable. Interestingly, both the actin polymerization inhibitor, cytochalasin D, and the microtubule disruptor, nocodazole, inhibit the increase in KCl-induced ROK colocalization with caveolin in rabbit artery (Fig. 6), and cytochalasins A, B, and D inhibit tonic but not early, phasic KCl-induced contraction in rat aorta (212). In this scenario, ROK need not be active for transport to occur because Y-27632, while strongly inhibiting KCl-induced tonic force, does not inhibit KCl-induced ROK colocalization with caveolin (202).

View larger version (177K): [in this window] [in a new window]   Fig. 5. Transmission electron micrograph of glutaraldehyde-fixed rabbit femoral artery showing close proximity of three caveolae (arrowhead shows left-most of the triplet of omega-shaped plasma membrane invaginations), microtubules (thin, long arrow) and microfilaments (thicker, shorter arrow). Caveolae, microtubules, and microfilaments also are very near the mitochondria in the left-center of the figure.

View larger version (30K): [in this window] [in a new window]   Fig. 6. KCl (110 mM; substituted isosmotically for Na+) causes an increase in ROK translocation to caveolae at 30 s of stimulation (202), and this increase was significantly inhibited by 1 µM cytochalasin D and 10 µM nocodazole, agents that can disrupt, respectively, actin and microtubule cables. Data are means ± SE, n = 3.

Perhaps the most interesting model was proposed by Morano (124), who speculated, based on smooth muscle myosin II knockout studies, that KCl-induced tonic bladder contraction in neonatal mice is maintained, at least in part, by ROK-induced activation of nonmuscle myosin (see Ref. 123 for review). However, using the same smooth muscle myosin II-deficient neonatal mice, Lohn et al. (115) showed that KCl elevates [Ca²⁺]_i but not force, whereas PKC activation using phorbol ester produces sustained cerebral artery contraction without elevating Ca²⁺. Thus nonmuscle myosin does not permit force maintenance in cerebral arteries stimulated with KCl. Moreover, because adult arteries do not express significant amounts of nonmuscle myosin (107), a major role for this motor protein in maintenance of strong KCl-induced tonic force in adult arterial muscle seems unlikely.

	UNRESOLVED ISSUES

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Tissues are heterogeneous mixtures of cell types, and KCl can cause release of neurotransmitters or factors from adjacent cells that activate GPCRs. If appropriate use of selective GPCR antagonists is considered, then the contribution of spurious GPCR activation can usually be eliminated. For example, in rabbit vas deferens, -adrenergic receptor blockade with prazosin abolishes the KCl-induced tonic increase in CPI-17 Thr38 phosphorylation and nearly abolishes tonic force (96), suggesting that KCl-induced Ca²⁺ sensitization in this tissue is largely the result of -adrenergic receptor stimulation by norepinephrine released from sympathetic nerves. Although not all studies exploring Ca²⁺ sensitization induced by KCl included the use of receptor antagonists, studies on the rabbit aorta (167) and femoral artery (150, 202), pig and cow airway smooth muscle (77), and rat caudal (121) and mesenteric (9) arteries did apply receptor blockers to eliminate any potential contribution of GPCR activation. The use of - and -adrenergic, H₁-histaminergic, AT₁, and thromboxane A₂ receptor blockers by Sakurada et al. (167) provides strong support for the conclusion that KCl acting through smooth muscle membrane depolarization can cause Ca²⁺ sensitization.

The use of primary single cell isolates can also eliminate the potential contribution of paracrine stimuli. However, integrin- and elastin-based signaling play important roles in the regulation of cell signaling (Refs. 88, 182, 183, 193, and reviewed in Ref. 217), and the loss of, or dramatic alteration in, focal contact tension on disruption of the extracellular matrix permitting isolation of single smooth muscle cells may dramatically alter cell signaling systems leading to contraction. Another complicating factor would arise if increases in [Ca²⁺]_i produced on membrane depolarization with KCl caused increased production of autocrine hormones such as sphingosine-1-phosphate and arachidonic acid metabolites (3, 110, 140, 144). If generated, autocrine agents could activate GPCRs, leading to Ca²⁺ sensitization, and this contribution to a KCl-induced response may not be eliminated even by isolation of single cells. These issues will be resolved only by use of appropriate tissue, cell and molecular models and protocol designs including the use of selective receptor blockers or inhibitors of the production of autocrine and paracrine agents.

Another unresolved issue is the precise identity of the molecular links bridging KCl stimulation with ROK activation. The definitive experiments in smooth muscle have not been done, and whether Ca²⁺ activates a molecular motor or some other system is not known. The binding partners involved in ROK transport and the signals that initiate and terminate translocation are not known. Also, whether Ca²⁺ or membrane depolarization plays the principal role in KCl-induced Ca²⁺ sensitization remains to be determined.

In summary, a general model (Fig. 7) for KCl-induced Ca²⁺ sensitization must at this time include several possible alternative mechanisms, including Ca²⁺-induced colocalization to plasma membrane site(s) such as caveolae or the dystroglycan complex, activation of ROK by RhoA or by arachidonic acid, activation of GPCRs by the generation of paracrine or autocrine agents, and activation of ROK by membrane depolarization independently of changes in [Ca²⁺]_i. In addition, the contribution made by other Ca²⁺-dependent and -independent enzymes on MLC kinase and phosphatase activities must also be considered in a comprehensive model describing KCl-induced Ca²⁺ sensitization.

View larger version (53K): [in this window] [in a new window]   Fig. 7. Diagram of a general model depicting several potential subcellular mechanisms by which KCl may cause Ca2+ sensitization (?s indicate some alternative hypotheses proposed in the literature). Increased [Ca2+]i produced upon membrane depolarization with KCl may cause ROK translocation to caveolin within distinct plasma membrane domains such as caveolae or the dystroglycan complex, where active RhoA (RhoA-GTP) or arachidonic acid activates ROK (ROK*). How ROK is translocated from the cytosol to plasma membrane (PM) sites is not known, but one possible mode of transport is as cargo carried on actin or tubulin cables. Membrane depolarization (EM) rather than increases in [Ca2+]i may be responsible for activation of ROK, but the molecular mechanism linking depolarization with ROK activation remains to be determined. Because GPCRs and KCl can both cause membrane depolarization and elevate [Ca2+]i, KCl-induced Ca2+ sensitization may represent a subset of GPCR-induced Ca2+ sensitization. Elevations in [Ca2+]i can also activate Ca2+ calmodulin-dependent protein kinase (CaMKII) and ERK, which may alter MLC kinase activity. To ensure that KCl-induced Ca2+ sensitization is due solely to increases in [Ca2+]i or membrane depolarization, the potential contribution of paracrine and autocrine agents that may cause Ca2+ sensitization by activation of GPCRs must be eliminated.

	SOME FUTURE DIRECTIONS IN KCL-INDUCED CA2+ SENSITIVITY RESEARCH

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View larger version (69K): [in this window] [in a new window]   Fig. 8. Example of a Western blot analysis of homogenates of rabbit renal arteries stimulated with 110 mM KCl (substituted isosmotically for Na+) ± 1 µM Y-27632 (ROK inhibitor), probed with site-selective antibodies for phospho-MYPT1. A: KCl increased MYPT1-Thr853-p (numbering is based on the human sequence), and Y-27632 inhibited not only KCl-stimulated but also basal phosphorylation. B: KCl slightly increased MYPT1-Thr696-p. C: all lanes were loaded with 50 µg of total protein, and loading was consistent across all lanes, as shown by the probe with anti-MYPT antibody.

In conclusion, KCl remains a valuable tool as a smooth muscle stimulus. Future studies designed to explore KCl-induced Ca²⁺ sensitization will undoubtedly provide new insights and a deeper understanding of spatiotemporal cell signaling paradigms involved in regulation of smooth muscle contraction in health and disease.

	GRANTS

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This study was supported by National Heart, Lung, and Blood Institute Grant R01-HL-61320.

	ACKNOWLEDGMENTS

 
We thank Natasha Purdie for expert technical assistance, Marta Ambrozevicz for the data shown in Fig. 4, and Dr. Jeffrey Dupree for consultation on electron microscopy.

Present address for N. H. Urban: Virginia Commonwealth University, Department of Orthopaedic Surgery, 1101 E. Marshall St., PO Box 980614, Richmond, VA 23289-0614.

	FOOTNOTES

 

Address for reprint requests and other correspondence: P. H. Ratz, Virginia Commonwealth Univ., School of Medicine, Depts. of Biochemistry and Pediatrics, 1101 E. Marshall St., PO Box 980614, Richmond, VA 23298-0614 (E-mail: phratz{at}vcu.edu)

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