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

Paul H. Ratz, Krystina M. Berg, Nicole H. Urban, and Amy S. Miner

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


THE UNIVERSAL CELLULAR regulator, Ca2+ (see Ref. 25 for a review), is the primary signal responsible for activation of smooth muscle contractile proteins (41, 125, 134). Cytosolic free Ca2+ concentration ([Ca2+]i) is finely tuned by multiple membrane-based Ca2+ channels, pumps, compartments, and intracellular buffers to suit the immediate contractile requirement of muscle cells (see Refs. 67, 85, 87, 108, 120, 147, and 204 for reviews). However, Ca2+ does not directly activate smooth muscle motor proteins. Rather, phosphorylation of 20-kDa regulatory myosin light chain (MLC) serves as the "switch" to "turn on" smooth muscle actomyosin ATPase activity (17, 26, 61, 162, 175, 199), increasing cross-bridge cycling rates and muscle contraction (35). Stimuli that increase [Ca2+]i elevate MLC phosphorylation levels by increasing the Ca2+-calmodulin-dependent MLC kinase-to-phosphatase activity ratio (see Ref. 81 for a review). Other kinases may serve the same role because a recent study (181) using MLC kinase knockout (–/–) mice shows that arteries still contract, and cells cultured from aorta still display increases in MLC phosphorylation. Nevertheless, the steady-state relationship between cross-bridge phosphorylation and force in swine carotid media stimulated by several contractile agonists is steeply hyperbolic (34, 153, 159), suggesting that a unique relationship often exists between these two parameters of muscle activation. Thus, based on a stimulus-response operational approach, if the number of cross bridges "switched on" to produce force is uniquely dependent on [Ca2+]i, then the sensitivity of contractile force to Ca2+ is invariant. However, studies beginning in the 1980s on intact ferret portal vein (127), ferret (32), and rat aorta (86, 169), rat (142) and swine carotid artery (22, 158), rabbit pulmonary artery (66), rabbit ear artery (64), rabbit femoral artery (150), guinea pig ileum (64), and canine trachea (53, 143) by using the photoprotein, aequorin, and fluorescent Ca2+ indicators, fura-2 and quin-2, show that K+-depolarization and G protein coupled receptor (GPCR) activation yield different [Ca2+]i-force curves and that cyclic nucleotides can cause relaxation with minimal reductions in [Ca2+]i (1, 126, 215). These results are supported by data obtained from Ca2+-clamped (permeabilized) tissues showing that, at constant Ca2+ concentrations, cyclic nucleotides can induce relaxation (163, 164), and stimuli activating contractile GPCRs can elevate force (66, 97, 103, 137) concomitant with increases in MLC phosphorylation (94).

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).



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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
 
Ca2+ sensitivity is regulated primarily at the level of MLC phosphorylation by modulation of MLC phosphatase activity (93, 94, 105), but may also be regulated by modulation of MLC kinase activity, and downstream from MLC phosphorylation by mechanisms involving thin filament regulation and possibly direct regulation by heat shock proteins of contractile proteins or load-bearing structures associated with them. Many excellent reviews cover these topics in depth (23, 57, 82, 87, 91, 145, 178, 211), and only the very general features and historical aspects focusing largely on MLC phosphatase regulation will be covered in this section.

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 Gq and G12/13 (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-{beta}-cyclodextrin, or by caveolin-1 knockout, reduces the ability of some GPCR stimuli, but not KCl, to produce contraction (36, 37, 78).



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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).

 
Thus the notion that GPCR stimuli cause Ca2+ sensitization by activation of kinases not "turned on" during K+ depolarization is an attractive hypothesis for several reasons, not the least of which is because it permits comparative cell physiological studies designed to reveal underlying mechanisms by which GPCR stimuli cause contraction. In support of the data from intact tissues, GPCR agonists and GTP{gamma}S were found to elevate force in Ca2+-clamped (permeabilized with {alpha}-toxin or saponin) smooth muscle tissues (46, 97, 137). These data together set the stage for work on mechanisms regulating Ca2+ sensitivity involving ROK, PKC, CPI-17 (the MLC phosphatase inhibitory protein), and myosin phosphatase targeting (MYPT1; the large regulatory subunit of MLC phosphatase).

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 {alpha}-adrenergic receptor blockade (95, 96).



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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 {vdash}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
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 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
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In 1997, Y-27632 was introduced as a Ser/Thr protein kinase inhibitor competitive with ATP at the catalytic site, selective for ROK over PKC, cAMP-dependent protein kinase and MLC kinase, and with an IC50 value ranging between 0.3 and 1 µM for inhibition of contractions produced by rabbit aorta stimulated with phenylephrine, histamine, acetylcholine, serotonin, endothelin, and U-46619 (200). The IC50 for inhibition of KCl-induced contraction was reported to be >30 µM, supporting the conclusion that Y-27632 is a specific and potent inhibitor of GPCR-induced smooth muscle contraction (200). Subsequent work showed that Y-27632 inhibits both ROK isotypes (ROK{alpha} and ROK{beta}) with nearly equivalent potencies and is ~16-fold more selective for ROK than citron kinase and PKN, other RhoA targets (74), but that PKC{delta} is only slightly less sensitive to Y-27632 than is ROK (40). At physiological ATP concentrations of 2 mM, Y-27632 inhibits PKC{delta} activity with an IC50 of 14 µM, whereas it inhibits ROK activity with an IC50 of 6 µM (40).

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 {alpha}-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 GTP{gamma}S 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 Ki 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 GTP{gamma}S-stimulated contraction of Ca2+-clamped ({beta}-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 GTP{gamma}S 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 {alpha}-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{alpha} (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).



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Fig. 4. Steady-state force produced by KCl and 0.1, 1, and 100 µM phenylephrine (PE), an {alpha}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
 
Because KCl bypasses GPCRs and activates smooth muscle by causing membrane depolarization, the realization that KCl can cause ROK-dependent Ca2+ sensitization is somewhat surprising, and precisely how KCl activates ROK is not yet fully understood. ROK was identified as a ubiquitously expressed, 160-kDa protein that specifically interacts with RhoA-GTP and induces stress fiber and focal adhesion formation (111, 112, 119). Before the discovery of ROK, Ridley and Hall showed in 1992 (160) that RhoA regulates stress fiber and focal adhesion formation in cells stimulated with extracellular ligands, such as lysophosphatidic acid, that activate GPCRs. ROK, an AGC Ser/Thr kinase family member, was the first RhoA effector identified (112), and is expressed in vertebrates as two isoforms, ROK{alpha} (ROCKII) and ROK{beta} (ROCKI). ROK{alpha} is abundant in muscle and brain and is distributed mostly, but not exclusively, in the cytosol. Stimuli that activate RhoA cause some ROK translocation to peripheral sites, most notably those associated with caveolin (reviewed by Ref. 187). Like other AGC Ser/Thr kinase family members, ROK exists in a closed, inactive conformation in which the COOH terminus (that includes a RhoA binding region and a plekstrin homology domain split by a Cys-rich region) acts as an autoinhibitory domain (4), and an open, active conformation (for reviews, see Refs. 5, 19, 161). RhoA induces a modest ~2-fold increase in ROK activity, whereas 30 µM arachidonic acid increases ROK activity by 5- to 6-fold independently of RhoA (43) by relieving autoinhibition (7). Arachidonic acid produces strong Ca2+ sensitization in Ca2+-clamped smooth muscle (7, 44), and several GPCR agonists generate arachidonic acid as a cellular messenger (55). High Ca2+ levels induce substantial arachidonic acid release in permeabilized rabbit femoral artery (55). However, although K+ depolarization of smooth muscle can cause significant increases in [Ca2+]i, KCl-induced contractions in rabbit thoracic aorta are not inhibited by the PLA2 inhibitor ONO-RS-082 (16, 122).

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 {beta}-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 {alpha}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{beta} 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).



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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.

 


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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.

 
On the basis of the current data, one hypothesis for regulation of KCl-induced contraction is a Ca2+-activated Ca2+ sensitivity model whereby the early increase in Ca2+ activates MLC kinase to activate contraction at myofilaments in the cell interior, but also activates a transport mechanism permitting movement of ROK to peripheral plasma membrane sites. Although speculative, this model is testable and consistent with the recent finding that Y-27632 does not, but both nifedipine and the Ca2+-calmodulin blocker, trifluoperazine, do inhibit KCl-induced ROK translocation to caveolin in rabbit artery (202). Moreover, the dihydropyridine Ca2+ channel agonist, BAY K 8644, and the Ca2+ ionophore, ionomycin, likewise cause increases in MLC phosphorylation and force that are inhibited by Y-27632 and increase the amount of ROK colocalization with caveolin that is inhibited by trifluoperazine, suggesting that Ca2+, rather than membrane depolarization, plays the predominant role in activating ROK, at least in rabbit artery (202). Any receptor agonist that increases [Ca2+]i would necessarily share this mechanism with KCl but additionally would produce a greater increase in Ca2+ sensitivity (150), presumably by activation of PKC (40, 95, 187) and by causing stronger increases ROK activity (reviewed by Ref. 177). This model will certainly require modification as new data are acquired, but it also assumes that Ca2+ is the primary stimulus for KCl-induced Ca2+ sensitization, and there is strong evidence consistent with the hypothesis that membrane depolarization, rather than increases in [Ca2+]i, can cause KCl-induced Ca2+ sensitization (214). It is also possible that both membrane potential and Ca2+ play roles in Ca2+ sensitivity because in rabbit aorta, the CaMKII inhibitor, KN-93, reduced KCl- but not ionomycin-induced contraction and increase in activated RhoA (167).

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.


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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, {alpha}-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 {alpha}-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 {alpha}- and {beta}-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.



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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 ({Delta}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.

 

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To more fully understand KCl-induced Ca2+ sensitization, some future challenges are to determine the precise mechanism(s) by which Ca2+ or membrane potential causes translocation of ROK to peripheral membrane sites, whether RhoA basally residing at peripheral sites is necessary and sufficient to activate newly translocated ROK, whether additional RhoA becomes activated during stimulation with KCl, and how activated ROK causes sustained activation of MLC phosphorylation to maintain tonic force. This last step presumes that ROK must either directly, through subsequent translocation back to the cytosol, or indirectly, through activation of additional mediators, such as ZIP-like kinase (117), phosphorylate MYPT1 on more centrally located myofilaments. In the rabbit aorta, KCl increases MYPT1-Thr696 phosphorylation by ~50% (167), and our laboratory provides supportive data showing that KCl increases MYPT1-Thr853 phosphorylation in rabbit renal artery (Fig. 8). Whether KCl-induced tonic contraction is uniquely due to sensitization of nonmuscle myosin (123) must also be resolved. There is a growing list of kinases that can phosphorylate MLCs and proteins involved in regulation of MLC phosphorylation (see Refs. 68, 82, and 178 for reviews). KCl can activate MLC kinase, ROK, CaMKII and ERK, but whether additional kinases are activated and participate in regulation of KCl-induced tonic contraction remains to be determined. Moreover, rigorous studies are required to verify that KCl-induced Ca2+ sensitization is not caused, in part, by GPCR stimulation caused by release of autocrine or paracrine agents.



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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.

 
Given the documented complexity of KCl-induced regulation of contraction, should K+ depolarization still be used as a tool to study smooth muscle physiology? My bias is to answer "yes." There is growing evidence that the mechanisms by which the many GPCR contractile and relaxant stimuli cause smooth muscle contraction differ on a scale ranging from subtle to dramatic. Smooth muscles from different organ systems often display different signaling patterns in response to the same stimulus. There is ample evidence that the type of contraction produced by each stimulus is dependent on stimulus concentration as well as on duration of muscle stimulation. Our laboratory has shown that smooth muscle retains a memory of GPCR activation such that both Ca2+ signaling and Ca2+ sensitization can be altered for minutes to hours after complete cessation of the stimulus (149, 150, 154, 155). Thus the cell signaling systems regulating smooth muscle contraction also display GPCR activation history dependence. For these reasons, despite its inherent complexity, KCl-induced smooth muscle contraction is still "simpler" than GPCR-induced contraction, and once the cell signaling and contractile protein regulatory systems induced by KCl are completely mapped, responses to KCl can quite accurately be applied for comparison to more fully understand the vastly more complex GPCR-induced regulation of smooth muscle contraction. Perhaps most importantly, just as KCl as a stimulus helped reveal Ca2+ entry signaling defects in hypertension (8, 10, 69, 213), KCl-induced Ca2+ sensitization is providing insights into signaling dysfunction in certain chronic disease conditions. For example, pig large coronary arteries distal to a chronic occlusion display enhanced KCl-induced, but not enhanced, endothelin-induced Ca2+ sensitivity (62). Y-27632 selectively reduces enhanced KCl-induced contraction in both large and small pulmonary arteries from pulmonary hypertensive rats (129). In rat mesenteric arteries, KCl produces greater Ca2+ sensitization in spontaneously hypertensive rats than in control normotensive animals (9). In the rat ileum, inhibition of KCl-induced Ca2+ sensitization by the proinflammatory interleukin-1{beta} identifies MYPT1 phosphorylation as a target for dysregulation of smooth muscle in intestinal motility disorders (139).

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.


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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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1. Abe A and Karaki H. Effect of forskolin on cytosolic Ca++ level and contraction in vascular smooth muscle. J Pharmacol Exp Ther 249: 895–900, 1989.[Abstract]

2. Abraham ST, Benscoter HA, Schworer CM, and Singer HA. A role for Ca2+/calmodulin-dependent protein kinase II in the mitogen-activated protein kinase signaling cascade of cultured rat aortic vascular smooth muscle cells. Circ Res 81: 575–584, 1997.[Abstract/Free Full Text]

3. Alemany R, Sichelschmidt B, zu Heringdorf DM, Lass H, van Koppen CJ, and Jakobs KH. Stimulation of sphingosine-1-phosphate formation by the P2Y2 receptor in HL-60 cells: Ca2+ requirement and implication in receptor-mediated Ca2+ mobilization, but not MAP kinase activation. Mol Pharmacol 58: 491–497, 2000.[Abstract/Free Full Text]

4. Amano M, Chihara K, Nakamura N, Kaneko T, Matsuura Y, and Kaibuchi K. The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity. J Biol Chem 274: 32418–32424, 1999.[Abstract/Free Full Text]

5. Amano M, Fukata Y, and Kaibuchi K. Regulation and functions of Rho-associated kinase. Exp Cell Res 261: 44–51, 2000.[CrossRef][ISI][Medline]

6. Anabuki J, Hori M, Hayakawa K, Akahane S, Ozaki H, and Karaki H. Muscarinic stimulation does not induce RhoA/ROCK-mediated Ca2+ sensitization of the contractile element in chicken gizzard smooth muscle. Pflügers Arch 441: 189–199, 2000.[CrossRef][ISI][Medline]

7. Araki S, Ito M, Kureishi Y, Feng J, Machida H, Isaka N, Amano M, Kaibuchi K, Hartshorne DJ, and Nakano T. Arachidonic acid-induced Ca2+ sensitization of smooth muscle contraction through activation of Rho-kinase. Pflügers Arch 441: 596–603, 2001.[CrossRef][ISI][Medline]

8. Arii T, Ohyanagi M, Shibuya J, and Iwasaki T. Increased function of the voltage-dependent calcium channels, without increase of Ca2+ release from the sarcoplasmic reticulum in the arterioles of spontaneous hypertensive rats. Am J Hypertens 12: 1236–1242, 1999.[CrossRef][ISI][Medline]

9. Asano M and Nomura Y. Comparison of inhibitory effects of Y-27632, a Rho kinase inhibitor, in strips of small and large mesenteric arteries from spontaneously hypertensive and normotensive Wistar-Kyoto rats. Hypertens Res 26: 97–106, 2003.[CrossRef][ISI][Medline]

10. Asano M, Nomura Y, Ito K, Uyama Y, Imaizumi Y, and Watanabe M. Increased function of voltage-dependent Ca++ channels and Ca++-activated K+ channels in resting state of femoral arteries from spontaneously hypertensive rats at prehypertensive stage. J Pharmacol Exp Ther 275: 775–783, 1995.[Abstract]

11. Asano T, Suzuki T, Tsuchiya M, Satoh S, Ikegaki I, Shibuya M, Suzuki Y, and Hidaka H. Vasodilator actions of HA1077 in vitro and in vivo putatively mediated by the inhibition of protein kinase. Br J Pharmacol 98: 1091–1100, 1989.[ISI][Medline]

12. Aullo P, Giry M, Olsnes S, Popoff MR, Kocks C, and Boquet P. A chimeric toxin to study the role of the 21 kDa GTP binding protein Rho in the control of actin microfilament assembly. EMBO J 12: 921–931, 1993.[Abstract]

13. Ayman S, Wallace P, Wayman CP, Gibson A, and McFadzean I. Receptor-independent activation of Rho-kinase-mediated calcium sensitisation in smooth muscle. Br J Pharmacol 139: 1532–1538, 2003.[CrossRef][ISI][Medline]

14. Babiychuk EB, Smith RD, Burdyga T, Babiychuk VS, Wray S, and Draeger A. Membrane cholesterol regulates smooth muscle phasic contraction. J Membr Biol 198: 95–101, 2004.[ISI][Medline]

15. Bain J, McLauchlan H, Elliott M, and Cohen P. The specificities of protein kinase inhibitors: an update. Biochem J 371: 199–204, 2003.[CrossRef][ISI][Medline]

16. Banga HS, Simons ER, Brass LF, and Rittenhouse SE. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci USA 83: 9197–9201, 1986.[Abstract]

17. Barron JT, Barany M, and Barany K. Phosphorylation of the 20,000-dalton light chain of myosin of intact arterial smooth muscle in rest and in contraction. J Biol Chem 254: 4954–4956, 1979.[Abstract]

18. Bergdahl A, Gomez MF, Dreja K, Xu SZ, Adner M, Beech DJ, Broman J, Hellstrand P, and Sward K. Cholesterol depletion impairs vascular reactivity to endothelin-1 by reducing store-operated Ca2+ entry dependent on TRPC1. Circ Res 93: 839–847, 2003.[Abstract/Free Full Text]

19. Bishop AL and Hall A. Rho GTPases and their effector proteins. Biochem J 348: 241–255, 2000.[CrossRef][ISI][Medline]

20. Bolton TB. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev 59: 606–718, 1979.[Free Full Text]

21. Boquet P, Popoff MR, Giry M, Lemichez E, and Bergez-Aullo P. Inhibition of p21 Rho in intact cells by C3 diphtheria toxin chimera proteins. Methods Enzymol 256: 297–306, 1995.[CrossRef][ISI][Medline]

22. Bradley AB and Morgan KG. Alterations in cytoplasmic calcium sensitivity during porcine coronary artery contractions as detected by aequorin. J Physiol 385: 437–448, 1987.[Abstract]

23. Brophy CM. Stress and vascular disease at the cellular and molecular levels. World J Surg 26: 779–782, 2002.[CrossRef][ISI][Medline]

24. Bulbring E and Tomita T. Catecholamine action on smooth muscle. Pharmacol Rev 39: 49–96, 1987.[ISI][Medline]

25. Campbell AK. Intracellular Calcium: Its Universal Role as Regulator. Chichester, UK: Wiley, 1983.

26. Cassidy P, Hoar PE, and Kerrick WG. Irreversible thiophosphorylation and activation of tension in functionally skinned rabbit ileum strips by [35S]ATP{gamma}S. J Biol Chem 254: 11148–11153, 1979.[Abstract]

27. Chiba Y, Takada Y, Miyamoto S, Mitsui Saito M, Karaki H, and Misawa M. Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats. Br J Pharmacol 127: 597–600, 1999.[CrossRef][ISI][Medline]

28. Curtis TM and Scholfield CN. Nifedipine blocks Ca2+ store refilling through a pathway not involving L-type Ca2+ channels in rabbit arteriolar smooth muscle. J Physiol 532: 609–623, 2001.[Abstract/Free Full Text]

29. Darblade B, Caillaud D, Poirot M, Fouque M, Thiers JC, Rami J, Bayard F, and Arnal JF. Alteration of plasmalemmal caveolae mimics endothelial dysfunction observed in atheromatous rabbit aorta. Cardiovasc Res 50: 566–576, 2001.[CrossRef][ISI][Medline]

30. Darby PJ, Kwan CY, and Daniel EE. Caveolae from canine airway smooth muscle contain the necessary components for a role in Ca2+ handling. Am J Physiol Lung Cell Mol Physiol 279: L1226–L1235, 2000.[Abstract/Free Full Text]

31. Davies SP, Reddy H, Caivano M, and Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem J 351: 95–105, 2000.[CrossRef][ISI][Medline]

32. DeFeo TT and Morgan KG. Calcium-force relationships as detected with aequorin in two different vascular smooth muscles of the ferret. J Physiol 369: 269–282, 1985.[Abstract]

33. DePina AS and Langford GM. Vesicle transport: the role of actin filaments and myosin motors. Microsc Res Tech 47: 93–106, 1999.[CrossRef][ISI][Medline]

34. Di Blasi P, Van Riper D, Kaiser R, Rembold CM, and Murphy RA. Steady-state dependence of stress on cross-bridge phosphorylation in the swine carotid media. Am J Physiol Cell Physiol 262: C1388–C1391, 1992.[Abstract/Free Full Text]

35. Dillon PF, Aksoy MO, Driska SP, and Murphy RA. Myosin phosphorylation and the cross-bridge cycle in arterial smooth muscle. Science 211: 495–497, 1981.[ISI][Medline]

36. Drab M, Verkade P, Elger M, Kasper M, Lohn M, Lauterbach B, Menne J, Lindschau C, Mende F, Luft FC, Schedl A, Haller H, and Kurzchalia TV. Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice. Science 293: 2449–2452, 2001.[Abstract/Free Full Text]

37. Dreja K, Voldstedlund M, Vinten J, Tranum-Jensen J, Hellstrand P, and Sward K. Cholesterol depletion disrupts caveolae and differentially impairs agonist-induced arterial contraction. Arterioscler Thromb Vasc Biol 22: 1267–1272, 2002.[Abstract/Free Full Text]

38. Driska SP, Aksoy MO, and Murphy RA. Myosin light chain phosphorylation associated with contraction in arterial smooth muscle. Am J Physiol Cell Physiol 240: C222–C233, 1981.[Abstract]

39. Eto M, Kitazawa T, and Brautigan DL. Phosphoprotein inhibitor CPI-17 specificity depends on allosteric regulation of protein phosphatase-1 by regulatory subunits. Proc Natl Acad Sci USA 101: 8888–8893, 2004.[Abstract/Free Full Text]

40. Eto M, Kitazawa T, Yazawa M, Mukai H, Ono Y, and Brautigan DL. Histamine-induced vasoconstriction involves phosphorylation of a specific inhibitor protein for myosin phosphatase by protein kinase C alpha and delta isoforms. J Biol Chem 276: 29072–29078, 2001.[Abstract/Free Full Text]

41. Fay FS, Shlevin HH, Granger WC Jr, and Taylor SR. Aequorin luminescence during activation of single isolated smooth muscle cells. Nature 280: 506–508, 1979.[ISI][Medline]

42. Feng J, Ito M, Ichikawa K, Isaka N, Nishikawa M, Hartshorne DJ, and Nakano T. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem 274: 37385–37390, 1999.[Abstract/Free Full Text]

43. Feng J, Ito M, Kureishi Y, Ichikawa K, Amano M, Isaka N, Okawa K, Iwamatsu A, Kaibuchi K, Hartshorne DJ, and Nakano T. Rho-associated kinase of chicken gizzard smooth muscle. J Biol Chem 274: 3744–3752, 1999.[Abstract/Free Full Text]

44. Fu X, Gong MC, Jia T, Somlyo AV, and Somlyo AP. The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTP{gamma}S-, and phorbol ester-induced Ca2+-sensitization of smooth muscle. FEBS Lett 440: 183–187, 1998.[CrossRef][ISI][Medline]

45. Fujihara H, Walker LA, Gong MC, Lemichez E, Boquet P, Somlyo AV, and Somlyo AP. Inhibition of RhoA translocation and calcium sensitization by in vitro ADP-ribosylation with chimeric toxin DC3B. Mol Biol Cell 8: 2437–2447, 1997.[Abstract/Free Full Text]

46. Fujiwara T, Itoh T, Kubota Y, and Kuriyama H. Effects of guanosine nucleotides on skinned smooth muscle tissue of the rabbit mesenteric artery. J Physiol 408: 535–547, 1989.[Abstract]

47. Fukuizumi Y, Kobayashi S, Nishimura J, and Kanaide H. Temporal changes in the Ca2+-force relationship during norepinephrine-, serotonin- and high K+-induced contractions in the rabbit femoral artery. Jpn J Pharmacol 58, Suppl 2: 277P, 1992.[Medline]

48. Fukuizumi Y, Kobayashi S, Nishimura J, and Kanaide H. Cytosolic calcium concentration-force relation during contractions in the rabbit femoral artery: time-dependency and stimulus specificity. Br J Pharmacol 114: 329–338, 1995.[ISI][Medline]

49. Gailly P, Gong MC, Somlyo AV, and Somlyo AP. Possible role of atypical protein kinase C activated by arachidonic acid in Ca2+ sensitization of rabbit smooth muscle. J Physiol 500: 95–109, 1997.[Abstract]

50. Ganitkevich V and Isenberg G. Membrane potential modulates inositol 1,4,5-trisphosphate-mediated Ca2+ transients in guinea-pig coronary myocytes. J Physiol 470: 35–44, 1993.[Abstract]

51. Ganitkevich V, Hasse V, and Pfitzer G. Ca2+-dependent and Ca2+-independent regulation of smooth muscle contraction. J Muscle Res Cell Motil 23: 47–52, 2002.[CrossRef][ISI][Medline]

52. Ganitkevich VY and Isenberg G. Depolarization-mediated intracellular calcium transients in isolated smooth muscle cells of guinea-pig urinary bladder. J Physiol 435: 187–205, 1991.[Abstract]

53. Gerthoffer WT, Murphey KA, and Gunst SJ. Aequorin luminescence, myosin phosphorylation, and active stress in tracheal smooth muscle. Am J Physiol Cell Physiol 257: C1062–C1068, 1989.[Abstract/Free Full Text]

54. Ghisdal P, Vandenberg G, and Morel N. Rho-dependent kinase is involved in agonist-activated calcium entry in rat arteries. J Physiol 551: 855–867, 2003.[Abstract/Free Full Text]

55. Gong MC, Kinter MT, Somlyo AV, and Somlyo AP. Arachidonic acid and diacylglycerol release associated with inhibition of myosin light chain dephosphorylation in rabbit smooth muscle. J Physiol 486: 113–122, 1995.[Abstract]

56. Gong MC, Fujihara H, Somlyo AV, and Somlyo AP. Translocation of RhoA associated with Ca2+ sensitization of smooth muscle. J Biol Chem 272: 10704–10709, 1997.[Abstract/Free Full Text]

57. Gusev NB. Some properties of caldesmon and calponin and the participation of these proteins in regulation of smooth muscle contraction and cytoskeleton formation. Biochemistry (Mosc) 66: 1112–1121, 2001.[CrossRef][ISI][Medline]

58. Hagiwara Y, Nishina Y, Yorifuji H, and Kikuchi T. Immunolocalization of caveolin-1 and caveolin-3 in monkey skeletal, cardiac and uterine smooth muscles. Cell Struct Funct 27: 375–382, 2002.[CrossRef][ISI][Medline]

59. Hartshorne DJ, Ito M, and Erdodi F. Myosin light chain phosphatase: subunit composition, interactions and regulation. J Muscle Res Cell Motil 19: 325–341, 1998.[CrossRef][ISI][Medline]

60. Hartshorne DJ, Ito M, and Erdodi F. Role of protein phosphatase type 1 in contractile functions: myosin phosphatase. J Biol Chem 279: 37211–37214, 2004.[Free Full Text]

61. Hathaway DR and Adelstein RS. Human platelet myosin light chain kinase requires the calcium-binding protein calmodulin for activity. Proc Natl Acad Sci USA 76: 1653–1657, 1979.[Abstract]

62. Heaps CL, Parker JL, Sturek M, and Bowles DK. Altered calcium sensitivity contributes to enhanced contractility of collateral-dependent coronary arteries. J Appl Physiol 97: 310–316, 2004.[Abstract/Free Full Text]

63. Hidaka H, Inagaki M, Kawamoto S, and Sasaki Y. Isoquinolinesulfonamides, novel and potent inhibitors of cyclic nucleotide dependent protein kinase and protein kinase C. Biochemistry 23: 5036–5041, 1984.[CrossRef][ISI][Medline]

64. Himpens B and Casteels R. Measurement by Quin2 of changes of the intracellular calcium concentration in strips of the rabbit ear artery and of the guinea-pig ileum. Pflügers Arch 408: 32–37, 1987.[CrossRef][Medline]

65. Himpens B and Somlyo AP. Free-calcium and force transients during depolarization and pharmacomechanical coupling in guinea-pig smooth muscle. J Physiol 395: 507–530, 1988.[Abstract]

66. Himpens B, Kitazawa T, and Somlyo AP. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflügers Arch 417: 21–28, 1990.[CrossRef][ISI][Medline]

67. Himpens R, Missiaen L, and Casteels R. Ca2+ homeostasis in vascular smooth muscle. J Vasc Res 32: 207–219, 1995.[ISI][Medline]

68. Hirano K, Derkach DN, Hirano M, Nishimura J, and Kanaide H. Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain. Mol Cell Biochem 248: 105–114, 2003.[CrossRef][ISI][Medline]

69. Holloway ET and Bohr DF. Reactivity of vascular smooth muscle in hypertensive rats. Circ Res 33: 678–685, 1973.[ISI][Medline]

70. Hori M and Karaki H. Regulatory mechanisms of calcium sensitiztion of contractile elements in smooth muscle. Life Sci 62: 1629–1633, 1998.[CrossRef][ISI][Medline]

71. Ichikawa K, Ito M, and Hartshorne DJ. Phosphorylation of the large subunit of myosin phosphatase and inhibition of phosphatase activity. J Biol Chem 271: 4733–4740, 1996.[Abstract/Free Full Text]

72. Imaizumi Y, Muraki K, Takeda M, and Watanabe M. Measurement and simulation of noninactivating Ca current in smooth muscle cells. Am J Physiol Cell Physiol 256: C880–C885, 1989.[Abstract/Free Full Text]

73. Inagaki M, Kawamoto S, and Hidaka H. Serotonin secretion from human platelets may be modified by Ca2+-activated, phospholipid-dependent myosin phosphorylation. J Biol Chem 259: 14321–14323, 1984.[Abstract/Free Full Text]

74. Ishizaki T, Uehata M, Tamechika I, Keel J, Nonomura K, Maekawa M, and Narumiya S. Pharmacological properties of Y-27632, a specific inhibitor of Rho-associated kinases. Mol Pharmacol 57: 976–983, 2000.[Abstract/Free Full Text]

75. Ito M, Nakano T, Erdodi F, and Hartshorne DJ. Myosin phosphatase: structure, regulation and function. Mol Cell Biochem 259: 197–209, 2004.[CrossRef][ISI][Medline]

76. Ito S, Kume H, Honjo H, Katoh H, Kodama I, Yamaki K, and Hayashi H. Possible involvement of Rho kinase in Ca2+ sensitization and mobilization by MCh in tracheal smooth muscle. Am J Physiol Lung Cell Mol Physiol 280: L1218–L1224, 2001.[Abstract/Free Full Text]

77. Janssen LJ, Tazzeo T, Zuo J, Pertens E, and Keshavjee S. KCl evokes contraction of airway smooth muscle via activation of RhoA and Rho kinase. Am J Physiol Lung Cell Mol Physiol 287: L852–L858, 2004.[Abstract/Free Full Text]

78. Je HD, Gallant C, Leavis PC, and Morgan KG. Caveolin-1 regulates contractility in differentiated vascular smooth muscle. Am J Physiol Heart Circ Physiol 286: H91–H98, 2004.[Abstract/Free Full Text]

79. Jezior JR, Brady JD, Rosenstein DI, McCammon KA, Miner AS, and Ratz PH. Dependency of detrusor contractions on calcium sensitization and calcium entry through LOE-908-sensitive channels. Br J Pharmacol 134: 78–87, 2001.[CrossRef][ISI][Medline]

80. Kamal A and Goldstein LS. Principles of cargo attachment to cytoplasmic motor proteins. Curr Opin Cell Biol 14: 63–68, 2002.[CrossRef][ISI][Medline]

81. Kamm KE and Stull JT. The function of myosin and myosin light chain kinase phosphorylation in smooth muscle. Annu Rev Pharmacol Toxicol 25: 593–620, 1985.[CrossRef][ISI][Medline]

82. Kamm KE and Stull JT. Dedicated myosin light chain kinases with diverse cellular functions. J Biol Chem 276: 4527–4530, 2001.[Free Full Text]

83. Kanaide H, Kobayashi S, Nishimura J, Hasegawa M, Shogakiuchi Y, Matsumoto T, and Nakamura M. Quin2 microfluorometry and effects of verapamil and diltiazem on calcium release from rat aorta smooth muscle cells in primary culture. Circ Res 63: 16–26, 1988.[Abstract]

84. Karaki H. Ca2+ localization and sensitivity in vascular smooth muscle. Trends Pharmacol Sci 10: 320–325, 1989.[CrossRef][ISI][Medline]

85. Karaki H. Historical techniques: cytosolic Ca2+ and contraction in smooth muscle. Trends Pharmacol Sci 25: 388–393, 2004.[CrossRef][ISI][Medline]

86. Karaki H, Sato K, and Ozaki H. Different effects of norepinephrine and KCl on the cytosolic Ca2+-tension relationship in vascular smooth muscle of rat aorta. Eur J Pharmacol 151: 325–328, 1988.[CrossRef][ISI][Medline]

87. Karaki H, Ozaki H, Hori M, Mitsui-Saito M, Amano K, Harada K, Miyamoto S, Nakazawa H, Won KJ, and Sato K. Calcium movements, distribution, and functions in smooth muscle. Pharmacol Rev 49: 157–230, 1997.[Abstract/Free Full Text]

88. Karnik SK, Wythe JD, Sorensen L, Brooke BS, Urness LD, and Li DY. Elastin induces myofibrillogenesis via a specific domain, VGVAPG. Matrix Biol 22: 409–425, 2003.[CrossRef][ISI][Medline]

89. Katoch SS and Moreland RS. Agonist and membrane depolarization induced activation of MAP kinase in the swine carotid artery. Am J Physiol Heart Circ Physiol 269: H222–H229, 1995.[Abstract/Free Full Text]

90. Kawamoto S and Hidaka H. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets. Biochem Biophys Res Commun 125: 258–264, 1984.[ISI][Medline]

91. Kawano Y, Yoshimura T, and Kaibuchi K. Smooth muscle contraction by small GTPase Rho. Nagoya J Med Sci 65: 1–8, 2002.[Medline]

92. Kim I, Je HD, Gallant C, Zhan Q, Riper DV, Badwey JA, Singer HA, and Morgan KG. Ca2+-calmodulin-dependent protein kinase II-dependent activation of contractility in ferret aorta. J Physiol 526: 367–374, 2000.[Abstract/Free Full Text]

93. Kitazawa T, Masuo M, and Somlyo AP. G protein-mediated inhibition of myosin light-chain phosphatase in vascular smooth muscle. Proc Natl Acad Sci USA 88: 9307–9310, 1991.[Abstract/Free Full Text]

94. Kitazawa T, Gaylinn BD, Denney GH, and Somlyo AP. G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation. J Biol Chem 266: 1708–1715, 1991.[Abstract/Free Full Text]

95. Kitazawa T, Eto M, Woodsome TP, and Brautigan DL. Agonists trigger G protein-mediated activation of the CPI-17 inhibitor phosphoprotein of myosin light chain phosphatase to enhance vascular smooth muscle contractility. J Biol Chem 275: 9897–9900, 2000.[Abstract/Free Full Text]

96. Kitazawa T, Eto M, Woodsome TP, and Khalequzzaman M. Phosphorylation of the myosin phosphatase targeting subunit and CPI-17 during Ca2+ sensitization in rabbit smooth muscle. J Physiol 546: 879–889, 2003.[Abstract/Free Full Text]

97. Kitazawa T, Kobayashi S, Horiuti K, Somlyo AV, and Somlyo AP. Receptor-coupled, permeabilized smooth muscle. Role of the phosphatidylinositol cascade, G-proteins, and modulation of the contractile response to Ca2+. J Biol Chem 264: 5339–5342, 1989.[Abstract/Free Full Text]

98. Klein U, Gimpl G, and Fahrenholz F. Alteration of the myometrial plasma membrane cholesterol content with beta-cyclodextrin modulates the binding affinity of the oxytocin receptor. Biochemistry 34: 13784–13793, 1995.[CrossRef][ISI][Medline]

99. Klemke RL, Cai S, Giannini AL, Gallagher PJ, de Lanerolle P, and Cheresh DA. Regulation of cell motility by mitogen-activated protein kinase. J Cell Biol 137: 481–492, 1997.[Abstract/Free Full Text]

100. Klockner U and Isenberg G. Calcium currents of cesium loaded isolated smooth muscle cells (urinary bladder of the guinea pig). Pflügers Arch 405: 340–348, 1985.[CrossRef][ISI][Medline]

101. Kobayashi S, Kanaide H, and Nakamura M. K+-depolarization induces a direct release of Ca2+ from intracellular storage sites in cultured vascular smooth muscle cells from rat aorta. Biochem Biophys Res Commun 129: 877–884, 1985.[CrossRef][ISI][Medline]

102. Kobayashi S, Kanaide H, and Nakamura M. Complete overlap of caffeine- and K+ depolarization-sensitive intracellular calcium storage site in cultured rat arterial smooth muscle cells. J Biol Chem 261: 15709–15713, 1986.[Abstract/Free Full Text]

103. Kobayashi S, Kitazawa T, Somlyo AV, and Somlyo AP. Cytosolic heparin inhibits muscarinic and {alpha}-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling. J Biol Chem 264: 17997–18004, 1989.[Abstract/Free Full Text]

104. Krutetskaia ZI, Lebedev OE, Krutetskaia NI, and Petrova TV. Organic and inorganic blockers of potential-dependent Ca2+ channels inhibit store-dependent entry of Ca2+ into rat peritoneal macrophages (in Russian). Tsitologiia 39: 1131–1141, 1997.[Medline]

105. Kubota Y, Nomura M, Kamm KE, Mumby MC, and Stull JT. GTP{gamma} S-dependent regulation of smooth muscle contractile elements. Am J Physiol Cell Physiol 262: C405–C410, 1992.[Abstract/Free Full Text]

106. Kupittayanant S, Burdyga T, and Wray S. The effects of inhibiting Rho-associated kinase with Y-27632 on force and intracellular calcium in human myometrium. Pflügers Arch 443: 112–114, 2001.[CrossRef][ISI][Medline]

107. Kuro-o M, Nagai R, Nakahara K, Katoh H, Tsai RC, Tsuchimochi H, Yazaki Y, Ohkubo A, and Takaku F. cDNA cloning of a myosin heavy chain isoform in embryonic smooth muscle and its expression during vascular development and in arteriosclerosis. J Biol Chem 266: 3768–3773, 1991.[Abstract/Free Full Text]

108. Lee CH, Poburko D, Kuo KH, Seow CY, and van Breemen C. Ca2+ oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Circ Physiol 282: H1571–H1583, 2002.[Abstract/Free Full Text]

109. Lenz T and Kleineke JW. Hormone-induced rise in cytosolic Ca2+ in axolotl hepatocytes: properties of the Ca2+ influx channel. Am J Physiol Cell Physiol 273: C1526–C1532, 1997.[Abstract/Free Full Text]

110. Leslie CC. Regulation of the specific release of arachidonic acid by cytosolic phospholipase A2. Prostaglandins Leukot Essent Fatty Acids 70: 373–376, 2004.[CrossRef][ISI][Medline]

111. Leung T, Manser E, Tan L, and Lim L. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem 270: 29051–29054, 1995.[Abstract/Free Full Text]

112. Leung T, Chen XQ, Manser E, and Lim L. The p160 RhoA-binding kinase ROK{alpha} is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol 16: 5313–5327, 1996.[Abstract]

113. Levent A and Buyukafsar K. Expression of Rho-kinase (ROCK-1 and ROCK-2) and its substantial role in the contractile activity of the sheep ureter. Br J Pharmacol 143: 431–437, 2004.[CrossRef][ISI][Medline]

114. Li L, Eto M, Lee MR, Morita F, Yazawa M, and Kitazawa T. Possible involvement of the novel CPI-17 protein in protein kinase C signal transduction of rabbit arterial smooth muscle. J Physiol 508: 871–881, 1998.[Abstract/Free Full Text]

115. Lohn M, Kampf D, Gui-Xuan C, Haller H, Luft FC, and Gollasch M. Regulation of arterial tone by smooth muscle myosin type II. Am J Physiol Cell Physiol 283: C1383–C1389, 2002.[Abstract/Free Full Text]

116. Lucius C, Arner A, Steusloff A, Troschka M, Hofmann F, Aktories K, and Pfitzer G. Clostidium difficile toxin B inhibits carbachol-induced force and myosin light chain phosphorylation in guinea-pig smooth muscle: role of Rho proteins. J Physiol 506: 83–93, 1998.[Abstract/Free Full Text]

117. MacDonald JA, Borman MA, Muranyi A, Somlyo AV, Hartshorne DJ, and Haystead TAJ. Identification of the endogenous smooth muscle myosin phosphatase-associated kinase. Proc Natl Acad Sci USA 98: 2419–2424, 2001.[Abstract/Free Full Text]

118. Makita T and Kiwaki S. Connection of microtubules, caveolae, mitochondria and sarcoplasmic reticulum in the taenia coli of guinea-pigs. Arch Histol Jpn 41: 167–176, 1978.[Medline]

119. Manser E, Leung T, and Lim L. Identification of GTPase-activating proteins by nitrocellulose overlay assay. Methods Enzymol 256: 130–139, 1995.[ISI][Medline]

120. McFadzean I and Gibson A. The developing relationship between receptor-operated and store-operated calcium channels in smooth muscle. Br J Pharmacol 135: 1–13, 2002.[ISI][Medline]

121. Mita M, Yanagihara H, Hishinuma S, Saito M, and Walsh MP. Membrane depolarization-induced contraction of rat caudal arterial smooth muscle involves Rho-associated kinase. Biochem J 364: 431–440, 2002.[CrossRef][ISI][Medline]

122. Miura M, Iwanaga T, Ito KM, Seto M, Sasaki Y, and Ito K. The role of myosin light chain kinase-dependent phosphorylation of myosin light chain in phorbol ester-induced contraction of rabbit aorta. Pflügers Arch 434: 685–693, 1997.[CrossRef][ISI][Medline]

123. Morano I. Tuning smooth muscle contraction by molecular motors. J Mol Med 81: 481–487, 2003.[CrossRef][ISI][Medline]

124. Morano I, Chai GX, Baltas LG, Lamounier-Zepter V, Lutsch G, Kott M, Haase H, and Bader M. Smooth-muscle contraction without smooth-muscle myosin. Nat Cell Biol 2: 371–375, 2000.[CrossRef][ISI][Medline]

125. Morgan JP and Morgan KG. Vascular smooth muscle: the first recorded Ca2+ transients. Pflügers Arch 395: 75–77, 1982.[CrossRef][ISI][Medline]

126. Morgan JP and Morgan KG. Alteration of cytoplasmic ionized calcium levels in smooth muscle by vasodilators in the ferret. J Physiol 357: 539–551, 1984.[Abstract]

127. Morgan JP and Morgan KG. Stimulus-specific patterns of intracellular calcium levels in smooth muscle of ferret portal vein. J Physiol 351: 155–167, 1984.[Abstract]

128. Morris RL and Hollenbeck PJ. Axonal transport of mitochondria along microtubules and F-actin in living vertebrate neurons. J Cell Biol 131: 1315–1326, 1995.[Abstract]

129. Nagaoka T, Morio Y, Casanova N, Bauer N, Gebb S, McMurtry I, and Oka M. Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 287: L665–L672, 2004.[Abstract/Free Full Text]

130. Nakahara T, Moriuchi H, Yunoki M, Sakamato K, and Ishii K. Y-27632 potentiates relaxant effects of {beta}2-adrenoceptor agonists in bovine tracheal smooth muscle. Eur J Pharmacol 389: 103–106, 2000.[CrossRef][ISI][Medline]

131. Nakayama S and Brading AF. Inactivation of the voltage-dependent Ca2+ channel current in smooth muscle cells isolated from the guinea-pig detrusor. J Physiol 471: 107–127, 1993.[Abstract]

132. Nakayama S and Brading AF. Long Ca2+ channel opening induced by large depolarization and Bay K 8644 in smooth muscle cells isolated from guinea-pig detrusor. Br J Pharmacol 119: 716–720, 1996.[ISI][Medline]

133. Neco P, Giner D, Viniegra S, Borges R, Villarroel A, and Gutierrez LM. New roles of myosin II during vesicle transport and fusion in chromaffin cells. J Biol Chem 279: 27450–27457, 2004.[Abstract/Free Full Text]

134. Neering IR and Morgan KG. Use of aequorin to study excitation-contraction coupling in mammalian smooth muscle. Nature 288: 585–587, 1980.[CrossRef][ISI][Medline]

135. Nelson MT, Standen NB, Brayden JE, and Worley JF. Noradrenaline contracts arteries by activating voltage-dependent calcium channels. Nature 336: 382–385, 1988.[CrossRef][ISI][Medline]

136. Niiro N, Koga Y, and Ikebe M. Agonist-induced changes in the phosphorylation of the myosin- binding subunit of myosin light chain phosphatase and CPI17, two regulatory factors of myosin light chain phosphatase, in smooth muscle. Biochem J 369: 117–128, 2003.[CrossRef][ISI][Medline]

137. Nishimura J, Kolber M, and van Breeman C. Norepinephrine and GTP-{gamma}-S increase myofilament Ca2+ sensitivity in {alpha}-toxin permeabilized arterial muscle. Biochem Biophys Res Commun 157: 677–683, 1988.[ISI][Medline]

138. North AJ, Galazkiewicz B, Byers TJ, Glenney JR Jr, and Small JV. Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle. J Cell Biol 120: 1159–1167, 1993.[Abstract]

139. Ohama T, Hori M, Sato K, Ozaki H, and Karaki H. Chronic treatment with interleukin-1{beta} attenuates contractions by decreasing the activities of CPI-17 and MYPT-1 in intestinal smooth muscle. J Biol Chem 278: 48794–48804, 2003.[Abstract/Free Full Text]

140. Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, and Spiegel S. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 147: 545–558, 1999.[Abstract/Free Full Text]

141. Otto B, Steusloff A, Just I, Aktories K, and Pfitzer G. Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle. J Physiol 496: 317–329, 1996.[Abstract]

142. Ozaki H, Sato K, Sakata K, and Karaki H. Endothelin dissociates muscle tension from cytosolic Ca2+ in vascular smooth muscle of rat carotid artery. Jpn J Pharmacol 50: 521–524, 1989.[ISI][Medline]

143. Ozaki H, Kwon SC, Tajimi M, and Karaki H. Changes in cytosolic Ca2+ and contraction induced by various stimulants and relaxants in canine tracheal smooth muscle. Pflügers Arch 416: 351–359, 1990.[CrossRef][ISI][Medline]

144. Pettus BJ, Bielawska A, Spiegel S, Roddy P, Hannun YA, and Chalfant CE. Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release. J Biol Chem 278: 38206–38213, 2003.[Abstract/Free Full Text]

145. Pfitzer G. Invited review: regulation of myosin phosphorylation in smooth muscle. J Appl Physiol 91: 497–503, 2001.[Abstract/Free Full Text]

146. Pfitzer G and Arner A. Involvement of small GTPases in the regulation of smooth muscle contraction. Acta Physiol Scand 164: 449–456, 1998.[ISI][Medline]

147. Poburko D, Kuo KH, Dai J, Lee CH, and van Breemen C. Organellar junctions promote targeted Ca2+ signaling in smooth muscle: why two membranes are better than one. Trends Pharmacol Sci 25: 8–15, 2004.[CrossRef][ISI][Medline]

148. Ratz PH. Effect of the kinase inhibitor, H-7, on stress, crossbridge phosphorylation, muscle shortening and inositol phosphate production in rabbit arteries. J Pharmacol Exp Ther 252: 253–259, 1990.[Abstract]

149. Ratz PH. Receptor activation induces short-term modulation of arterial contractions: memory in vascular smooth muscle. Am J Physiol Cell Physiol 269: C417–C423, 1995.[Abstract/Free Full Text]

150. Ratz PH. Dependence of Ca2+ sensitivity of arterial contractions on history of receptor activation. Am J Physiol Heart Circ Physiol 277: H1661–H1668, 1999.[Abstract/Free Full Text]

151. Ratz PH. Regulation of ERK phosphorylation in differentiated arterial muscle of the rabbit. Am J Physiol Heart Circ Physiol 281: H114–H123, 2001.[Abstract/Free Full Text]

152. Ratz PH and Murphy RA. Contributions of intracellular and extracellular Ca2+ pools to activation of myosin phosphorylation and stress in swine carotid media. Circ Res 60: 410–421, 1987.[Abstract]

153. Ratz PH, Hai CM, and Murphy RA. Dependence of stress on cross-bridge phosphorylation in vascular smooth muscle. Am J Physiol Cell Physiol 256: C96–C100, 1989.[Abstract/Free Full Text]

154. Ratz PH, Lattanzio FA Jr., and Salomonsky PM. Memory of arterial receptor activation involves reduced [Ca2+]i and desensitization of cross bridges to [Ca2+]i. Am J Physiol Cell Physiol 269: C1402–C1407, 1995.[Abstract/Free Full Text]

155. Ratz PH, Salomonsky PM, and Lattanzio FA Jr. Memory of previous receptor activation induces a delay in Ca2+ mobilization and decreases [Ca2+]i sensitivity of arterial contractions. J Vasc Res 33: 489–498, 1996.[ISI][Medline]

156. Ratz PH, Meehl JT, and Eddinger TJ. RhoA kinase and protein kinase C participate in regulation of rabbit stomach fundus smooth muscle contraction. Br J Pharmacol 137: 983–992, 2002.[CrossRef][ISI][Medline]

157. Razani B, Woodman SE, and Lisanti MP. Caveolae: from cell biology to animal physiology. Pharmacol Rev 54: 431–467, 2002.[Abstract/Free Full Text]

158. Rembold CM and Murphy RA. Myoplasmic [Ca2+] determines myosin phosphorylation in agonist-stimulated swine arterial smooth muscle. Circ Res 63: 593–603, 1988.[Abstract]

159. Rembold CM, Wardle RL, Wingard CJ, Batts TW, Etter EF, and Murphy RA. Cooperative attachment of cross bridges predicts regulation of smooth muscle force by myosin phosphorylation. Am J Physiol Cell Physiol 287: C594–C602, 2004.[Abstract/Free Full Text]

160. Ridley AJ and Hall A. The small GTP-binding protein Rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70: 389–399, 1992.[CrossRef][ISI][Medline]

161. Riento K and Ridley AJ. Rocks: multifunctional kinases in cell behaviour. Nat Rev Mol Cell Biol 4: 446–456, 2003.[CrossRef][ISI][Medline]

162. Rosenfeld SS, Xing J, Cheung HC, Brown F, Kar S, and Sweeney HL. Structural and kinetic studies of phosphorylation-dependent regulation in smooth muscle myosin. J Biol Chem 273: 28682–28690, 1998.[Abstract/Free Full Text]

163. Ruegg JC and Paul RJ. Vascular smooth muscle. Calmodulin and cyclic AMP-dependent protein kinase after calcium sensitivity in porcine carotid skinned fibers. Circ Res 50: 394–399, 1982.[Abstract]

164. Ruegg JC and Pfitzer G. Modulation of calcium sensitivity in guinea pig taenia coli: skinned fiber studies. Experientia 41: 997–1001, 1985.[ISI][Medline]

165. Sah VP, Seasholtz TM, Sagi SA, and Brown JH. The role of Rho in G protein-coupled receptor signal transduction. Annu Rev Pharmacol Toxicol 40: 459–489, 2000.[CrossRef][ISI][Medline]

166. Sakamoto K, Hori M, Izumi M, Oka T, Kohama K, Ozaki H, and Karaki H. Inhibition of high K+-induced contraction by the ROCKs inhibitor Y-27632 in vascular smooth muscle: possible involvement of ROCKs in a signal transduction pathway. J Pharm Sci 92: 56–69, 2003.[CrossRef]

167. Sakurada S, Takuwa N, Sugimoto N, Wang Y, Seto M, Sasaki Y, and Takuwa Y. Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction. Circ Res 93: 548–556, 2003.[Abstract/Free Full Text]

168. Sanders KM. Invited review: mechanisms of calcium handling in smooth muscles. J Appl Physiol 91: 1438–1449, 2001.[Abstract/Free Full Text]

169. Sato K, Ozaki H, and Karaki H. Changes in cytosolic calcium level in vascular smooth muscle strip measured simultaneously with contraction using fluorescent calcium indicator fura 2. J Pharmacol Exp Ther 246: 294–300, 1988.[Abstract]

170. Savineau JP and Marthan R. Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications. Fundam Clin Pharmacol 11: 289–299, 1997.[ISI][Medline]

171. Seasholtz TM, Majumdar M, and Brown JH. Rho as a mediator of G protein-coupled receptor signaling. Mol Pharmacol 55: 949–956, 1999.[Free Full Text]

172. Shabir S, Borisova L, Wray S, and Burdyga T. Rho-kinase inhibition and electromechanical coupling in phasic smooth muscle: Ca2+-dependent and independent mechanisms. J Physiol 560: 839–855, 2004.[Abstract/Free Full Text]

173. Shaul PW and Anderson RG. Role of plasmalemmal caveolae in signal transduction. Am J Physiol Lung Cell Mol Physiol 275: L843–L851, 1998.[Abstract/Free Full Text]

174. Smart EJ, Graf GA, McNiven MA, Sessa WC, Engelman JA, Scherer PE, Okamoto T, and Lisanti MP. Caveolins, liquid-ordered domains, and signal transduction. Mol Cell Biol 19: 7289–7304, 1999.[Free Full Text]

175. Sobieszek A. Ca-linked phosphorylation of a light chain of vertebrate smooth-muscle myosin. Eur J Biochem 73: 477–483, 1977.[Abstract]

176. Somlyo AP and Somlyo AV. From pharmacomechanical coupling to G-proteins and myosin phosphatase. Acta Physiol Scand 164: 437–448, 1998.[CrossRef][ISI][Medline]

177. Somlyo AP and Somlyo AV. Signal transduction by G-proteins, Rho-kinase and protein phosphatase to smooth muscle and non-muscle myosin II. J Physiol 522: 177–185, 2000.[Abstract/Free Full Text]

178. Somlyo AP and Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 83: 1325–1358, 2003.[Abstract/Free Full Text]

179. Somlyo AP, Xuqiong W, Walker L, and Somlyo A. Pharmacomechanical coupling: the role of calcium, G-proteins, kinases and phosphatase. Rev Physiol Biochem Pharmacol 440: 183–187, 1999.

180. Somlyo AV and Somlyo AP. Electromechanical and pharmacomechanical coupling in vascular smooth muscle. J Pharmacol Exp Ther 159: 129–145, 1968.[ISI][Medline]

181. Somlyo AV, Wang H, Choudhury N, Khromov AS, Majesky M, Owens GK, and Somlyo AP. Myosin light chain kinase knockout. J Muscle Res Cell Motil 25: 241–242, 2004.[CrossRef][ISI][Medline]

182. Spofford CM and Chilian WM. The elastin-laminin receptor functions as a mechanotransducer in vascular smooth muscle. Am J Physiol Heart Circ Physiol 280: H1354–H1360, 2001.[Abstract/Free Full Text]

183. Spofford CM and Chilian WM. Mechanotransduction via the elastin-laminin receptor (ELR) in resistance arteries. J Biomech 36: 645–652, 2003.[CrossRef][ISI][Medline]

184. Stull JT, Hsu LC, Tansey MG, and Kamm KE. Myosin light chain kinase phosphorylation in tracheal smooth muscle. J Biol Chem 265: 16683–16690, 1990.[Abstract/Free Full Text]

185. Sward K, Dreja K, Susnjar M, Hellstrand P, Hartshorne DJ, and Walsh MP. Inhibition of Rho-associated kinase blocks agonist-induced Ca2+ sensitization of myosin phosphorylation and force in guinea-pig ileum. J Physiol 522: 33–49, 2000.[Abstract/Free Full Text]

186. Taggart MJ. Smooth muscle excitation-contraction coupling: a role for caveolae and caveolins? News Physiol Sci 16: 61–65, 2001.[ISI][Medline]

187. Taggart MJ, Lee YH, and Morgan KG. Cellular redistribution of PKC{alpha}, RhoA, and ROK{alpha} following smooth muscle agonist stimulation. Exp Cell Res 251: 92–101, 1999.[CrossRef][ISI][Medline]

188. Taggart MJ, Leavis P, Feron O, and Morgan KG. Inhibition of PKC{alpha} and RhoA translocation in differentiated smooth muscle by a caveolin scaffolding domain peptide. Exp Cell Res 258: 72–81, 2000.[CrossRef][ISI][Medline]

189. Takayasu M, Suzuki Y, Shibuya M, Asano T, Kanamori M, Okada T, Kageyama N, and Hidaka H. The effects of HA compound calcium antagonists on delayed cerebral vasospasm in dogs. J Neurosurg 65: 80–85, 1986.[ISI][Medline]

190. Takizawa S, Hori M, Ozaki H, and Karaki H. Effects of isoquinoline derivatives, HA1077 and H-7, on cytosolic Ca2+ level and contraction in vascular smooth muscle. Eur J Pharmacol 250: 431–437, 1993.[CrossRef][ISI][Medline]

191. Takuwa Y, Takuwa N, and Rasmussen H. Measurement of cytoplasmic free Ca2+ concentration in bovine tracheal smooth muscle using aequorin. Am J Physiol Cell Physiol 253: C817–C827, 1987.[Abstract/Free Full Text]

192. Tanaka H, Hijikata T, Murakami T, Fujimaki N, and Ishikawa H. Localization of plectin and other related proteins along the sarcolemma in smooth muscle cells of rat colon. Cell Struct Funct 26: 61–70, 2001.[CrossRef][ISI][Medline]

193. Tang D, Mehta D, and Gunst SJ. Mechanosensitive tyrosine phosphorylation of paxillin and focal adhesion kinase in tracheal smooth muscle. Am J Physiol Cell Physiol 276: C250–C258, 1999.[Abstract/Free Full Text]

194. Tansey MG, Luby-Phelps K, Kamm KE, and Stull JT. Ca2+-dependent phosphorylation of myosin light chain kinase decreases the Ca2+ sensitivity of light chain phosphorylation within smooth muscle cells. J Biol Chem 269: 9912–9920, 1994.[Abstract/Free Full Text]

195. Tansey MG, Word RA, Hidaka H, Singer HA, Schworer CM, Kamm KE, and Stull JT. Phosphorylation of myosin light chain kinase by the multifunctional calmodulin-dependent protein kinase II in smooth muscle cells. J Biol Chem 267: 12511–12516, 1992.[Abstract/Free Full Text]

196. Thomsen P, Roepstorff K, Stahlhut M, and van Deurs B. Caveolae are highly immobile plasma membrane microdomains, which are not involved in constitutive endocytic trafficking. Mol Biol Cell 13: 238–250, 2002.[Abstract/Free Full Text]

197. Tomomasa T, Takahashi A, Kaneko H, Watanabe T, Tabata M, Kato M, and Morikawa A. Y-27632 inhibits gastric motility in conscious rats. Life Sci 66: PL29–34, 2000.[CrossRef][ISI][Medline]

198. Torihashi S, Fujimoto T, Trost C, and Nakayama S. Calcium oscillation linked to pacemaking of interstitial cells of Cajal: requirement of calcium influx and localization of TRP4 in caveolae. J Biol Chem 277: 19191–19197, 2002.[Abstract/Free Full Text]

199. Trybus KM, Waller GS, and Chatman TA. Coupling of ATPase activity and motility in smooth muscle myosin is mediated by the regulatory light chain. J Cell Biol 124: 963–969, 1994.[Abstract]

200. Uehata M, Ishizaki T, Satoh H, Ono T, Kawahara T, Morishita T, Tamakawa H, Yamagami K, Inui J, Maekawa M, and Narumiya S. Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension. Nature 389: 990–994, 1997.[CrossRef][ISI][Medline]

201. Urban NH and Ratz PH. Vascular smooth muscle (VSM) cell memory involves inhibition of ROK signaling and caveolin internalization. FASEB J 18: A1089–A1089, 2004.

202. Urban NH, Berg KM, and Ratz PH. K+ depolarization induces RhoA kinase translocation to caveolae and Ca2+ sensitization of arterial muscle. Am J Physiol Cell Physiol 285: C1377–C1385, 2003.[Abstract/Free Full Text]

203. Van Breemen C, Hwang O, and Meisheri KD. The mechanism of inhibitory action of diltiazem on vascular smooth muscle contractility. J Pharmacol Exp Ther 218: 459–463, 1981.[Abstract]

204. Van Breemen C, Chen Q, and Laher I. Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends Pharmacol Sci 16: 98–105, 1995.[CrossRef][ISI][Medline]

205. Van Breemen C, Farinas BR, Gerba P, and McNaughton ED. Excitation-contraction coupling in rabbit aorta studied by the lanthanum method for measuring cellular calcium influx. Circ Res 30: 44–54, 1972.[ISI][Medline]

206. Velasco G, Armstrong C, Morrice N, Frame S, and Cohen P. Phosphorylation of the regulatory subunit of smooth muscle protein phosphatase 1M at Thr850 induces its dissociation from myosin. FEBS Lett 527: 101–104, 2002.[CrossRef][ISI][Medline]

207. Welte MA. Bidirectional transport along microtubules. Curr Biol 14: R525–R537, 2004.[CrossRef][ISI][Medline]

208. Wettschureck N and Offermanns S. Rho/Rho-kinase mediated signaling in physiology and pathophysiology. J Mol Med 80: 629–638, 2002.[CrossRef][ISI][Medline]

209. Willmott NJ, Choudhury Q, and Flower RJ. Functional importance of the dihydropyridine-sensitive, yet voltage-insensitive store-operated Ca2+ influx of U937 cells. FEBS Lett 394: 159–164, 1996.[CrossRef][ISI][Medline]

210. Wollert T, DePina AS, Sandberg LA, and Langford GM. Reconstitution of active pseudo-contractile rings and myosin-II-mediated vesicle transport in extracts of clam oocytes. Biol Bull 201: 241–243, 2001.[Free Full Text]

211. Woodrum DA and Brophy CM. The paradox of smooth muscle physiology. Mol Cell Endocrinol 177: 135–143, 2001.[CrossRef][ISI][Medline]

212. Wright G and Hurn E. Cytochalasin inhibition of slow tension increase in rat aortic rings. Am J Physiol Heart Circ Physiol 267: H1437–H1446, 1994.[Abstract/Free Full Text]

213. Yamada K, Goto A, Matsuoka H, and Sugimoto T. Alterations of calcium channels in vascular smooth muscle cells from spontaneously hypertensive rats. Jpn Heart J 33: 727–734, 1992.[ISI][Medline]

214. Yanagisawa T and Okada Y. KCl depolarization increases Ca2+ sensitivity of contractile elements in coronary arterial smooth muscle. Am J Physiol Heart Circ Physiol 267: H614–H621, 1994.[Abstract/Free Full Text]

215. Yanagisawa T, Kawada M, and Taira N. Nitroglycerin relaxes canine coronary arterial smooth muscle without reducing intracellular Ca2+ concentrations measured with fura-2. Br J Pharmacol 98: 469–482, 1989.[ISI][Medline]

216. Young RC, Schumann R, and Zhang P. Nifedipine block of capacitative calcium entry in cultured human uterine smooth-muscle cells. J Soc Gynecol Investig 8: 210–215, 2001.[CrossRef][ISI][Medline]

217. Zamir E and Geiger B. Molecular complexity and dynamics of cell-matrix adhesions. J Cell Sci 114: 3583–3590, 2001.[ISI][Medline]