Cyclic GMP-dependent protein kinase Ialpha inhibits thrombin receptor-mediated calcium mobilization in vascular smooth muscle cells

Vascular smooth muscle contractile state is regulated by intracellular calcium levels. Nitric oxide causes vascular relaxation by stimulating production of cyclic GMP, which activates type I cGMP-dependent protein kinase (PKGI) in vascular smooth muscle cells (VSMC), inhibiting agonist-induced intracellular Ca2+ mobilization ([Ca2+]i). The relative roles of the two PKGI isozymes, PKGIalpha and PKGIbeta, in cyclic GMP-mediated inhibition of [Ca2+]i in VSMCs are unclear. Here we have investigated the ability of PKGI isoforms to inhibit [Ca2+]i in response to VSMC activation. Stable Chinese hamster ovary cell lines expressing PKGIalpha or PKGIbeta were created, and the ability of PKGI isoforms to inhibit [Ca2+]i in response to thrombin receptor stimulation was examined. In Chinese hamster ovary cells stably expressing PKGIalpha or PKGIbeta, 8-Br-cGMP activation suppressed [Ca2+]i by thrombin receptor activation peptide (TRAP) by 98 +/- 1 versus 42 +/- 5%, respectively (p <0.002). Immunoblotting studies of cultured human VSMC cells from multiple sites using PKGIalpha- and PKGIbeta-specific antibodies showed PKGIalpha is the predominant VSMC PKGI isoform. [Ca2+]i following thrombin receptor stimulation was examined in the absence or presence of cyclic GMP in human coronary VSMC cells (Co403). 8-Br-cGMP significantly inhibited TRAP-induced [Ca2+]i in Co403, causing a 4-fold increase in the EC50 for [Ca2+]i. In the absence of 8-Br-cGMP, suppression of PKGIalpha levels by RNA interference (RNAi) led to a significantly greater TRAP-stimulated rise in [Ca2+]i as compared with control RNAi-treated Co403 cells. In the presence of 8-Br-cGMP, the suppression of PKGIalpha expression by RNAi led to the complete loss of cGMP-mediated inhibition of [Ca2+]i. Adenoviral overexpression of PKGIbeta in Co403 cells was unable to alter TRAP-stimulated Ca2+ mobilization either before or after suppression of PKGIalpha expression by RNAi. These results support that PKGIalpha is the principal cGMP-dependent protein kinase isoform mediating inhibition of VSMC activation by the nitric oxide/cyclic GMP pathway.     

Cyclic GMP-dependent protein kinase stimulates the plasmalemmal Ca2+ pump of smooth muscle via phosphorylation of phosphatidylinositol.

The effect of phosphorylation by cyclic GMP-dependent protein kinase (G-kinase) on the activity of the plasmalemmal Ca2+-transport ATPase was studied on isolated plasma membranes and on the ATPase purified from pig erythrocytes and from the smooth muscle of pig stomach and pig aorta. Incubation with G-kinase resulted, in both smooth-muscle preparations, but not in the erythrocyte ATPase, in a higher Ca2+ affinity and in an increase in the maximal rate of Ca2+ uptake. Cyclic AMP-dependent protein kinase (A-kinase) did not exert such an effect. The stimulation of the (Ca2+ + Mg2+)-dependent ATPase activity of the purified Ca2+ pump reconstituted in liposomes depended on the phospholipid used for reconstitution. The stimulation of the (Ca2+ + Mg2+)-ATPase activity by G-kinase was only observed in the presence of phosphatidylinositol (PI). G-kinase, but not A-kinase, stimulated the phosphorylation of PI to phosphatidylinositol phosphate (PIP) in a preparation of (Ca2+ + Mg2+)-ATPase obtained by calmodulin affinity chromatography from smooth muscle, but not in a similar preparation from erythrocytes. Adenosine inhibited both the phosphorylation of PI and the stimulation of the (Ca2+ + Mg2+)-ATPase by G-kinase. In the absence of G-kinase the (Ca2+ + Mg2+)-ATPase was stimulated by the addition of PIP, but not by PI. In contrast with previous results of Furukawa & Nakamura [(1987) J. Biochem (Tokyo) 101, 287-290], no convincing evidence for a phosphorylation of the (Ca2+ + Mg2+)-ATPase was found. Evidence is presented showing that the apparent phosphorylation occurs in a contaminant protein, possibly myosin light-chain kinase. It is proposed that G-kinase stimulates the plasmalemmal Ca2+ pump of smooth-muscle cells indirectly via the phosphorylation of an associated PI kinase.     

Cyclic GMP-dependent protein kinase stimulates the plasma membrane Ca2+ pump ATPase of vascular smooth muscle via phosphorylation of a 240-kDa protein.

The plasma membrane Ca2+ pump ATPase from porcine aorta was isolated by the calmodulin affinity chromatographic method of Kosk-Kosicka et al. (Kosk-Kosicka, D., Scaillet, S., and Inesi, G. (1986) J. Biol. Chem. 261, 3333-3338). Its activity was restored by adding either phosphatidylcholine or phosphatidylserine. Cyclic GMP-dependent protein kinase (G-kinase) stimulated the enzyme in a concentration-dependent manner. However, phosphatidylinositol kinase (PI-kinase) activity was not detected in the enzyme preparation, and the presence of phosphatidylinositol was not necessary for stimulation by G-kinase. Furthermore, adenosine, a potent PI-kinase inhibitor, did not affect the stimulation. The enzyme preparation contained three major proteins, with molecular masses of 240, 145, and 135 kDa, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 240- and 135-kDa proteins were phosphorylated in association with the stimulation by G-kinase, but only the phosphorylation of the 240-kDa protein was dependent on the G-kinase concentration. A purified enzyme without the 240-kDa protein, prepared by our previous method (Imai, S., Yoshida, Y., and Sun, H.-T. (1990) J. Biochem. (Tokyo) 107, 755-761), was not activated by G-kinase. Immunoblotting with an antibody against the human erythrocyte Ca2+ pump revealed that the 135-kDa protein corresponded to one of the isoforms of the plasma membrane Ca2+ pump. These results suggest that the phosphorylation of the 240-kDa protein is responsible for stimulation of the plasma membrane Ca2+ pump ATPase by G-kinase.     

Ca2+-independent phospholipase A2 is required for agonist-induced Ca2+ sensitization of contraction in vascular smooth muscle

Excitatory agonists can induce significant smooth muscle contraction under constant free Ca(2+) through a mechanism called Ca(2+) sensitization. Considerable evidence suggests that free arachidonic acid plays an important role in mediating agonist-induced Ca(2+)-sensitization; however, the molecular mechanisms responsible for maintaining and regulating free arachidonic acid level are not completely understood. In the current study, we demonstrated that Ca(2+)-independent phospholipase A(2) (iPLA(2)) is expressed in vascular smooth muscle tissues. Inhibition of the endogenous iPLA(2) activity by bromoenol lactone (BEL) decreases basal free arachidonic acid levels and reduces the final free arachidonic acid level after phenylephrine stimulation, without significant effect on the net increase in free arachidonic acid stimulated by phenylephrine. Importantly, BEL treatment diminishes agonist-induced Ca(2+) sensitization of contraction from 49 +/- 3.6 to 12 +/- 1.0% (p < 0.01). In contrast, BEL does not affect agonist-induced diacylglycerol production or contraction induced by Ca(2+), phorbol 12,13-dibutyrate (a protein kinase C activator), or exogenous arachidonic acid. Further, we demonstrate that adenovirus-mediated overexpression of exogenous iPLA(2) in mouse portal vein tissue significantly potentiates serotonin-induced contraction. Our data provide the first evidence that iPLA(2) is required for maintaining basal free arachidonic acid levels and thus is essential for agonist-induced Ca(2+)-sensitization of contraction in vascular smooth muscle.     

Tyrosine phosphorylation increases Ca2+ sensitivity of vascular smooth muscle contraction

In order to elucidate the role of tyrosine phosphorylation in vasoconstriction, we investigated the effects of inhibitors of tyrosine kinase (genistein, 30 microM) and phosphatase (sodium o-vanadate, 5 microM) on the contraction of aorta isolated from guinea pig. Genistein significantly inhibited norepinephrine-induced contraction, but it did not affect that induced by KCI. Thus, tyrosine phosphorylation may not be involved in the contractile response to KCI alone. The aortic contraction elicited by KCl was significantly augmented by sodium o-vanadate, which increased both the maximum force and pD2 values of KCl contraction. In the presence of verapamil, KCl-induced contraction was abolished even after pretreatment with sodium o-vanadate. Sodium o-vanadate also augmented Ca2+-induced contraction in the aortic strips depolarized with KCl, increasing both its maximum force and pD2 values. Neither basal 45Ca2+ uptake nor verapamil-sensitive 45Ca2+ uptake induced by KCl were affected by pretreatment with sodium o-vanadate. These results suggest that tyrosine phosphorylation is involved in the contraction of guinea-pig aorta not through transplasmalemmal Ca2+ entry but through increased Ca2+ sensitivity of the intracellular contractile pathway.     

Characterization of protein kinase pathways responsible for Ca2+ sensitization in rat ileal longitudinal smooth muscle

We investigated the protein kinases responsible for myosin regulatory light chain (LC20) phosphorylation and regulation of myosin light chain phosphatase (MLCP) activity during microcystin (phosphatase inhibitor)-induced contraction at low Ca2+ concentrations of rat ileal smooth muscle stretched in the longitudinal axis. Application of 1 microM microcystin induced LC20 diphosphorylation and contraction of beta-escin-permeabilized rat ileal smooth muscle at pCa 9. The PKC inhibitor GF-109203x, the MEK inhibitor PD-98059, and the p38 MAPK inhibitor SB-203580 significantly reduced this contraction. These inhibitory effects were abolished when the microcystin concentration was increased to 10 muM, indicating that application of these kinase inhibitors generated an increase in MLCP activity. GF-109203x and PD-98059, but not SB-203580, significantly decreased the phosphorylation level of the myosin-targeting subunit of MLCP, MYPT1, at Thr-697 (rat sequence) during microcystin-induced contraction at pCa 9. On the other hand, SB-203580, but not GF-109203x or PD-98059, significantly reduced the phosphorylation level of the PKC-potentiated phosphatase inhibitor of 17 kDa (CPI-17). A zipper-interacting protein kinase (ZIPK) inhibitor (SM1 peptide) and a Rho-associated kinase inhibitor (Y-27632) had little effect on microcystin-induced contraction at pCa 9. In conclusion, PKC, ERK1/2, and p38 MAPK pathways facilitate microcystin-induced contraction at low Ca2+ concentrations by contributing to the inhibition of MLCP activity either through phosphorylation of MYPT1 or CPI-17 [probably mediated by integrin-linked kinase (ILK)]. ILK and not ZIPK is likely to be the protein kinase responsible for LC20 diphosphorylation during microcystin-induced contraction of rat ileal smooth muscle at pCa 9, similar to its recently described role in vascular smooth muscle. The negative regulation of MLCP by PKC and MAPKs during microcystin-induced contraction at pCa 9, which is not observed in vascular smooth muscle, may be unique to phasic smooth muscle.     

G-protein-mediated Ca2+ sensitization of smooth muscle contraction through myosin light chain phosphorylation.

The Ca2+ sensitivities of tonic (pulmonary and femoral artery) and phasic (portal vein and ileum) smooth muscles and the effects of guanosine 5'-O-(gamma-thiotriphosphate) (GTP gamma S) and norepinephrine on Ca2+ sensitivity of force development and myosin light chain (MLC20) phosphorylation were determined in permeabilized preparations that retained coupled receptors and endogenous calmodulin. The Ca2+ sensitivity of force was higher (approximately 3-fold) in the tonic than in the phasic smooth muscles. The nucleotide specificity of Ca2+ sensitization was: GTP gamma S much greater than GTP greater than ITP much greater than CTP = UTP. Baseline phosphorylation (7% at pCa greater than 8) and maximal phosphorylation (58% at pCa 5.0) were both lower in portal vein than in femoral artery (20 and 97%). Norepinephrine and GTP gamma S increased phosphorylation at constant [Ca2+] (pCa 7.0-6.5). MLC20 phosphorylation induced by norepinephrine was completely inhibited by guanosine 5'-O-(beta-thiodiphosphate) (GDP beta S). In portal vein at pCa 5, GTP gamma S increased phosphorylation from 58%, the maximal Ca2(+)-activated value, to 75%, and at pCa greater than 8, from 7 to 13%. In femoral artery at pCa 5, neither phosphorylation (97%) nor force was affected by GTP gamma S, while at pCa greater than 8, GTP gamma S caused an increase in force (16% of maximum) with a borderline increase in MLC20 phosphorylation (from 20 to 27%). MLC20 phosphorylation (up to 100%) was positively correlated with force. The major results support the hypothesis that the G-protein coupled Ca2(+)-sensitizing effect of agonists on force development is secondary to increased MLC20 phosphorylation.     

Ca2+-independent contraction of uterine smooth muscle

Rat uterine smooth muscle shows sustained contraction to oxytocin in Ca2+-free medium with EGTA, that is called "Ca-free contraction"(1). Participation of the rise in cytosolic free Ca2+ in this Ca-free contraction was tested. In Ca-free contraction, the cytosolic free Ca2+ level was not changed at all as measured with fura-2. Further, the chelation of cytosolic free Ca2+ with quin-2 did not at all affect Ca-free contraction. These results strongly suggest that Ca-free contraction is not triggered by Ca2+.     

Regulation of intracellular Ca2+ levels in cultured vascular smooth muscle cells. Reduction of Ca2+ by atriopeptin and 8-bromo-cyclic GMP is mediated by cyclic GMP-dependent protein kinase

Primary rat aortic cells, when treated with arginine vasopressin or depolarizing concentrations of K+, responded to atriopeptin II and 8-bromo-cGMP (8-Br-cGMP) with decreases in intracellular Ca2+ levels. The effects of atriopeptin and 8-Br-cGMP were diminished in cells which had been passaged many times. Low levels of cGMP-dependent protein kinase were present in soluble extracts prepared from the unresponsive cells in later passage compared with extracts from responsive cells. Unresponsive cells, when induced to incorporate cGMP-dependent protein kinase into the cytoplasm using the osmotic lysis procedure of Okada and Rechsteiner (Okada, C. Y., and Rechsteiner, M. (1982) Cell 29, 33-41), responded to atriopeptin and 8-Br-cGMP with reductions in peak Ca2+ levels in response to vasopressin and depolarizing concentrations of K+. Cells which were furnished with affinity-purified antibody to the cGMP-dependent protein kinase after the introduction of the kinase remained unresponsive to the effects of atriopeptin. In addition, antibody furnished to responsive primary cultured cells inhibited the effects of atriopeptin and 8-Br-cGMP on Ca2+ levels. These data suggest that repetitively passaged cultured rat aortic smooth muscle cells lose their responsiveness to cGMP concurrently with the loss of cGMP-dependent protein kinase. Restoration of kinase to the cells results in the restoration of responsiveness to cGMP. Thus cGMP-dependent protein kinase appears to be the mediator of the reduction in Ca2+ levels upon elevation of intracellular cGMP.     

Cyclic GMP-dependent protein kinase activation in canine tracheal smooth muscle by methacholine and sodium nitroprusside

Methacholine (3 μM) and sodium nitroprusside (300 μM) increased cGMP-dependent protein kinase activity ratios (activity without cGMP divided by activity with 2 μM cGMP) in canine tracheal smooth muscle from a control value of 0.47 to 0.55 and 0.71, respectively. This correlates with 3-fold and 6-fold increases in cGMP concentrations in response to methacholine and sodium nitroprusside, respectively. Addition of charcoal to the homogenizing buffer prior to homogenization had no significant effect on the cGMP-dependent protein kinase response to either agent, suggesting that activation of the enzyme was not occurring as a result of cGMP release during homogenization. In order to limit cGMP dissociation from cGMP-dependent protein kinase during the assay procedure, it was necessary to perform assays at a reduced temperature (0°C) and with an abbreviated incubation time (2.5 min). When assayed at 30°C, activated cGMP-dependent protein kinase rapidly lost activity. This inactivation occurred whether the enzyme had been activated exogenously, by exposing a supernatant fraction of canine trachealis to 0.1 μM cGMP, or endogenously, by treating intact canine trachealis with methacholine or sodium nitroprusside. By assaying instead at 0°C, the inactivation of cGMP-dependent protein kinase was minimized. Therefore, the activity ratio obtained by this new modified assay provided an estimate of the endogenous activation state of cGMP-dependent protein kinase. The data indicate that cGMP responses in canine trachealis to both methacholine and sodium nitroprusside are functionally linked to activation of cGMP-dependent protein kinase and are consistent with the hypothesis that cGMP, via cGMP-dependent protein kinase activation, regulates smooth muscle contractility.     

Ca2+ regulation of vascular smooth muscle

Regulation of intracellular free Ca2+ concentrations in vascular smooth muscle is accomplished mainly by Ca2+ channels and ATP-dependent Ca2+ pumps in the plasmalemma and sarcoplasmic reticulum (SR). Ca2+ entry through the plasmalemma is apparently mediated by four different pathways: leak; receptor-operated Ca2+ channels; potential sensitive Ca2+ channels; and stretch-activated channels. The agonist releasable intracellular Ca2+ store appears to be identical with the SR. Evidence for the involvement of Ca2+-induced Ca2+ release and inositol-1,4,5-trisphosphate in the release of SR Ca2+ is discussed. Smooth muscle contractions induced by certain agonists may be further enhanced by inhibition of Ca2+ uptake by the SR and of active Ca2+ extrusion across the plasmalemma. At the moment it is not clear from a consideration of the Ca2+ regulatory mechanisms present in vascular smooth muscle how dietary Ca2+ affects vascular tone. The increased Ca2+ permeation through smooth muscle cell membranes of resistance arteries taken from spontaneously hypertensive rats may be relevant to this problem.     

Down-regulation of G-protein-mediated Ca2+ sensitization in smooth muscle.

Prolonged treatment with guanosine 5'-[gamma-thio]triphosphate (GTP gamma S; 5-16 h, 50 microM) of smooth muscle permeabilized with Staphylococcus aureus alpha-toxin down-regulated (abolished) the acute Ca2+ sensitization of force by GTP gamma S, AIF-4, phenylephrine, and endothelin, but not the response to phorbol dibutyrate or a phosphatase inhibitor, tautomycin. Down-regulation also abolished the GTP gamma S-induced increase in myosin light chain phosphorylation at constant [Ca2+] and was associated with extensive translocation of p21rhoA to the particulate fraction, prevented its immunoprecipitation, and inhibited its ADP ribosylation without affecting the immunodetectable content of G-proteins (p21rhoA, p21ras, G alpha q/11, G alpha i3, and G beta) or protein kinase C (types alpha, beta 1, beta 2, delta, epsilon, eta, theta, and zeta). We conclude that the loss of GTP gamma S- and agonist-induced Ca2+ sensitization through prolonged treatment with GTP gamma S is not due to a decrease in the total content of either trimeric (G alpha q/11, G alpha i3, and G beta) or monomeric (p21rhoA and p21ras) G-protein or protein kinase C but may be related to a structural change of p21rhoA and/or to down-regulation of its (yet to be identified) effector.     

Ca2+-independent smooth muscle contraction. a novel function for integrin-linked kinase

Smooth muscle contraction follows an increase in cytosolic Ca(2+) concentration, activation of myosin light chain kinase, and phosphorylation of the 20-kDa light chain of myosin at Ser(19). Several agonists acting via G protein-coupled receptors elicit a contraction without a change in [Ca(2+)](i) via inhibition of myosin light chain phosphatase and increased myosin phosphorylation. We showed that microcystin (phosphatase inhibitor)-induced contraction of skinned smooth muscle occurred in the absence of Ca(2+) and correlated with phosphorylation of myosin light chain at Ser(19) and Thr(18) by a kinase distinct from myosin light chain kinase. In this study, we identify this kinase as integrin-linked kinase. Chicken gizzard integrin-linked kinase cDNA was cloned, sequenced, expressed in E. coli, and shown to phosphorylate myosin light chain in the absence of Ca(2+) at Ser(19) and Thr(18). Subcellular fractionation revealed two distinct populations of integrin-linked kinase, including a Triton X-100-insoluble component that phosphorylates myosin in a Ca(2+)-independent manner. These results suggest a novel function for integrin-linked kinase in the regulation of smooth muscle contraction via Ca(2+)-independent phosphorylation of myosin, raise the possibility that integrin-linked kinase may also play a role in regulation of nonmuscle motility, and confirm that integrin-linked kinase is indeed a functional protein-serine/threonine kinase.     

Ca2+ regulation in vascular smooth muscle.

Regulation of aorta smooth muscle contraction by Ca ion requires the collaboration of the 80,000 dalton factor and tropomyosin. A method for preparing pure actin from aorta smooth muscle is described.     

Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization

Endothelin-1 (ET) induces increases in intracellular Ca(2+) concentration ([Ca(2+)](i)), Ca(2+) sensitization, and contraction of both bronchiole and pulmonary arteriole smooth muscle cells (SMCs) and may play an important role in the pathophysiology of asthma and pulmonary hypertension. However, because it remains unclear how changes in [Ca(2+)](i) and the Ca(2+) sensitivity regulate SMC contraction, we have studied mouse lung slices with phase-contrast and confocal microscopy to correlate the ET-induced contraction with the changes in [Ca(2+)](i) and Ca(2+) sensitivity of bronchiole and arteriole SMCs. In comparison with acetylcholine (ACh) or serotonin (5-HT), ET induced a stronger and long-lasting contraction of both bronchioles and arterioles. This ET-induced contraction was associated with prominent asynchronous Ca(2+) oscillations that were propagated as Ca(2+) waves along the SMCs. These Ca(2+) oscillations were mediated by cyclic intracellular Ca(2+) release and required external Ca(2+) for their maintenance. Importantly, as the frequency of the Ca(2+) oscillations increased, the extent of contraction increased. ET-induced contraction was also associated with an increase in Ca(2+) sensitivity. In "model" slices in which the [Ca(2+)](i) was constantly maintained at an elevated level by pretreatment of slices with caffeine and ryanodine, the addition of ET increased bronchiole and arteriole contraction. These results indicate that ET-induced contraction of bronchiole and arteriole SMCs is regulated by the frequency of Ca(2+) oscillations and by increasing the sensitivity of the contractile machinery to Ca(2+).     

Ca2+-independent stimulation of cyclic GMP-dependent protein kinase by calmodulin

Calmodulin purified from bovine brain markedly stimulated cyclic GMP-dependent protein kinase from pig lung in the presence of cyclic GMP. This stimulation by calmodulin did not require Ca²⁺ and was dose-dependent up to optimal amounts, but the extent of stimulation decreased at concentrations over the optimal condition. The concentrations of cyclic GMP and cyclic AMP producing half-maximal stimulation were 4.5 × 10^?8 M and 5.0 × 10^?6 M respectively, under optimal conditions. Calmodulin increased maximum velocity without altering the Km for ATP. These effects of calmodulin on cyclic GMP-dependent protein kinase were similar to those of the stimulatory modulator described by Kuo and Kuo (J. Biol. Chem. $\text{251}$, 4283–4286, 1976). Ouf findings indicate that calmodulin regulates enzyme activity both Ca²⁺-dependently and independently.     

Extracellular signal-regulated kinase1/2 in contraction of vascular smooth muscle

Smooth muscle contractility is regulated by both intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ sensitivity of the contractile apparatus. Extracellular signal-regulated kinases1/2 (ERK1/2) have been implicated in modulating Ca2+ sensitivity of smooth muscle contraction but mechanisms of action remain elusive. This study investigated the roles of ERK1/2 in modulating [Ca2+]i, calcium sensitivity and the 20-kDa myosin light chain (MLC20) phosphorylation during contraction activated by alpha1-adrenoceptor agonist phenylephrine and thromboxane A2 mimetic U46619 in rat tail artery strips. A specific inhibitor for ERK1/2 activation, U0126, inhibited phenylephrine- and U46619-induced contraction, shifting both concentration-response curves rightward. During phenylephrine-stimulated contraction, U0126 exhibited concentration-dependent inhibition towards force but significant decreases in [Ca2+]i were detected only at higher concentration. Both phenylephrine and U46619 induced a transient activation of ERK1/2 which was abolished by U0126 but unaffected by a general tyrosine kinase inhibitor genistein or Rho kinase inhibitor Y27632 at concentrations inhibiting more than 50% force. Interestingly, U0126 had no effect on steady-state MLC20 phosphorylation levels stimulated by both receptor agonists. These results indicated that during contraction of rat tail artery smooth muscle activated by alpha1-adrenoceptor agonist or thromboxane A2 analogue, ERK1/2 increase Ca2+ sensitivity that does not involve the modulation of MLC20 phosphorylation.