Urocortin-Induced Decrease in Ca 2+ Sensitivity of Contraction in Mouse Tail Arteries Is Attributable to cAMP-Dependent Dephosphorylation of MYPT1 and Activation of Myosin Light Chain Phosphatase (original) (raw)
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Biophysics, 2006
Urocortin is a peptide hormone related to corticotrophin-releasing factor. It is assumed that urocortin is involved in blood pressure regulation by dilating the peripheral blood vessels. In rat tail arteries, urocortin-induced vasodilation is caused by a decrease in the myofilament Ca 2+ sensitivity, the mechanism of which is still unclear. In this study, the hypothesis was tested that the decrease in the Ca 2+ sensitivity in mouse tail arteries results from the activation of myosin light chain phosphatase. The relaxation of KCl (42 mM) precontracted intact mouse tail arteries by 1 and 10 nM urocortin was significantly inhibited by 1 μM antisauvagine-30, a CRF-2 receptor antagonist (p < 0.05, n = 3). The addition of 1 μM KT 5720, a protein kinase A inhibitor, to intact rat tail arteries did not affect the KCl-induced force but significantly attenuated the urocortin-induced relaxation (n = 5). In α-toxin-permeabilized mouse tail arteries, urocortin relaxed activated preparations at constant pCa 6.1 by 37.6 ± 8.2% (n = 5) as compared with reference vessels (n = 5, p < 0.001). The relaxation of vessels with impaired membranes was inhibited by pretreatment with 30 μM Rp-8-CPT-cAMPS, an inactive analog of cAMP. In permeabilized mouse arteries, treatment with 100 nM urocortin was related to dephosphorylation of MLC 20 Ser 19 and MYPT1 Thr696/Thr850. The effect of urocortin on MYPT1 dephosphorylation was completely abolished by 30 μM Rp-8-CPT-cAMPS and mimicked by Sp-5,6-DCl-cBiMPS, an active cAMP analog. On the basis of these findings, it was assumed that the urocortin-induced relaxation is a consequence of a decrease in the calcium sensitivity mediated by a cAMP-dependent increase in the activity of myosin light chain phosphatase.
British Journal of Pharmacology, 2001
1 Urocortin is an endogenous vasodilator although the mechanism of vasorelaxation is not completely understood. The hypothesis that an alteration of smooth muscle calcium concentration is involved was tested using isometric tension recording and calcium¯uorimetry. The relationship between contraction and intracellular calcium was also estimated. 2 Urocortin produced a concentration dependent relaxation (pD 2 8.59+0.06, n=6) of vessels precontracted with a physiological salt solution containing 42 mM KCl (42 mM K-PSS).
The Journal of Physiology, 2009
Ca 2+ sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure-induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) required to sustain this view is not currently available. Here, we determined whether Ca 2+ sensitization pathways involving Rho kinase (ROK)and PKC-dependent phosphorylation of MYPT1 and CPI-17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03-10 μmol l −1 ; H1152, 0.001-0.3 μmol l −1) and PKC inhibitors (GF109203X, 3 μmol l −1 ; Gö6976; 10 μmol l −1) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3-step Western blot method was developed for detection and quantification of MYPT1 and CPI-17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine-855 (T855) and myosin light chain (LC 20). Phosphorylation of MYPT1 at threonine-697 (T697) and CPI-17 were not affected by pressure. Pressure-evoked elevations in MYPT1-T855 and LC 20 phosphorylation were reduced by H1152, but MYPT1-T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC 20 phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca 2+ sensitization pathway involving ROK-dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC 20 phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca 2+ sensitization mediated by CPI-17 or MYPT1 phosphorylation.
The Journal of biological chemistry, 2014
Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PK...
Cardiovascular Research, 2011
The role of Ca 2+ sensitization induced by a Ca 2+-independent myosin light chain kinase (MLCK) in hypertension has not been determined. The aim of this study was to clarify the role of possible Ca 2+-independent MLCK activity in hypertension. Methods and results We compared increases in contractile force and phosphorylation of myosin light chain (MLC) evoked by calyculin A, a phosphatase inhibitor, in b-escin-permeabilized mesenteric arteries at pCa 9.0 between spontaneously hypertensive rat (SHR) and Wistar Kyoto rat (WKY). We found that there was no detectable phosphorylation of MLC at pCa 9.0, but that the administration of 1 mM calyculin A gradually increased force and mono-and di-phosphorylation of MLC. This contraction was inhibited by staurosporine but not by wortmannin, Y-27632, or calphostin-C. The calyculin A-induced contraction was significantly greater in the SHR than in the WKY and was associated with an increase in mono-and di-phosphorylation of MLC. SM-1, a zipper-interacting protein kinase (ZIPK)-inhibiting peptide, significantly inhibited the amplitude of the calyculin A-induced contraction and di-phosphorylation. Total ZIPK expression (54 + 32 kDa) was greater in the SHR than in the WKY. Phosphorylation of myosin phosphatase target subunit at Thr 697 , but not at Thr 855 , was consistently stronger in the SHR than in the WKY in calyculin A-treated tissues at pCa 9.0. Conclusions Our results suggest that Ca 2+-independent MLCK activity is enhanced in the SHR, and that ZIPK plays, at least in part, an important role as a candidate for this kinase in rat mesenteric arteries.
The Journal of Physiology, 2010
Our understanding of the cellular signalling mechanisms contributing to agonist-induced constriction is almost exclusively based on the study of conduit arteries. Resistance arteries/arterioles have received less attention as standard biochemical approaches lack the necessary sensitivity to permit quantification of phosphoprotein levels in these small vessels. Here, we have employed a novel, highly sensitive Western blotting method to assess: (1) the contribution of Ca 2+ sensitization mediated by phosphorylation of myosin light chain phosphatase targeting subunit 1 (MYPT1) and the 17 kDa PKC-potentiated protein phosphatase 1 inhibitor protein (CPI-17) to serotonin (5-HT)-induced constriction of rat middle cerebral arteries, and (2) whether there is any interplay between pressure-induced myogenic and agonist-induced mechanisms of vasoconstriction. Arterial diameter and levels of MYPT1 (T697 and T855), CPI-17 and 20 kDa myosin light chain subunit (LC 20) phosphorylation were determined following treatment with 5-HT (1 μmol l −1) at 10 or 60 mmHg in the absence and presence of H1152 or GF109203X to suppress the activity of Rho-associated kinase (ROK) and protein kinase C (PKC), respectively. Although H1152 and GF109203X suppressed 5-HT-induced constriction and reduced phospho-LC 20 content at 10 mmHg, we failed to detect any increase in MYPT1 or CPI-17 phosphorylation. In contrast, an increase in MYPT1-T697 and MYPT1-T855 phosphorylation, but not phospho-CPI-17 content, was apparent at 60 mmHg following exposure to 5-HT, and the phosphorylation of both MYPT1 sites was sensitive to H1152 inhibition of ROK. The involvement of MYPT1 phosphorylation in the response to 5-HT at 60 mmHg was not dependent on force generation per se, as inhibition of cross-bridge cycling with blebbistatin (10 μmol l −1) did not affect phosphoprotein content. Taken together, the data indicate that Ca 2+ sensitization owing to ROK-mediated phosphorylation of MYPT1 contributes to 5-HT-evoked vasoconstriction only in the presence of pressure-induced myogenic activation. These findings provide novel evidence of an interplay between myogenic-and agonist-induced vasoconstriction in cerebral resistance arteries.
American Journal of Physiology-heart and Circulatory Physiology, 2012
The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na ϩ reabsorption. Recently we demonstrated that Ca 2ϩ signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na ϩ /Ca 2ϩ exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca 2ϩ-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca 2ϩ signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 Ϯ 1 mmHg) than in MNS (112 Ϯ 1 mmHg; P Ͻ 0.001; n ϭ 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca 2ϩ signals in response to 5 M PE or ATP in the absence and presence of extracellular Ca 2ϩ. These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca 2ϩ release and increased Ca 2ϩ entry, respectively. The increased SR Ca 2ϩ release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ϳ70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca 2ϩ signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca 2ϩ signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension. adducin; ouabain; myogenic tone; hypertension; Milan normotensive rats PRIMARY (ESSENTIAL) HYPERTENSION is a multifactorial disorder that leads to severe cardiovascular and renal complications (39). Hypertension is caused by the complex interplay between genetic predisposition (genetic heritability ϳ30%) and multi
Autonomic Neuroscience, 2009
increased expression of myosin light chain kinase, myosin light chain phosphatase targeting subunit and h-caldesmon, i.e. the proteins that mediate Ca 2+-dependent regulation. In contrast, expression of Rho-kinase, ERK and p38 MAP-kinases, that are involved in Ca 2+sensitization, was gradually diminished. The opposite changes in the expression profiles of these proteins, associated with increased Ca 2+sensitivity of contraction, were observed upon denervation of ASM in the adult rats. We also showed, for the first time, the critical role of Rho-kinase in regulation of blood pressure during early postnatal development and after chronic sympathetic denervation. The pressor response to α-adrenoceptor agonist was reduced by Rho-kinase inhibitors, Y27632 and fasudil, much stronger in newborn and chemically sympathectomised rats than in adult rats with intact sympathetic system. These differences were also observed after ganglionic blockade with chlorisondamine as well as in experiments on isolated vessels, indicating mainly peripheral action of the inhibitors. Altogether, these data suggest that sympathetic nerves exert trophic function in establishment of rapid contractile phenotype of ASM. The results may contribute to development of strategies for treatment of circulatory disorders in different forms of autonomic neuropathy.