title: Ca2+-dependent activation of Rho and Rho kinase in membrane depolarization-induced and receptor stimulation-induced vascular smooth muscle contraction.
abstract: Ca2+ sensitization of vascular smooth muscle (VSM) contraction involves Rho-dependent and Rho-kinase-dependent suppression of myosin phosphatase activity. We previously demonstrated that excitatory agonists in fact induce activation of RhoA in VSM. In this study, we demonstrate a novel Ca2+-dependent mechanism for activating RhoA in rabbit aortic VSM. High KCl-induced membrane depolarization as well as noradrenalin stimulation induced similar extents of sustained contraction in rabbit VSM. Both stimuli also induced similar extents of time-dependent, sustained increases in the amount of an active GTP-bound form of RhoA. Consistent with this, the Rho kinase inhibitors HA1077 and Y27632 inhibited both contraction and the 20-kDa myosin light chain phosphorylation induced by KCl as well as noradrenalin, with similar dose-response relations. Either removal of extracellular Ca2+ or the addition of a dihydropyridine Ca2+ channel antagonist totally abolished KCl-induced Rho stimulation and contraction. The calmodulin inhibitor W7 suppressed KCl-induced Rho activation and contraction. Ionomycin mimicked W7-sensitive Rho activation. The expression of dominant-negative N19RhoA suppressed Ca2+-induced Thr695 phosphorylation of the 110-kDa regulatory subunit of myosin phosphatase and phosphorylation of myosin light chain in VSM cells. Finally, either the combination of extracellular Ca2+ removal and depletion of the intracellular Ca2+ store or the addition of W7 greatly reduced noradrenalin-induced and the thromboxane A2 analogue-induced Rho stimulation and contraction. Taken together, these results indicate the existence of the thus-far unrecognized Ca2+-dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho.
authors.affiliation: Department of Physiology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa, Japan.
authors.forename: Sotaro
authors.initials: S
authors.lastname: Sakurada
authors.forename: Noriko
authors.initials: N
authors.lastname: Takuwa
authors.forename: Naotoshi
authors.initials: N
authors.lastname: Sugimoto
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Minoru
authors.initials: M
authors.lastname: Seto
authors.forename: Yasuharu
authors.initials: Y
authors.lastname: Sasaki
authors.forename: Yoh
authors.initials: Y
authors.lastname: Takuwa
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001011:Aorta / Q000166:cytology; D002118:Calcium / Q000502:physiology*; D002147:Calmodulin / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D046508:Culture Techniques; D004789:Enzyme Activation; D047908:Intracellular Signaling Peptides and Proteins; D015759:Ionomycin / Q000494:pharmacology; D009119:Muscle Contraction; D009131:Muscle, Smooth, Vascular / Q000201:enzymology / Q000378:metabolism / Q000502:physiology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D009638:Norepinephrine / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011817:Rabbits; D017482:Receptors, Thromboxane / Q000819:agonists; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism* / Q000502:physiology
chemical_list: D000316:Adrenergic alpha-Agonists; D002147:Calmodulin; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D017482:Receptors, Thromboxane; D015759:Ionomycin; D011189:Potassium Chloride; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D020742:rhoA GTP-Binding Protein; D002118:Calcium; D009638:Norepinephrine