Ca -Dependent Activation of Rho and Rho Kinase in Membrane Depolarization–Induced and Receptor Stimulation–Induced Vascular Smooth Muscle Contraction

Ca 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 Ca -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 Ca or the addition of a dihydropyridine Ca 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 NRhoA suppressed Ca -induced Thr 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 Ca removal and depletion of the intracellular Ca 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 Ca -dependent Rho stimulation mechanism in VSM. Excitatory receptor agonists are suggested to use this pathway for simulating Rho. (Circ Res. 2003; 93:548-556.)