Abstract-Two mechanisms are proposed to account for the inhibition of myosin phosphatase (MP) involved in Ca 2ϩ sensitization of vascular muscle, ie, phosphorylation of either MYPT1, a target subunit of MP or CPI-17, an inhibitory phosphoprotein. In cultured vascular aorta smooth muscle cells (VSMCs), stimulation with angiotensin II activated RhoA, and this was blocked by pretreatment with 8-bromo-cGMP. VSMCs stimulated by angiotensin II, endothelin-1, or U-46619 significantly increased the phosphorylation levels of both MYPT1 (at Thr696) and CPI-17 (at Thr38). The angiotensin II-induced phosphorylation of MYPT1 was completely blocked by 8-bromo-cGMP or Y-27632 (a Rho-kinase inhibitor), but not by GF109203X (a PKC inhibitor). In contrast, phosphorylation of CPI-17 was inhibited only by GF109203X. Y-27632 dramatically corrected the hypertension in N -nitro-L-arginine methyl ester (L-NAME)-treated rats, and this hypertension also was sensitive to isosorbide mononitrate. The level of the active form of RhoA was significantly higher in aortas from L-NAME-treated rats. Expression of RhoA, Rho-kinase, MYPT1, CPI-17, and myosin light chain kinase were not significantly different in aortas from L-NAME-treated and control rats. Activation of RhoA without changes in levels of other signaling molecules were observed in three other rat models of hypertension, ie, stroke-prone spontaneously hypertensive rats, renal hypertensive rats, and DOCA-salt rats. These results suggest that independent of the cause of hypertension, a common point in downstream signaling and a critical component of hypertension is activation of RhoA and subsequent activation of Rho-kinase. A lthough the mechanisms underlying development of hypertension are not established, one critical feature observed in most cases of hypertension is increased peripheral resistance, implying enhanced constriction of the relevant vessels. 1 A major determinant of vascular tone is the level of myosin light chain (MLC) phosphorylation that is controlled by the Ca 2ϩ /calmodulin-dependent myosin light chain kinase (MLCK) and by myosin phosphatase (MP). 2 However, there is no fixed relationship between cytosolic Ca 2ϩ and MLC phosphorylation and this can vary depending on conditions. An increased phosphorylation and tension can occur at suboptimal Ca 2ϩ levels and is referred to as Ca 2ϩ sensitization and occurs frequently after stimulation by many agonists. 3 A major contributor to this effect is inhibition of MP and over the last decade this was shown to be operating via a small GTPase RhoA-linked pathway. [3][4][5][6] There is a strong evidence to implicate Rho-kinase as a downstream target in the RhoA-linked pathway, 6,7 and it has been shown that Rho-kinase inhibitors, such as Y-27632, block the agonist-induced Ca 2ϩ sensitization in smooth muscle. 8 At a molecular level, one mechanism responsible for inhibition of MP is that phosphorylation by Rho-kinase of the target subunit of MP, MYPT1, at Thr696 (for the human isoform) inhibits activity of the catalytic subunit. 7...