Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

Objective To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT(2)R), clownregulates RhoA/Rho kinase, which plays a role in AT(1) receptor (AT(1)R)-mediated function. Methods In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar-Kyoto (WKY) and strokeprone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10(-7) Mol/l), CGP42112A (10(-7) mol/l, a selective AT(2)R agonist) valsartan (10(-5) mol/l, an AT, R antagonist), or the Rho kinase inhibitor fasudil (10-5 mol/l). AT, R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT(2)R, RhoA/Rho kinase and vasoclilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasoclilatory responses to Ang II (10(-9)-10(-6) Mol/l) were performed in norepinephrine pre-contracted vessels +/- valsartan (10(-6) Mol/l), PD123319 (10-6 mol/l, an AT(2)R antagonist) or fasudil (10(-6) mol/l). Results A10 VSMC expressed AT(1)R and AT(2)R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 +/- 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 +/- 7%). PD123319 blocked these effects. Fasudil increased Angli-induced relaxation in SHRSP vessels. AT(2)R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased twofold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. Conclusions Ang II/AT(2)R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasoclilation in conditions associated with vascular AT(2)R upregulation, such as in SHRSP chronically treated with AT(1)R blockers, which may contribute to blood pressure lowering by these antihypertensive agents. (c) 2005 Lippincott Williams & Wilkins.