Mechanism of RhoA/Rho kinase activation in endothelin-1- induced contraction in rabbit basilar artery.

This study was undertaken to demonstrate the role of the RhoA/Rho kinase pathway in endothelin-1 (ET-1)-induced contraction of the rabbit basilar artery. Isometric tension and Western blot were used to examine ET-1-induced contraction and RhoA activation. The upstream effect on ET-1-induced RhoA activity was determined by using ET(A) and ET(B) receptor antagonists, protein kinase C (PKC), tyrosine kinase, and phosphatidylinositol-3 kinase inhibitors. The downstream effect of ET-1-induced contraction and RhoA activity was studied in the presence of the Rho kinase inhibitor Y-27632. The effect of Rho kinase inhibitor on ET-1-induced myosin light chain (MLC) phosphorylation was investigated by using urea-glycerol-PAGE immunoblotting. We found 1) ET-1 increased RhoA activity (membrane binding RhoA) in a concentration-dependent manner; 2) ET(A), but not ET(B), receptor antagonist abolished the effect of ET-1 on RhoA activation; 3) phosphodylinositol-3 kinase inhibitor, but not PKC and tyrosine kinase inhibitors, reduced ET-1-induced RhoA activation; 4) Rho kinase inhibitor Y-27632 (10 microM) inhibited ET-1-induced contraction; and 5) ET-1 increased the level of MLC phosphorylation. Rho kinase inhibitor Y-27632 reduced the effect of ET-1 on MLC phosphorylation. This study demonstrated that RhoA/Rho kinase activation is involved in ET-1-induced contraction in the rabbit basilar artery. Phosphodylinositol-3 kinase and MLC might be the upstream and downstream factors of RhoA activation.