Oxidative species increase arginase activity in endothelial cells through the RhoA/Rho kinase pathway

BACKGROUND AND PURPOSE NO produced by endothelial NOS is needed for normal vascular function. During diabetes, aging and hypertension, elevated levels of arginase can compete with NOS for available l-arginine, reducing NO and increasing superoxide (O2.-) production via NOS uncoupling. Elevated O2.- combines with NO to form peroxynitrite (ONOO-), further reducing NO. Oxidative species increase arginase activity, but the mechanism(s) involved are not known. Our study determined the mechanism involved in peroxynitrite and hydrogen peroxide-induced enhancement in endothelial arginase activity. We hypothesized that oxidative species increase arginase activity through PKC-activated RhoA/Rho kinase (ROCK) pathway. EXPERIMENTAL APPROACH Arginase activity/expression was analysed in bovine aortic endothelial cells (BAEC) treated with an ONOO(-) generator (SIN-1) or H(2)O(2). Pretreatment with inhibitors of Rho kinase (Y-27632) or PKC (Go6976) was used to investigate the mechanism involved in arginase activation. KEY RESULTS Exposure to SIN-1 (25 mu M, 24 h) or H(2)O(2) (25 mu M, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Go6976 or siRNA to p115-Rho GEE. There was an early activation of p115-Rho GEE (SIN-1, 2 h; H(2)O(2), 1 h) and Rho A (SIN-1, 4 h; H(2)O(2), 1 h) that was prevented by using the PKC inhibitor. Exposure to SIN-1 and H(2)O(2) also reduced NOS activity, which was blocked by pretreatment with p115-RhoGEF siRNA. CONCLUSIONS AND IMPLICATIONS Our data indicate that the oxidative species ONOO(-) and H(2)O(2) increase arginase activity/expression through PKC-mediated activation of RhoA/Rho kinase pathway.