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 (O(2) (.-)) production via NOS uncoupling. Elevated O(2) (.-) 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 (Gö6976) was used to investigate the mechanism involved in arginase activation. KEY
RESULTS: Exposure to SIN-1 (25 µM, 24 h) or H(2) O(2) (25 µM, 8 h) increased arginase I expression and arginase activity (35% and 50%, respectively), which was prevented by ROCK inhibitor, Y-27632, PKC inhibitor, Gö6976 or siRNA to p115-Rho GEF. There was an early activation of p115-Rho GEF (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.