Abstract 1692: Peroxynitrite Mediates Diabetes-induced Endothelial Dysfunction by Reducing eNOS Expression: Possible Role of Rho Kinase (ROCK) Activation
Endothelial dysfunction is characterized by reduced bioavailability of NO, which could be caused by reduced expression of eNOS and increased inactivation of NO by oxidative stress. Our previous studies have shown that vessels from diabetic rats showed decreases in acetylcholine (ACh)-mediated maximal relaxation compared to controls. This effect was associated with decreases in eNOS expression and increases in oxidative stress and protein tyrosine nitration suggesting a critical role for peroxynitrite in decreasing NO availability. Our studies in bovine aortic endothelial cells (BAEC) show that high glucose (25 mM) or peroxynitrite (25 μM) increases the activity of Rho kinase (ROCK) and decreases eNOS expression. Here, we test the hypothesis that peroxynitrite mediates diabetes-induced endothelial dysfunction by a mechanism that involves decreasing eNOS expression via activation of ROCK. Diabetes was induced in rats by STZ injection. After 4 weeks, vessels were isolated from control, diabetic, and diabetic rats treated with the specific peroxynitrite decomposition catalyst (FeTTPs 15 mg/Kg/day) and vasorelaxation to acetylcholine (ACh, 0.1nM-10μM), ROCK activity and eNOS expression were determined. Coronary arteries from diabetic rats showed reduction in ACh-mediated maximal relaxation to 34.7%±3.2 compared to 81%±2.5 in control. Also, the dose-response curve to ACh was shifted to the right with an EC50value of 94±0.2 nM compared to 40±1 nM of control. These effects were associated with significant increases in ROCK activity in diabetic aorta and decreases in eNOS expression. Treatment of diabetic rats with FeTTPs significantly improved Emax relaxation to ACh to 58.8%±2.8 and EC50 was decreased to 70±1.3 nM. In addition, treatment with FeTPPs normalized ROCK activity in diabetic rats and in BAEC maintained in high glucose. Furthermore, treatment with the ROCK inhibitor Y-27632 (10 μM) reversed the effects of peroxynitrite in reducing eNOS expression in BAEC. Taken together, these data suggest that peroxynitrite mediates diabetes-induced endothelial dysfunction by suppressing eNOS expression via activation of ROCK and that inhibiting peroxynitrite would be a novel therapeutic target for the treatment of vascular dysfunction.