A novel role for caveolin-1 in regulating endothelial nitric oxide synthase activation in response to H2O2 and shear stress

Abstract

Previous studies have shown that acute increases in oxidative stress induced by the addition of hydrogen peroxide (H2O2) can increase endothelial nitric oxide synthase (eNOS) catalytic activity via an increase in the phosphorylation of eNOS at serine 1177. However, it is unclear how increased H2O2 affects nitric oxide (NO) signaling when endothelial cells are exposed to biomechanical forces. Thus, the purpose of this study was to evaluate the acute effects of H2O2 on NO signaling in the presence or absence of laminar shear stress. We found that acute sustained increases in cellular H2O2 levels in bovine aortic endothelial cells did not alter basal NO generation but the NO produced in response to shear stress was significantly increased. This amplification in NO signaling was found to correlate with an H2O2-induced increase in eNOS localized to the plasma membrane and an increase in total caveolin-1 protein levels. We further demonstrated that overexpressing caveolin-1 increased eNOS localized to the plasma membrane again without altering total eNOS protein levels. We also found that caveolin-1 overexpression increased NO generation in response to shear stress but only in the presence of H2O2. Conversely, depleting caveolin-1 with an siRNA decreased eNOS localized to the plasma membrane and abolished the enhanced NO generation. Finally, we found that expressing a caveolin-1 binding-site deletion mutant of eNOS in COS-7 cells decreased its plasma membrane localization and resulted in attenuated NO production in response to calcium activation. In conclusion, we have identified a new role for caveolin-1 in enhancing eNOS trafficking to the plasma membrane that seems to be involved in priming eNOS for flow-mediated activation under conditions of oxidative stress. To our knowledge, this is the first report that H2O2 modulates eNOS activity by altering its subcellular location and that caveolin-1 can play a stimulatory role in NO signaling.