In the vasculature, nitric oxide is generated by eNOS in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor BH4, NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH4 oxidation product, BH2, is the molecular trigger for eNOS uncoupling rather than BH4 depletion alone. In the current study, we investigated the effects of both BH4 depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH4 does not result in eNOS oxidase activity while BH4 oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using siRNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH4 oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH4 depletion increased eNOS-derived superoxide to 165% of that observed with BH4 oxidation. This study indentifies a critical role of Caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease associated changes in Cav-1 expression may contribute to endothelial dysfunction.
- nitric oxide
- eNOS uncoupling
- Copyright © 2011, American Journal of Physiology - Heart and Circulatory Physiology