Suppression of eNOS-derived superoxide by caveolin-1: A biopterin-dependent mechanism

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/ calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (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, dihydrobiopterin, 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, whereas 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 small interfering RNA 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 identifies 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 caveolin-1 expression may contribute to endothelial dysfunction.