title: Suppression of eNOS-derived superoxide by caveolin-1: a biopterin-dependent mechanism.
abstract: 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 (BH(4)), 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 BH(4) oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH(4) depletion alone. In the current study, we investigated the effects of both BH(4) 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 BH(4) does not result in eNOS oxidase activity, whereas BH(4) 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 BH(4) oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH(4) depletion increased eNOS-derived superoxide to 165% of that observed with BH(4) 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.
authors: Department of Anesthesiology, Ohio State University, Columbus, Ohio, USA.
authors: Kanchana
authors: K
authors: Karuppiah
authors: Lawrence J
authors: LJ
authors: Druhan
authors: Chun-an
authors: CA
authors: Chen
authors: Travis
authors: T
authors: Smith
authors: Jay L
authors: JL
authors: Zweier
authors: William C
authors: WC
authors: Sessa
authors: Arturo J
authors: AJ
authors: Cardounel
mesh_terms: D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives* / Q000378:metabolism; D002417:Cattle; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D015536:Down-Regulation; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D019255:NADPH Oxidases / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010084:Oxidation-Reduction; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D015398:Signal Transduction; D013481:Superoxides / Q000378:metabolism*
chemical_list: D051242:Caveolin 1; D047428:Protein Kinase Inhibitors; D013481:Superoxides; D001708:Biopterins; D052250:Nitric Oxide Synthase Type III; D019255:NADPH Oxidases; D051057:Proto-Oncogene Proteins c-akt; C003402:sapropterin