Tetrahydrobiopterin Oxidation and Reactive Oxygen Species Contribute to H2O2-Induced Endothelial Nitric Oxide Synthase Dysfunction

An oxidative stress in the form of H2O2 exposure previously has been shown to cause a transient increase in NO??oduction and a chronic increase in eNOS protein levels. Nevertheless, oxidative stress can cause an uncoupling of catalytic activity resulting in decreased NO??d increased O2??roduction from eNOS. This uncoupling seems to be mediated predominantly by oxidation of tetrahydrobiopterin (BH4), an eNOS required cofactor. To study how these phenomena regulate the physiological balance of reactive oxygen species (ROS), H2O2-induced NO??oduction was measured in bovine aortic endothelial cells (BAECs) using an NO??ecific electrode. Following H2O2 exposure, NO??ncentrations initially increased; however, if cells were challenged a second time with H2O2, the increase in NO??oduction was attenuated. We postulated that the decline in NO??oduction after H2O2 exposure resulted from BH4 oxidation and tested this by supplementing cells with BH4 prior to the second H2O2 exposure. This resulted in a recovery of NO??oduction. Since H2O2 also activates NADPH oxidase to produce superoxide (O2?? we tested whether the decrease in NO??oduction during the second H2O2 exposure could be explained by increased NADPH oxidase-dependent oxygen free radical production, including O2??peroxynitrite (ONOO-), and hydroxyl radicals (??. A reduction in H2O2-induced NO??lease was prevented in apocynin-and PEG-SOD-treated cells and in p47phox-knockout mouse aortic endothelial cells (MAECs), which lack a critical subunit of the NADPH oxidase. These results suggest that O2??roduced by NADPH oxidase leads to eNOS dysfunction. Scavenging ONOO- resulted in a full recovery of NO??oduction, and scavenging ??resulted in a partial recovery of NO??oduction. This implies roles for these O2??erivatives in the reduced NO??sponse to repeated H2O2 exposures.M.S.Committee Chair: Samuel C. Dudley, Jr.; Committee Member: Hanjoong Jo; Committee Member: W. Robert Taylo