Early determinants of H₂O₂-induced endothelial dysfunction

Reactive oxygen species (ROS) can stimulate nitric oxide (NO^{{radical dot}}) production from the endothelium by transient activation of endothelial nitric oxide synthase (eNOS). With continued or repeated exposure, NO^{{radical dot}} production is reduced, however. We investigated the early determinants of this decrease in NO^{{radical dot}} production. Following an initial H₂O₂ exposure, endothelial cells responded by increasing NO^{{radical dot}} production measured electrochemically. NO^{{radical dot}} concentrations peaked by 10 min with a slow reduction over 30 min. The decrease in NO^{{radical dot}} at 30 min was associated with a 2.7-fold increase in O₂^{{radical dot}-} production (p < 0.05) and a 14-fold reduction of the eNOS cofactor, tetrahydrobiopterin (BH₄, p < 0.05). Used as a probe for endothelial dysfunction, the integrated NO^{{radical dot}} production over 30 min upon repeated H₂O₂ exposure was attenuated by 2.1-fold (p = 0.03). Endothelial dysfunction could be prevented by BH₄ cofactor supplementation, by scavenging O₂^{{radical dot}-} or peroxynitrite (ONOO^-), or by inhibiting the NADPH oxidase. Hydroxyl radical (^{{radical dot}}OH) scavenging did not have an effect. In summary, early H₂O₂-induced endothelial dysfunction was associated with a decreased BH₄ level and increased O₂^{{radical dot}-} production. Dysfunction required O₂^{{radical dot}-}, ONOO^-, or a functional NADPH oxidase. Repeated activation of the NADPH oxidase by ROS may act as a feed forward system to promote endothelial dysfunction.