| The vascular endothelium forms an interface between circulating blood and the vascular wall, but more importantly, synthesizes and releases numerous factors that regulate blood pressure, vascular tone, and hemostasis. A key endothelial-derived factor is nitric oxide (NO), a small, lipohillic molecule synthesized by the constitutively expressed enzyme endothelial nitric oxide (eNOS) synthase whose actions include but are not limited to vasodilation of vascular smooth muscle cells. NO's importance is underscored by the link between pathologies such as hypertension and atherosclerosis and reductions in endothelial-derived NO. Associated with the reduction in NO is increased production of reactive oxygen species (ROS) and decreased antioxidant defenses. The imbalance in ROS and antioxidants reflects oxidative stress, and is closely related to decreased NO bioavailability. To examine the interactions between oxidative stress and endothelial cell function, NO production capacity by human EA.hy926 cells, as a cell model for vascular endothelium was examined. NO-specific difluorofluorescein dyes, DAF-FM and DAF-FM DA were used to detect NO and western blotting was used to confirm eNOS expression. Oxidative stress was verified by increased dicholorofluorescein fluorescence using the ROS-sensitive indicator H2-DCF DA. EA.hy926 cells produce low basal levels of NO and express eNOS comparable to human umbilical vein endothelial cells, another commonly used human endothelial cell. Induction of cellular oxidative stress by glutathione depletion, hydrogen peroxide, increased intracellular NO, whereas the superoxide anion generating compound, menadione, decreased NO. Furthermore, glutathione depletion-induced increased intracellular NO was completely preventable by repletion with glutathione. The studies presented here suggest that EA.hy926 cell-derived NO, evaluated under conditions of oxidative stress, is a useful model for studying the relationships between ROS, antioxidants, and NO. |
