This grant is designed to enable the applicant to continue his training to become an independent investigator in the study of basic mechanisms of cardiovascular disease and aging. The scientific goal of this proposal is to characterize oxygen free radical generation from endothelial nitric oxide synthase (eNOS) and study the role of this oxygen radical generation in the process of aging-associated vascular endothelial dysfunction and injury. Endothelium-dependent relaxation is impaired in aging vasculature. This age-associated endothelial dysfunction has been attributed to loss of NO activity and increased oxidant production. However, the mechanism underlying these changes is poorly understood. NO is synthesized from L-arginine by a family of NOSs. Low levels of L-arginine trigger NOS to switch from NO to superoxide (.O2-) generation. Previous studies showed that L-arginine supplement reverses endothelial dysfunction in aged vessels. Our preliminary studies indicated that increased .O2- production in aged vessels was largely prevented by eNOS blockade. These two lines of evidence suggest that NO decrease/oxidant increase in aged vasculature may be caused by an age associated functional switch of eNOS from NO to .O2- production. This hypothesis will be tested at three levels. First, the generation of .O2- and NO from purified eNOS will be directly measured and characterized by electron paramagnetic resonance spin-trapping techniques. Second, we will compare NO and .O2- production in young and aged cells. Chemiluminescence assay and immunocytochemistry will be used to test whether eNOS-derived .O2- and NO interact to form the potent oxidant peroxynitrite. Finally, we will measure and compare NO and .O2- formation in young and aged vascular rings and define the role of eNOS-mediated .O2- and peroxynitrite in the process of aging-related endothelial dysfunction. These studies will provide new insight into the enzymatic function of eNOS and the alterations in the regulation and control of this critical enzyme during aging. This will greatly increase our understanding of the basic mechanisms of aging-associated endothelial dysfunction and injury and may lead to new therapeutical approaches.
