DESCRIPTION: Diminished production of nitric oxide (NO) by endothelial NO synthase (eNOS) plays a key role in the early pathogenesis of atherosclerosis. eNOS appears to be acylated and thereby targeted to cholesterol-enriched, plasmalemmal signal transduction domains called caveolae. Localization to caveolae is critical to eNOS function. The objectives of this proposal are to define the molecular mechanisms normally regulating eNOS trafficking to caveolae, and to determine if changes in cholesterol balance attenuate eNOS function by modifying eNOS trafficking to caveolae. Aim 1 will test the hypothesis that eNOS is first targeted to the Golgi apparatus by myristoylation, and then targeted to caveolae by palmitoylation. eNOS trafficking will be assessed in COS-7 cells transfected with wild-type or mutant eNOS cDNAs by immunoprecipitation of biosynthetically-labelled eNOS in subcellular fractions, and by immunofluorescence and, immunoelectron microscopy. Aim 2 will test the hypothesis that the integral membrane protein caveolin is necessary for eNOS trafficking to caveolae and for optimal eNOS function. Studies will be performed in permanently-transfected cell lines that are caveolin (+) or caveolin (-). Aim 3 will employ endothelial cells to test the hypothesis that components of the eNOS activation system are colocalized in caveolae. Receptor-mediated eNOS stimulation will be evaluated in isolated caveolae, and the existence of an eNOS activation complex will be assessed by coprecipitation. Aim 4 will test the hypothesis that eNOS trafficking to caveolae and eNOS function are attenuated when cholesterol content is abnormally low or high; experiments will be performed in transfected COS-7 cells, endothelial cells, and isolated plasma membranes. The studies planned will increase our basic knowledge of the regulation of endothelial NO production under normal conditions and during altered cholesterol balance, thereby revealing the pathogenetic link between hypercholesterolemia and endothelial dysfunction. In doing so, it is anticipated that the proposed work will lead to new preventative measures and treatment modalities for atherosclerosis that complement approaches aimed at cholesterol control.