Hypertension is an age-related condition, affecting fewer than 10% of young adults but more than 70% of those in their 7th and 8th decades. The disease predisposes its victims to cardiovascular disease and stroke. Interestingly, among those over 75, hypertension affects significantly more women than men. In both men and women, age-related hypertension has been linked to endothelial dysfunction, associated with decreased availability of nitric oxide (NO), an important regulator of vascular smooth muscle tone. Endothelial nitric oxide synthase (eNOS) cycles from membrane (inactive) to cytoplasm (active) and back, a process facilitated by cyclic depalmitoylation/repalmitoylation. The PI recently identified a long-chain fatty acyl CoA synthetase that is highly expressed in the endothelium (eLCFACoAS). Preliminary evidence shows that eLCFACoAS expression and activity is elevated in ovariectomized female rats, suggesting that it plays a role in post-menopausal hypertension. Triacsin C, an inhibitor of eLCFACoAS, inhibits eNOS palmitoylation, increases NO synthesis in cultured endothelial cells, and enhances both release of NO and vascular smooth muscle relaxation in rat aortic rings. The proposed work tests the hypothesis that palmitoyl CoA availability is rate-limiting in eNOS repalmitoylation, and that endothelial long chain fatty acyl CoA synthetase (as opposed to other LCFACoASs) is the enzyme that supplies palmitoyl CoA. The hypothesis suggests that inhibiting eLCFACoAS will enhance eNOS activity in vivo, opening a new therapeutic category for treating hypertension. The hypothesis will be tested by: 1. Elucidating the reaction mechanism of eLCFACoAS by defining a) the substrate preference of eLCFACoAS using fatty acids having varying cis/trans double bond arrangements and b) the rank order of a series of known LCFACoAS inhibitors to inhibit eLCFACoAS catalytic activity; 2. Using known LCFACoAS inhibitors to define the physiologic role of eLCFACoAS in eNOS palmitoylation in cultured cells; 3. Using known LCFACoAS inhibitors to define the physiologic role of eLCFACoAS in agonist-stimulated eNOS function in intact blood vessels. Accomplishing these aims will establish the role for eLCFACoAS mediated palmitoylation in eNOS function, and provide lead information for the synthesis of highly specific, potent inhibitors of eLCFACoAS enabling future studies of eLCFACoAS inhibitors as potential therapeutic agents. ? ? ?
