Endothelium-derived relaxing factor (EDRF), identified as nitric oxide (NO), is a potent vasodilator and anti-thrombotic autocoid that is important for the maintenance of systemic blood pressure. When activated by changes in local blood flow or circulating hormones, NO is synthesized by a membrane bound, calcium-requiring enzyme, nitric oxide synthase (ECNOS) that converts L-arginine to NO and L-citrulline. Once generated, NO is released from the endothelium causing relaxation of vascular smooth muscle. In order to elucide how the endothelial production of NO is regulated we have cloned the cDNA for ECNOS and have shown that it contains an N-myristoylation consensus sequence, not found in neuronal or macrophage forms of NOS (both soluble isoforms). In addition, we have shown by site-directed mutagenesis, that mutation of glycine 2 (the putative myristic acid acceptor site) in the N-myristoylation consensus sequence converts membrane-associated ECNOS to a cytosolic form. Therefore, the purpose of this proposal is to test the hypothesis that N- myristoylation directs ECNOS towards the membrane where the enzyme is most responsive to changes in flow and to local hormones. To this end, we will: 1. Characterize the N-terminal fatty acid modification of ECNOS and examine its functional relevance by site-directed mutagenesis and the generation of chimeric NOS proteins; 2. Localize ECNOS protein (immunohistochemically) in EC and in cells lines stably expressing either wild-type (membrane) or mutant ECNOS (soluble); 3. Test the functional significance of N-myristoylation of ECNOS by examining the release of NO in response to calcium-mobilizing agonists or flow in endothelial cells stably transfected with either wild-type (membrane) or mutant (soluble) ECNOS. and 4. Examine in endothelial cells, if the rate of N- myristoylation or localization of myristoylated ECNOS is affected by calcium-mobilizing hormones or flow. If ECNOS is myristoylated and if myristoylation is necessary for ECNOS localization and activity, then understanding the importance of this protein modification will aid in the understanding how NO is regulated in vivo.
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