The overall goal of these studies is to understand the endothelial nitric o de synthase signaling system in health and disease. Nitric oxide (NO) is an important signaling molecular subserving diverse role in a variety of tissu . NO is synthesized in the vasculature by the endothelial isoform of nitric o de synthase (ecNOS). Regulation of endothelial NO production appears to be a critical pathway for the maintenance of blood pressure homeostasis, and is important determinant of platelet aggregation. NO is also the major bioact e metabolite of clinically important organic nitrate vasodilator drugs such a nitroglycerin. During the initial funding period of this grant, we isolate and characterized ecNOS cDNA and genomic clones; developed highly specific anti dies and recombinant expression systems which have allowed investigations of ecN biosynthesis and intracellular regulation; and discovered that ecNOS underg s a series of complex post-translational modifications, including phosphoryla on, myristoylation and palmitoylation. Using in vitro mutagenesis, we have identified specific amino acid residues affected by these covalent modifica ons. In the proposed studies, we will extend these findings to explore the role, regulation, and interrelationships of post-translational modifications in c trol of the ecNOS signaling system and its modulation in pathophysiological stat . We will create and characterize mutant ecNOS enzymes deficient in these modifications, which will be studied from the standpoints of pharmacologica and intracellular regulation. We will characterize the endothelial protein kin e responsible for ecNOS phosphorylation, as well as the regulation of ecNOS palmitoylation by agonists and drugs. The domain and subunit structure of NOS will be explored, and we will investigate the sites of subunit interaction well as the factors controlling enzyme assembly. Spectroscopic analyses of purified recombinant wild-type and mutant ecNOS will be performed in order more fully characterize ecNOS enzymatic mechanisms and identify sites for pharmacological intervention. Abnormalities in the NO signaling pathway have been implicated in the pathogenesis of the impaired endothelial-dependent relaxation observed in diseases such as hypertension, atherosclerosis and diabetes, as well as in pharmacological tolerance to organic nitrate vasodilator drugs. Our characterizations of cellular and molecular mechanisms that regulate endoth ial production of NO may lead to new interventions for the amelioration of thes disorders.
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