L-Arginine/nitric oxide pathway plays a key role in regulation of arterial tone. Biosynthesis of nitric oxide requires activation of nitric oxide synthase in the presence of tetrahydrobiopterin as a cofactor. The proposition to be tested is that in vascular endothelial cells suboptimal concentrations of tetrahydrobiopterin may favor nitric oxide synthase- catalyzed production of hydrogen peroxide. Protocols are designed to determine if in coronary arteries with impaired tetrahydrobiopterin biosynthesis, hydrogen peroxide may replace nitric oxide as mediator of endothelium-dependent relaxations. Mechanisms of relaxations to hydrogen peroxide will be studied and levels of cyclic GMP will be measured in order to characterize the role of soluble guanylate cyclase. Hydrogen peroxide is a potent oxidant and it is expected that these studies may provide new information concerning the role of dysfunctional nitric oxide synthase in endothelial injury. Since de novo synthesis of tetrahydrobiopterin is regulated by enzymatic activity of GTP cyclohydrolase I, molecular mechanisms regulating expression of mRNA isoforms encoding for this protein will be studied. Activity of GTP cyclohydrolase I and concentrations of tetrahydrobiopterin in endothelial cells will be measured. Preliminary studies demonstrated that type 1 GTP cyclohydrolase I is expressed in human aortic endothelial cells. Identification of signal transduction pathways involved in control of gene expression, as well as activity of GTP cyclohydrolase I, will provide a molecular basis for understanding of mechanisms responsible for impairment of tetrahydrobiopterin biosynthesis and dysfunction of nitric oxide synthase. In order to determine whether dysfunction could be corrected by exogenous pterins, vascular effects of tetrahydrobiopterin and 6-methyl- tetrahydropterin on mechanisms of endothelium-dependent relaxations in control arteries and arteries with impaired tetrahydrobiopterin biosynthesis will be investigated. Canine basilar arteries will be used as a model to study the inhibitory effects of pterins on endothelium- dependent contractions. The effect of pterins on production of cyclic GMP, cyclic AMP as well as on prostanoid production associated with endothelium-dependent contractions will be measured. These experiments will reveal whether pterins may affect balance between relaxing and contracting factors released from endothelium. It is anticipated that results of this proposal will define the role of tetrahydrobiopterin in regulation of endothelial function. The proposed studies may also help to identity if impaired tetrahydrobiopterin biosynthesis may contribute to initial endothelial injury known to be a key event in the pathogenesis of vascular diseases.
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