NO is constitutively produced by vascular endothelial cells (EC) where it plays a key physiological role in the moment-to-moment regulation of blood pressure and vascular tone. Deficient NO bioactivity, described as endothelial dysfunction, contributes to the pathogenesis of numerous vascular diseases and conditions; these include coronary artery disease, atherosclerosis, hypertension and diabetic vasculopathy. May recent studies have demonstrated that administration of the endothelial NO synthase (eNOS) cofactor tetrahydrobiopterin Many recent studies have restore endothelial function in animals models of vascular disease and patients. Despite the efficiency of BH4 in these cases, measurements of BH4 tissue content has not revealed overt biochemical deficiency. This research will evaluate the possibility that altered biopterin redox balance, not amount, provides a common molecular basis for chronic vascular diseases that manifest with endothelial dysfunction. Indeed, BH4 is readily oxidized to dihydrobiopterin (BH2) under the oxidative conditions that can predominate in atherosclerotic, hypertensive and diabetic blood vessels. In preliminary studies we have discovered that eNOS binds BH4 and BH2 with equal affinity. Importantly, whereas BH4-bound eNOS exclusively produces superoxide anion. Superoxide reacts with an inactivates NO in a near diffusion-limited reaction; the product peroxynitrite (OONO-) is a more potent oxidant than either parent molecule and can further oxidize BH4. Thus any stimulus which initiates BH4 oxidation in EC (e.g.., oxidized LDL) can potentially initiate a fee-forward cascade wherein eNOS- derived superoxide perpetuates BH4 insufficiency, attenuated NO bioactivity and chronic endothelial dysfunction. Our overall hypothesis is that the redox status of bound pterin cofactor constitutes a phenotypic switch that determines eNOS function. Further regulation of eNOS is imposed by two autoinhibitory control elements that we have identified and confirmed to control catalytic rate and dependence of activity on free intracellular calcium concentration. The overall research plan is to elucidate the role of pterin oxidation in the eNOS phenotype switch, from NO-producing to superoxide-producing (AIM 1) define fundamental control mechanisms that modulate the rate of NOS catalysis-NO or superoxide production (AIM 2), and elucidate novel therapeutic strategies with potential to remedy endothelial dysfunction and restore NO synthesis to chronically superoxide-producing endothelial cells (AIM 3). PPG resources and effective collaborations with other PPG investigators (D. Hajjar, R. Upmacis and R. Silverstein) are essential to the success of this research.
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