The long-term objectives of the proposed research are to increase our knowledge of the various mechanisms of vasodilation in general, and that of endothelium-dependent vasodilation in particular.
Specific aims i nclude: (1) investigate whether endothelium-derived relaxing factor (EDRF) released by various agents is nitric oxide (NO) or some labile nitrosyl precursor of NO; (2) to determine whether certain nitrovasodilators stimulate soluble guanylate cyclase (sGC) directly rather than indirectly by releasing NO; (3) to determine whether photorelaxation of vascular smooth muscle depends on release of an intracellular product of photoactivation that stimulates sCG; (4) to determine the action spectrum for photorelaxation of blood vessels with and without sensitizing agents (e.g. Bay K 8644); (5) to reinvestigate the inhibition of endothelium-dependent relaxation by unsaturated fatty acids (UFA). Among the preparations to be used will be isolated rings and strips of blood vessels with and without endothelial cells in organ chambers, for quantifying changes in contractile tone and accompanying changes in certain biochemical parameters such as levels of cyclic nucleotides. Perfusion-bioassay procedures will be used for investigating the properties of EDRF released from endothelial cells of perfused arteries or from cultured endothelial cells. In pursuing aim 1, agents that are highly effective in inhibiting NO-induced relaxation but poorly effective in inhibiting endothelium-dependent relaxation or arteries in organ chambers will be tested against infused NO and released EDRF in a perfusion-bioassay system. If a real difference between EDRF and NO is established, various labile nitrosyl compounds will be tested in the attempt to identify EDRF. Spectrophotometry in the visible and UV will be used extensively for quantitative analysis of many substances (e.g., superoxide, hydrogen peroxide, NO by reaction with hemoglobin, labile nitrosothiols), and for following reaction kinetics of labile substances. In pursuing aim 2, the activation of sCG (purified from bovine lung) by various nitrovasodilators with and without cofactors added will be tested under aerobic and anaerobic conditions. In pursuing aim 3, a modified perfusion-bioassay system will be used to determine whether photorelaxation of a perfused artery is accompanied by release of a photo-induced relaxing factor (PIRF). In pursuing aim 4, UV and visible light at various wavelengths and intensities from a double grating monochromator with xenon source will be used to obtain action spectra before and after sensitization of smooth muscle to light. In pursuing aim (5), an attempt will be made to determine if inhibition of endothelium-dependent relaxation by UFA is due to generation of superoxide oxidation of sulfhydryl groups, or block of sCG; and if it is related to the inhibition of endothelium-dependent relaxation reported for oxidized LDL.
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