During the past two years of funding we pursued two goals related to mechanisms of vascular control. Experiments were performed examining regulation of the coronary microcirculation under normal conditions and in the setting of disease. Additionally we have examined the chemical nature of the endothelium-derived relaxing factor and how its synthesis may be altered by atherosclerosis. During the upcoming funding period we plan to pursue two separate projects related to this prior work. The first is related to regulation of the coronary microcirculation. During the past several years we have gained substantial expertise in in-vitro studies of the coronary microvasculature and have recently developed the capacity to study coronary microvessels removed from the subendocardium. During this funding period we will use this approach to compare several aspects of vascular control between arterioles of the subendocardium versus the subepicardium. We have recently shown that in large epicardial coronary arteries endothelium-dependent relaxation occurs via two very independent but additive mechanisms. The first is dependent on activation of vascular smooth muscle of guanylate cyclase and the second appears to be due to activation of an ATP sensitive potassium channel. Studies are planned to examine the relative importance of these two mechanisms in the coronary microcirculation both on subepicardial and subenodcardial vessels. We have recently shown that S-nitrosocysteine more closely resembles the biologic activity of EDRF than does nitric oxide. We plan studies to determine if S-nitrosocysteine has effects on coronary vascular smooth muscle other than activation of guanylate cyclase. Specifically we plan to determine if S- nitrosocysteine or other nitrosothiols hyperpolarize vascular smooth muscle. A second goal of this work is to examine factors underlying abnormal endothelium-dependent vascular relaxation in atherosclerosis. We have recently shown that the release of EDRF (as assessed by bioassay) is markedly impaired. Studies will be performed to examine possible mechanisms underlying this discrepancy. Specifically, we plan to test the hypothesis that EDRF is inactivated by oxygen free radicals within the vascular endothelium. Secondly, we plan to test the hypothesis that atherosclerosis does not prevent the production of nitric oxide but prevents its incorporation into a more potent parent vasodilator. We have recently shown that at least two nitrosylated and as yet unidentified compounds is impaired in atherosclerosis. We also hope to determine if chronic administration of an intracellular oxygen radical scavenger can correct this abnormality. Additional studies will be performed to determine if endothelium-dependent smooth muscle hyperpolarization is abnormal in atherosclerosis. Such an abnormality might account for altered endothelium-dependent relaxation in the presence of normal endothelial derived nitric oxide release. Finally, we plan studies of cultured endothelial cells to test the hypothesis that endothelial cell cyclic GMP regulates the enzymatic process responsible for the oxidation of the guanidino nitrogen of arginine. Overall the experiments planned during the next five years should provide substantial new information regarding regulation of vascular smooth muscle by the endothelium and how this is altered by atherosclerosis. Because of our past experience in this field and a variety of unique experimental preparations and technology available in our laboratories, we are in a unique position to accomplish these goals.
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