Our long term interest is in understanding the regulation of the endothelium-derived relaxing factor/nitric oxide (EDRF/NO) signalling pathway as it relates to pulmonary vascular physiology and pathophysiology. This proposal will investigate basic cellular and molecular aspects of the regulation of NO signalling which may be directly related to the pathophysiology of several pulmonary disease processes including acute and chronic hypoxia, pulmonary hypertension, and the transplanted lung in which endothelial cell dysfunction has been clearly demonstrated and EDRF/NO-dependent vasodilation is impaired. By studying NO synthase activity, the mechanisms of acute hypoxia and hyperoxia inhibition of isolated pulmonary endothelial NO synthase and the importance of this oxygen tension regulation of NO synthase in the intact pulmonary endothelial cell at oxygen levels which physiologically regulate the pulmonary circulation will be determined. Unlike acute hypoxia, chronic hypoxia causes persistent inhibition of NO signalling suggesting more permanent regulation of this pathway at the level of protein synthesis or gene expression. This may be important in the pulmonary hypertension accompanying chronic hypoxia from several pulmonary disease states. The regulation of endothelial NO synthase and vascular smooth muscle soluble guanylyl cyclase activity by chronic hypoxia at the pre and post translation and transcription levels of expression of these two proteins will be investigated as mechanisms of chronic hypoxia inhibition of NO signalling using both cell culture and whole animal models. Evidence suggests that interactions between endothelial and vascular smooth muscle cells may influence the expression of NO synthase or guanylyl cyclase. This has significant implications in pulmonary disease states where one or the other cell type may be dysfunctional. The effect of coculture versus isolated culture of these two cell types on the activity, translation and transcription of NO synthase and of soluble guanylyl cyclase will be determined. NO and cyclic GMP appear to regulate NO synthase via a feedback action. The Ki for NO inhibition of endothelial NO synthase, the importance of this action in the intact cell, and the mechanism and nature of the interaction will be studied using enzyme assay, spectrophotometric, and electron paramagnetic resonance techniques. The existence of a soluble guanylyl cyclase in endothelial cells to serve as an effector site for NO derived from endothelial NO synthase, and thus provide a feedback regulatory mechanism, will be definitively determined by Northern blot and polymerase chain reaction techniques using cDNA's for the soluble guanylyl cyclase subunits. the importance of NO signalling and these regulatory actions to pulmonary physiology and pathophysiology is clearly dependent on the presence and distribution of NO synthase and guanylyl cyclase throughout the pulmonary circulation. Thus, the longitudinal distribution of these two enzymes in the pulmonary arterial and venous vasculature will be determined using immunohistochemical and in situ hybridization techniques in lung sections.
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