Hypoxia stimulates endothelium dependent vasoconstriction in the pulmonary artery and enhances expression of the gene for endothelin-1 (ET-1) in human umbilical vein endothelial cells in culture and in rat lung, but not in organs of the rat that are supplied by the systemic arterial bed. The general hypothesis of the current proposal is that ET-1 gene expression and release of big ET-1 from endothelial cells of the pulmonary vasculature are enhanced by exposure to hypoxia, and that the ET-1 so generated plays an etiologic role in hypoxia induced pulmonary vasoconstriction and pulmonary hypertension. A subhypothesis is that inhibitors of endothelin converting enzyme (ECE) such as phosphoramidon and/or ET-1 receptor antagonists such as [Dpr-1 Asp-15I ET-1 inhibit hypoxic pulmonary vasoconstriction and protect against the development of hypoxic pulmonary hypertension.
The Specific Aims are: 1) To test the hypothesis that ET-1 gene expression, ET-1 receptor mechanisms and ET-1 clearance are altered in the rat in response to acute (24, 48 hrs) and chronic (7, 14, 28 das) normobaric hypoxia and relate these alterations to hypoxia induced increases in pulmonary artery pressure. 2) To examine the cellular basis of selectivity of the hypoxic response of ET-1 for lung. 3) To define the cis-regulatory element(s) involved in amplifying transcription of the ET-1 gene in endothelial cells in response to hypoxia and to identify and clone the hypoxic transcription activator protein(s). 4) To test the hypothesis that enhanced ET-1 gene expression and, thereby, increased endogenous ET-1 levels mediate hypoxic pulmonary hypertension in the rat. The effect of lowering endogenous ET-1 levels by inhibiting ECE with the neutral metalloendopeptidase inhibitor phosphoramidon and of blocking ET-1 receptors with the ET-1 antagonist [Dpr-1, Asp15] ET-1 will be examined. The effects of phosphoramidon and [Dpr-1, Aspl5] ET-1 treatment on acute hypoxic pulmonary vasoconstriction and on chronic hypoxia-induced pulmonary hypertension will be tested. Sprague Dawley rats exposed to normobaric hypoxia (10% O2 at 1 atmosphere) will be studied in Specific Aims 1 and 4. Endothelial and smooth muscle cells in low passage derived from porcine main pulmonary artery and aorta and from precapillary arterioles of porcine lung will be studied in Specific Aim 2. Porcine aortic endothelial cells transfected with fragments of human ET-1 genomic DNA containing putative hypoxia response element(s) will be used in Specific Aim 3.
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