ENDOTHELIN AND RECEPTOR GENE EXPRESSION IN HYPOXIA

低氧刺激大鼠血管内皮依赖性收缩 增强内皮素-1基因的表达 (ET-1)在体外培养和大鼠脐静脉内皮细胞中的表达 肺，但不是在大鼠的器官中，这些器官是由全身器官供应的 动脉床。目前提案的总体假设是 血管内皮细胞ET-1基因表达及BIG-1释放 暴露在低氧环境中会增强肺血管系统，而且 由此产生的ET-1在低氧诱导中起病因学作用 肺血管收缩和肺动脉高压。一个子假说 内皮素转换酶的抑制物，如 磷酰胺和/或ET-1受体拮抗剂，如[DPR-1 Asp-15I ET-1抑制缺氧性肺血管收缩 缺氧性肺动脉高压的发展。 其具体目的是：1)检验ET-1基因的假设 ET-1表达、受体机制和清除ET-1的改变 大鼠对急性(24，48小时)和慢性(7，14，28小时)的反应 常压低氧及其与低氧诱导的改变之间的关系 肺动脉压升高。2)检查细胞基础 ET-1对肺的低氧反应的选择性。3)定义 参与转录扩增的顺式调节元件(S) ET-1基因对内皮细胞缺氧的响应及鉴定 克隆低氧转录激活蛋白(S)。4)测试 增强ET-1基因表达并因此增加的假说 内源性ET-1水平在大鼠缺氧性肺动脉高压中的作用 血管紧张素转换酶抑制剂降低内源性ET-1水平的实验研究 中性金属内肽酶抑制剂磷酰胺及其阻断作用 ET-1受体与ET-1拮抗剂[DPR-1，Asp15]ET-1将 检查过了。磷酰胺和[DPR-1，Aspl5]ET-1的作用 急性缺氧性肺血管收缩和慢性阻塞性肺疾病的治疗 将对缺氧性肺动脉高压进行测试。 常压低氧(10%O2，1h)暴露SD大鼠 大气)将在具体目标1和4中进行研究。 猪主肺来源的低传代细胞中的平滑肌细胞 从猪肺的动脉、大动脉和毛细血管前小动脉 针对性研究2.转染猪主动脉内皮细胞 用含有假定缺氧的人ET-1基因组DNA片段 具体目标3中将使用反应要素(S)。

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.