HYPOXIA/REOXYGENATION AND REGULATORY MECHANISMS

缺氧/复氧和调节机制

• 批准号: 6151551
• 负责人: RALPH G DACEY
• 金额: $ 29.26万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 2002-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6151551
• 关键词: arterioles; biological signal transduction; brain circulation; calcium channel blockers; calcium flux; cerebral ischemia /hypoxia; flavonoids; laboratory mouse; laboratory rat; nitric oxide synthase; tissue /cell culture; vascular endothelium; vascular smooth muscle; vasoconstriction; vasodilation; vasodilators; video microscopy

The goal of this project is to test the hypothesis that short term hypoxia and reoxygenation (H/RO)-induced impairment of vascular regulation in isolated cerebral penetrating arterioles is due to specific damage of the endothelial nitric oxide synthase pathway. H/RO causes vasoconstriction in large isolated cerebral blood vessels. The mechanism for this construction it is not understood and it is not known if penetrating arterioles are directly damaged after H/RO. Since cerebral arterioles are the last regulator of blood flow to the tissue, even a small vasoconstriction would greatly increase vascular resistance. This could contribute to cerebral damage due to postischemic hypoperfusion after ischemia/ reperfusion (I/R) in vivo. Experiments will be performed in vitro. Rat cerebral arterioles and arterioles of endothelial nitric oxide synthase deficient (eNOS) mice will be isolated, cannulated, and video-microscopically observed. The vessels will be subjected to hypoxia (p02 of 1mm Hg) of up to two hours. Cell cultures of rat cerebral microvascular endothelial (EC) and smooth muscle cells (SMC) will be used in parallel experiments. (1) It will be determined if only the endothelium is impaired after H/RO. Measurements of the vessel diameter, physiological vessel responses, membrane potentials (MP) in SMC as well as intracellular calcium (Ca++) in SMC and EC will be used as indicators of injury after H/RO. Selective impairment of the endothelium will be used to investigate whether the EC is the target of H/RO induced damage. Measurement of MP and Ca++ in cultured EC and SMC will identify the cell type affected by H/RO. (2) We will determine where the eNOS dependent metabolic pathway is affected in the endothelial cell. We will test for changes in the signal transduction pathway in both isolated vessels and in cell culture using specific agonists and measuring transient changes in vessel diameter, MP, and Ca++. Detection of signal transduction proteins in cell culture will indicate the impaired step in the signal transduction chain. (3) We will demonstrate the feasibility of pharmacologically alleviating vascular impairment due to H/RO. Treatments with calcium blockers, eNOS independent vasodilators or protective flavonoid will indicate if endothelial impairment due to H/RO can be alleviated or prevented in both isolated vessel and culture ECS. These studies will give as crucial information on the cellular target of H/RO inducted vascular microvessel injury and possible treatments targeted precisely to either prevent a specific cell damage or to take advantage of using regulatory mechanisms resistant to H/RO to improve vascular perfusion in vivo.

该项目的目的是检验短期的假设 缺氧和重氧（H/RO）诱导的血管损伤 孤立的脑穿透性小动脉中的调节是由于特定的 内皮一氧化氮合酶途径的损伤。 H/RO原因 大型分离的脑血管的血管收缩。 这 这种结构的机制尚不理解，也不是 已知是否穿透动脉在H/RO后直接损坏。 由于脑小动物是血液流向的最后一个调节剂 组织，即使是小血管收缩也会大大增加血管 反抗。 这可能会导致大脑损伤 缺血/再灌注（I/ R）体内缺血后灌注不足。 实验将在体外进行。 大鼠脑动脉和 内皮一氧化氮合酶缺乏（ENOS）小鼠的小动脉 将观察到孤立，插管和视频微观观察。 这 容器将受到多达两种的缺氧（1mm Hg的P02） 小时。 大鼠脑微血管内皮（EC）的细胞培养物 平滑肌细胞（SMC）将用于并行实验。 （1）将确定是否仅在 H/ro。 血管直径，生理血管的测量值 SMC和细胞内的响应，膜电位（MP） SMC和EC中的钙（CA ++）将用作损伤的指标 H/RO之后。 内皮的选择性损害将用于 研究EC是否是H/RO诱导损伤的靶标。 培养的EC和SMC中MP和CA ++的测量将识别 受H/RO影响的细胞类型。 （2）我们将确定 eNOS依赖性代谢途径在内皮细胞中受到影响。 我们将测试两者中信号转导途径的变化 孤立的血管和在细胞培养中使用特定的激动剂和 测量血管直径，MP和Ca ++的瞬态变化。 在细胞培养中检测信号转导蛋白的检测将表明 信号转导链中的步骤受损。 （3）我们会的 证明药理学减轻血管的可行性 由于H/RO造成的损害。用钙阻滞剂，eNOS处理 独立的血管扩张剂或防护类黄酮将指示是否是 H/RO引起的内皮损伤可以缓解或阻止 孤立的容器和培养EC。 这些研究将至关重要 有关H/RO感应血管的细胞靶标的信息 微血管损伤和可能针对的治疗方法 防止特定的细胞损坏或利用使用调节 对H/RO的机制可改善体内血管灌注。