AMINO ACID RELEASE AND HYPOXIC NEURONAL DAMAGE

氨基酸释放和缺氧神经元损伤

• 批准号: 3478126
• 负责人: JAMES E MADL
• 金额: $ 9.52万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3478126
• 关键词: adenosine triphosphate; aminoacid transport; aspartate; calcium; calcium indicator; digital imaging; electron microscopy; excitatory aminoacid; fluorescence microscopy; glia; glutamates; high performance liquid chromatography; hippocampus; hypoxia; immunocytochemistry; injury; laboratory rat; membrane transport proteins; neurons; ouabain; potassium; sodium; sodium potassium exchanging ATPase; tissue /cell culture; transport proteins

A growing body of evidence suggests that release of glutamate (Glu), aspartate (Asp) and other excitatory amino acids (EAAS) play important roles in many disease processes of the mammalian CNS (see reviews by Rothman and Olney, 1986; Cotman et al., 1989), including ischemia (Simon et al., 1984), hypoglycemia (Weiloch, 1985), epilepsy (Nadler et al., 1978), Huntington's disease (Coyle and Schwartz, 1976) and Alzheimer's disease (Maragos et al., 1987). The concept that neuronal damage is mediated through the release of EAAs and subsequent stimulation of EAA receptors is of great clinical importance (Meldrum, 1985), including the development of therapy with drugs that decrease EAA release. Although much has been leamed in recent years about the toxicity of EAAs to neurons (Choi et al.,1988), the processes producing the release of EAAs remain largely unknown. The goal of this proposal is to test the following hypotheses: At least two pools of EAAs in the CNS can be released independently during metabolic insults. These pools differ in their location, the calcium-dependence of their release and also the degree of ATP depletion required to produce their release. Pool 1 is released from neurons in a Ca-independent manner by reversal of the Na-EAA cotransporter during severe metabolic insults that perturb Na+ concentrations. Pool 2 is selectively released from terminals in a Ca-dependent manner during mild ATP depletion. To test these hypotheses we will accomplish the following abbreviated Specific Aims: 1. To test that Pool 1 is released in a Ca-independent manner from neurons by reversal of the Na-EAA cotransporter, we will show that reversal of the cotransporter by either (i) replacement of extracellular Na+ or (ii) inhibition of the Na/K ATPase with ouabain will increase extracellular EAAs while decreasing intracellular EAAS. We will determine whether severe ATP depletion releases EAAs in a Ca-independent manner consistent with reversal of the Na-EAA cotransporter. 2. To test that Pool 2 is released from terminals during less severe ATP depletion, we will confirm that mild ATP depletion results in a Ca-dependent release, that mild ATP depletion selectively decreases terminal Glu compared to non-terminal Glu, and that mild ATP depletion produces a rise of intracellular Ca++ correlated with release of terminal Glu. 3. To examine the independence of the two pools, slices and cultures enriched for neurons and glia will be exposed to conditions that selectively release EAAs from each pool to determine if the combined treatment causes inereased release.

越来越多的证据表明，谷氨酸（Glu）的释放， 天冬氨酸（Asp）和其他兴奋性氨基酸（EAAS）起重要作用， 在哺乳动物CNS的许多疾病过程中的作用（参见 Rothman和Olney，1986年; Cotman等人，1989），包括缺血（Simon 例如，1984）、低血糖（Weiloch，1985）、癫痫（Nadler等人， 1978年）、亨廷顿氏病（Coyle和Schwartz，1976年）和阿尔茨海默氏病 疾病（Maragos等，1987年）。神经元损伤的概念是 通过释放EAA和随后刺激EAA介导 受体具有重要的临床意义（Meldrum，1985），包括 开发减少EAA释放的药物治疗。虽然 近年来，人们对EAA的毒性有了很多了解， 神经元（Choi等人，1988年），生产EAA释放的过程 但基本上仍不为人所知。本提案的目的是测试 以下假设：CNS中至少有两个EAA库， 在代谢损伤期间独立释放。这些池的不同之处在于 它们的位置，它们释放的钙依赖性， 产生释放所需的ATP消耗程度。游泳池1是 以钙非依赖性方式从神经元释放，通过逆转 干扰Na+的严重代谢损伤期间的Na-EAA协同转运蛋白 浓度的池2选择性地从 轻度ATP耗竭时的Ca依赖性方式。来验证这些假设 我们将实现以下简要的具体目标：1。测试 池1以不依赖钙的方式从神经元释放， 逆转的Na-EAA协同转运，我们将表明，逆转的 协同转运蛋白（i）替代细胞外Na+或（ii） 哇巴因抑制Na/K ATP酶将增加细胞外 EAAs，同时降低细胞内EAAS。我们将决定 严重的ATP耗竭以不依赖钙的方式释放EAA， 与Na-EAA协同转运蛋白的逆转有关。2.为了测试池2是否 在不太严重的ATP耗竭期间从终端释放，我们将 证实轻度ATP耗竭导致Ca依赖性释放， 轻度ATP耗竭选择性降低末端Glu， 非末端Glu，轻度ATP耗竭会导致 细胞内Ca ~（++）与末端Glu的释放有关。3.到 检查两个池、切片和培养物的独立性， 神经元和神经胶质细胞将暴露在选择性 从每个池中释放EAA，以确定联合治疗是否 导致释放增加。