AMINO ACID RELEASE AND HYPOXIC NEURONAL DAMAGE

越来越多的证据表明，谷氨酸(Glu)， 天冬氨酸(Asp)和其他兴奋性氨基酸(EAAs)起重要作用 哺乳动物中枢神经系统在许多疾病过程中的作用(见 Rothman和Olney，1986；Cotman等人，1989)，包括缺血(Simon 低血糖(Weiloch，1985)、癫痫(Nadler等， 1978年)、亨廷顿病(Coyle和Schwartz，1976)和阿尔茨海默氏症 疾病(Maragos等人，1987年)。神经元损伤的概念是 通过释放EaS和随后刺激EAA来调节 受体具有重要的临床意义(梅尔德鲁姆，1985)，包括 减少EAA释放的药物治疗的发展。虽然 近年来，人们对EaaS的毒性有了很多了解 神经元(Choi等人，1988)，产生EaS释放的过程 在很大程度上仍然不为人知。这项提案的目标是测试 以下假设：CNS中至少有两个EaaS池可以 在新陈代谢的侮辱中独立释放。这些池的不同之处在于 它们的位置，它们的释放对钙的依赖，以及 产生其释放所需的ATP消耗程度。池1是 以钙非依赖性的方式从神经元释放，通过逆转 严重代谢损伤扰乱Na+时的Na-EAA共转运体 浓度。池2被有选择地从终端释放 轻度ATP耗竭时的钙依赖方式。为了检验这些假说 我们将实现以下缩写的具体目标：1.测试 Pool 1以一种不依赖于钙的方式从神经元释放，通过 Na-EAA共转运蛋白的逆转，我们将展示 (I)替换胞外Na+或(Ii)的共转运蛋白 哇巴因抑制Na/K-ATPase使胞外增加 EAAS，同时降低细胞内EAAS。我们将确定是否 严重的ATP耗竭以一致的钙非依赖性方式释放EaS Na-EAA共转运蛋白的逆转。2.要测试Pool 2是否 在不太严重的ATP耗尽期间从终端释放，我们将 确认轻度的ATP消耗会导致钙依赖的释放，即 与以下情况相比，轻度ATP耗竭选择性地降低末端谷氨酸 非末端谷氨酸，这种轻微的ATP耗竭会导致 细胞内钙离子与末端谷氨酸的释放呈正相关。3.至 检查两个池、切片和丰富的培养的独立性 因为神经元和神经胶质细胞将暴露在选择性 从每个池中释放EaAs以确定联合治疗是否 导致释放增加。

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.