K + EFFLUX--ROLE IN CA++ HOMEOSTASIS AND EXCITOTOXICITY

K EFFLUX--CA 稳态和兴奋性毒性的作用

• 批准号: 6343887
• 负责人: LECH Kiedrowski
• 金额: $ 10.68万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6343887
• 关键词: NMDA receptors; calcium flux; calcium ion; confocal scanning microscopy; electrophysiology; excitatory aminoacid; fluorescence microscopy; fluorescent dye /probe; glutamate receptor; glutamates; gramicidin; homeostasis; ischemia; laboratory rat; membrane potentials; neurotoxins; potassium channel; potassium ion; reperfusion; sodium ion; tissue /cell culture

Within only 2-3 min of brain ischemia, extracellular K+ concentrations ([K+])o reach 60 - 80 mM, which implies that the excitotoxic action of glutamate during ischemia is executed at highly elevated [K+]o. The proposed research will study whether such high [K+]o affects mechanisms of glutamate excitotoxicity. The hypothesis "High [K+]o reduces the electrochemical Ca2+ driving force (ECDF) in neurons exposed to glutamate receptor agonists" will be tested in Aim 1: "Study the effects of various [K+]o on the plasma membrane potential (Em) and the cytoplasmic Ca2+ concentration ([Ca2+]c) in cultured cortical and cerebellar neurons exposed to glutamate receptor agonists". To this end, neurons will be loaded with Em- and Ca2+-sensitive fluorescent probes. This hypothesis, if true, predicts that high [K+]o may prevent excitotoxicity by decreasing the glutamate mediated Ca2+ influx across the plasma membrane. It is also possible, however, that glutamate may collapse the Na+ and K+ concentration gradients across the plasma membrane and, consequently, set Em close to zero regardless of [K+]o. To test whether this is the case, the cytoplasmic Na+ and K+ concentrations will be measured in parallel experiments in neurons loaded with Na+- and K+-sensitive fluorescent probes. An alternative hypothesis, will be tested in Aim 2: "Determine whether increasing [K+]o may inhibit the glutamate-mediated Ca2+ influx in a manner not related to the plasma membrane depolarization". To this end, the effects of [K+]o on Ca2+ accumulation stimulated by glutamate receptor agonists will be studied in neurons depolarized by a Na+ and K+ ionophore, gramicidin. Finally, in Aim 3: "Test whether restoration of low [K+]o following ischemia in vitro causes a delayed Ca2+ accumulation and compromises neuronal survival", it will be studied whether [K+]o during the postischemic period affects Ca2+ homeostasis and is related to improved or compromised neuronal survival during the next 24 hours. These studies may yield a new target for pharmacological intervention to decrease neuronal death following ischemia or hypoglycemia, namely, to prevent excessive K+ loss from the brain during reperfusion.

仅在脑缺血2-3分钟内，细胞外K+浓度 ([K+])_0达到60-80 mM，表明其兴奋毒性作用 缺血时谷氨酸在高水平的[K+]o下执行。 拟议的研究将研究如此高的[K+]O是否会影响机制 谷氨酸的兴奋性毒性。假说“高[K+]o降低 电化学钙驱动力(ECDF)在神经细胞中的作用 谷氨酸受体激动剂“将在目标1中进行测试：”研究 不同[K+]o对质膜电位(Em)和膜电位的影响 培养的大脑皮层细胞内钙离子浓度([Ca~(2+)]c) 小脑神经元暴露于谷氨酸受体激动剂。 最终，神经元将被加载Em和钙敏感的荧光 探测器。如果这一假设为真，则预测高[K+]o可能会阻止 减少谷氨酸介导的钙离子跨膜内流的兴奋性毒性 质膜。然而，谷氨酸也有可能 横跨等离子体的Na+和K+浓度梯度的崩溃 因此，无论[K+]o如何，都将Em设置为接近于零。 为了测试情况是否如此，细胞质中的Na+和K+ 浓度将在神经元的平行实验中进行测量。 装有对Na+和K+敏感的荧光探针。另一种选择 假设，将在目标2中进行检验：“确定增加[K+]o 可能以一种无关的方式抑制谷氨酸介导的钙内流 至质膜去极化“。为此， [K+]o对谷氨酸受体激动剂刺激的钙蓄积的影响 将在由Na+和K+离子载体去极化的神经元中进行研究， 格拉米丁。最后，在目标3中：“测试低[K+]o是否恢复 体外缺血后引起延迟的钙蓄积和 损害神经元存活“，将研究[K+]o是否在 缺血后时间影响钙离子内稳态，并与 在接下来的24小时内改善或损害神经元的存活率。 这些研究可能为药物干预提供一个新的靶点。 减少缺血或低血糖引起的神经元死亡，即， 以防止再灌流过程中脑组织K+的过量流失。