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

脑缺血仅 2-3 分钟内，细胞外 K 浓度 ([K])o 达到 60 - 80 mM，这意味着 缺血期间谷氨酸在高度升高的 [K ]o 下执行。 的 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。假设“高 [K ]o 会降低 神经元暴露的电化学 Ca2 驱动力 (ECDF) 谷氨酸受体激动剂”将在目标 1 中进行测试：“研究效果 不同 [K ]o 对质膜电位 (Em) 和 培养皮质中的细胞质 Ca2 浓度 ([Ca2 ]c) 暴露于谷氨酸受体激动剂的小脑神经元”。 对此 最后，神经元将加载 Em 和 Ca2 敏感荧光 探针。 如果这个假设成立，则预测高 [K ]o 可能会阻止 通过减少谷氨酸介导的 Ca2 内流来抑制兴奋性毒性 质膜。 然而，谷氨酸也有可能 破坏等离子体中的 Na 和 K 浓度梯度 膜，因此，无论 [K ]o 如何，都将 Em 设置为接近于零。 为了测试是否是这种情况，细胞质 Na 和 K 将在神经元的平行实验中测量浓度 装载有 Na 和 K 敏感荧光探针。 另一种选择 假设，将在目标 2 中进行检验：“确定是否增加 [K ]o 可能以不相关的方式抑制谷氨酸介导的 Ca2 流入 质膜去极化”。 为此，效果 [K ]o 对谷氨酸受体激动剂刺激的 Ca2+ 积累的影响 将在由 Na 和 K 离子载体去极化的神经元中进行研究， 短杆菌肽。 最后，在目标 3 中：“测试是否恢复低 [K ]o 体外缺血后会导致 Ca2 积聚延迟， 损害神经元存活”，将研究 [K ]o 期间是否 缺血后时期影响 Ca2 稳态并与 在接下来的 24 小时内改善或损害神经元的存活。 这些研究可能会产生药物干预的新目标 减少缺血或低血糖后的神经元死亡，即 以防止再灌注期间大脑中钾流失过多。

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.