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

K EFFLUX--CA 稳态和兴奋性毒性的作用

基本信息

批准号：
6627670
负责人：
LECH Kiedrowski
金额：
$ 11.33万
依托单位：
UNIVERSITY OF ILLINOIS AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
1999
资助国家：
美国
起止时间：
1999-01-01 至 2005-12-31
项目状态：
已结题

项目摘要

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.

在脑缺血的2-3分钟内， ([K+]）。达到60 - 80 mM，这意味着，在缺血期间谷氨酸在高度升高的[K+]o下被执行。的拟议的研究将研究这种高[K+]O是否会影响机制谷氨酸兴奋性毒性假设“高[K+]O降低了电化学钙驱动力（ECDF）在神经元暴露于谷氨酸受体激动剂”将在目标1：“研究不同[K+]o对质膜电位（Em）的影响，培养皮层细胞胞浆Ca ~（2+）浓度（[Ca ~（2+）]c），暴露于谷氨酸受体激动剂的小脑神经元”。本最后，神经元将负载Em-和Ca 2 +-敏感的荧光 probes. 这一假设，如果是真的，预测高[K+]O可能会阻止通过减少谷氨酸介导的跨膜Ca 2+内流，质膜。然而，谷氨酸也可能使血浆中的Na+和K+浓度梯度崩溃膜，并因此将Em设置为接近零，而不管[K+]o。为了验证这一点，细胞质中的Na+和K+ 浓度将在神经元中的平行实验中测量装载Na+和K+敏感的荧光探针。一个替代假设，将在目标2中进行检验：“确定增加[K+]o 可能抑制谷氨酸介导的Ca 2+内流，到质膜去极化”。为此， [K+]o对谷氨酸受体激动剂刺激的Ca 2+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，短杆菌肽最后，在目标3中：“测试是否恢复低[K+]o 体外缺血后引起延迟的Ca 2+积累， “损害神经元存活”，将研究[K+]o是否在缺血后时期影响Ca 2+稳态，并与在接下来的24小时内改善或损害神经元存活。这些研究可能为药物干预提供新的靶点为了减少局部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的K+过度流失。