K + EFFLUX--ROLE IN CA++ HOMEOSTASIS AND EXCITOTOXICITY
K + EFFLUX--ROLE IN CA++ HOMEOSTASIS AND EXCITOTOXICITY
批准号:
2762014
负责人:
LECH Kiedrowski
金额:
$10.65万
依托单位国家:
美国
项目类别:
财政年份:
1999
资助国家:
美国
项目状态:
已结题
起止时间:
1999-01-01 至 2003-12-31
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
中文摘要
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。
英文摘要
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.
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海外基金