IP3 METABOLISM AND CA2+ HOMEOSTASIS IN CEREBRAL ISCHEMIA

细胞内Ca ~（2+）稳态机制的紊乱 脑缺血性损伤与不可逆损伤有关 导致神经元细胞死亡。 尽管一些已知的因素可能 有助于在早期阶段[Ca 2 +]i的初始增加， 缺血性损伤，导致进行性神经元损伤的机制 长期缺血和再循环后的损伤尚不清楚。 由于 Ins（1，4，5）P3作为细胞内第二信使重要作用 [Ca2+]i动员，假设长时间缺血- 再灌注导致不可逆的改变，无论是对酶， 负责Ins（1，4，5）P3的代谢或其与 胞内受体 反过来，Ins（1，4，5）P3代谢和/或 结合可能是导致细胞内Ca ~（2+）改变的重要机制 与神经元细胞死亡相关的稳态。 这个假设将是 使用良好建立的大鼠局灶性缺血模型（闭塞 大脑中动脉）类似于人类中风。 具体目标 （1）体内和体外实验，以确定 短暂（15分钟）或长期（60分钟）缺血性损伤， 磷酸肌醇分解和肌醇磷酸的释放。 （二） 研究缺血-再灌注对血管内皮细胞代谢的影响的实验 Ins（1，4，5）P3通过5-磷酸酶和3-激酶表达。 由于有强大的 有证据表明，Ins（1，4，5）P3 3-激酶，一种关键的调节酶， Ins（1，4，5）P3代谢，可能被许多不可逆的修饰， 包括Ca 2 +/钙调蛋白和Ca 2+蛋白酶（钙蛋白酶）在内的因子，研究 将重点关注缺血对蛋白水解程度和水平的影响， 这种酶的mRNA。 (3)为了检测缺血对 Ins（1，4，5）P3受体结合活性和编码该受体的mRNA水平 受体蛋白 (4)在建立这些实验方案后，我们 将检查非NMDA拮抗剂（NBQX）和蛋白酶 抑制剂（亮抑酶肽）对它们抑制缺血诱导的 Ins（1，4，5）P3代谢和结合的变化。 长期目标是 了解局部缺血损伤对分子和细胞的影响， 导致细胞内Ca 2+稳态变化的机制， 多磷酸肌醇信号通路的改变。 信息 这一项目的成果将在制定战略方面发挥重要作用， 治疗干预，以减轻病理生理 缺血性组织损伤

A disturbance of the mechanism for intracellular Ca2+ homeostasis due to cerebral ischemic insult has been implicated in the irreversible damage leading to neuronal cell death. Although several known factors may contribute to the initial increase in [Ca2+]i during the early phase of an ischemic insult, the mechanism(s) leading to the progressive neuronal damage after prolonged ischemia and recirculation is not known. Due to the important role of Ins(1,4,5)P3 as second messenger for intracellular [Ca2+]i mobilization, it is hypothesized that prolonged ischemia- reperfusion results in irreversible modifications, either on the enzymes responsible for metabolism of Ins(1,4,5)P3 or its binding to the intracellular receptor. In turn, changes in Ins(1,4,5)P3 metabolism and/or binding may be an important mechanism leading to the altered cellular Ca2+ homeostasis associated with neuronal cell death. this hypothesis will be tested using a well established rat focal ischemia model (occlusion of middle cerebral artery) that resembles stroke in humans. Specific aims are: (1) In vivo and in vitro experiments to determine the effects of transient (15 min) or prolonged (60 min) ischemic insult on poly- phosphoinositide breakdown and release of inositol phosphates. (2) Experiments to examine the effects of ischemia-reperfusion on metabolism of Ins(1,4,5)P3 by the 5-phosphatase and 3-kinase. Since there is strong evidence that Ins(1,4,5)P3 3-kinase, a key regulatory enzyme for Ins(1,4,5)P3 metabolism, may be irreversibly modified by a number of factors including Ca2+/calmodulin and Ca2+-proteases (calpain), studies will focus on effects of ischemia on the extent of proteolysis and levels of mRNA for this enzyme. (3) To examine the effect of ischemia on Ins(1,4,5)P3 receptor binding activity and the levels of mRNA encoding this receptor protein. (4) Upon establishing these experimental protocols, we will examine the effects of non-NMDA antagonists (NBQX) and protease inhibitors (leupeptin) on their ability to inhibit the ischemia-induced changes in Ins(1,4,5)P3 metabolism and binding. The long term goal is to understand the effects of focal ischemic insult on molecular and cellular mechanisms leading to the changes in intracellular Ca2+ homeostasis through alteration of the poly-phosphoinositide signaling pathways. Information resulted from this project will be important in designing strategies for therapeutic intervention towards alleviating the pathophysiology of ischemic tissue injury.