IP3 METABOLISM AND CA2+ HOMEOSTASIS IN CEREBRAL ISCHEMIA

心肌梗死后细胞内钙稳态机制的紊乱 脑缺血损伤与不可逆转的损伤有关 导致神经细胞死亡。尽管几个已知因素可能 在急性心肌梗死的早期阶段参与了细胞内钙离子的初始升高。 缺血性损伤--导致进行性神经元的机制(S) 长时间缺血和再循环后的损害尚不清楚。由于 INS(1，4，5)P3作为细胞内第二信使的重要作用 [Ca~(2+)]i动员，推测长时间的缺血- 再灌流导致不可逆的修饰，无论是在酶上 负责INS(1，4，5)P3的代谢或其与 细胞内受体。反过来，INS(1，4，5)P3代谢和/或 结合可能是导致细胞内钙离子变化的重要机制 动态平衡与神经细胞死亡有关。这一假设将是 使用已建立的大鼠局灶性缺血模型进行测试(阻断 大脑中动脉)，类似于人类的中风。具体目标 主要有：(1)体内和体外实验，以确定其作用效果 短暂性(15分钟)或长时间(60分钟)的脑缺血损伤 磷脂酰肌醇磷酸盐的分解和释放。(2) 脑缺血再灌流对心肌细胞代谢影响的实验研究 Ins(1，4，5)P3由5-磷酸酶和3-激酶组成。既然有很强的 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，可能被一些不可逆的修饰 包括钙/钙调蛋白和钙蛋白水解酶(Calain)在内的因素，研究 将重点放在缺血对蛋白质降解程度和水平的影响上 这种酶的信使核糖核酸。(3)观察脑缺血对心肌细胞的影响。 INS(1，4，5)P3受体结合活性及编码该受体的mRNA水平 受体蛋白。(4)在建立这些实验方案后，我们 将研究非NMDA拮抗剂(NBQX)和蛋白酶的作用 抑制剂(亮肽素)对其抑制缺血诱导的能力的影响 INS(1，4，5)P3代谢和结合的变化。长期目标是 了解局灶性脑缺血对分子和细胞的影响 细胞内钙离子动态平衡变化的机制 多聚磷脂酰肌醇信号通路的改变。信息 这一项目的结果将在制定战略时发挥重要作用 治疗干预对缓解心绞痛的病理生理作用 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.