ASSEMBLY OF ION PROTEINS IN GLIAL CELLS

这个项目的目的是为了阐明 阿米洛利对大鼠神经胶质细胞钠氢交换的激活作用 中枢神经系统。我们认为这个运输系统是 脑内神经胶质细胞的离子缓冲活性及其测定 控制其激活状态的进程对于 对癫痫和脑水肿相关缺陷的理解。我们的 方法是首先通过监测来分析活化的动力学 使用自动滴定方法的质子外流，即氢氧化氢的量 保持恒定pH值所需的离子。我们将使用C6细胞在 暂停；我们将利用Na/H交换是 在这些细胞中表达但不活跃的缺乏适当的 刺激物。我们会用缓激肽、佛波酯刺激系统 醋酸盐(PMA)和钙离子载体A23187。这些代理代表一个 膜受体介导的激素--蛋白激酶C的直接激活剂 以及一种专门提高细胞内钙离子的试剂， 分别进行了分析。我们不仅将监测特定的动力学参数，而且还将监测 依赖外部钙激活运输，有能力 去掉激活剂后停用转运及其动力学 重新刺激。因为数据已经表明聚磷脂酰肌醇 周转参与活化，其循环受氯化锂的影响， 将研究这种盐对再活化过程的影响。我们的 假设Na/H交换的激活水平是受控制的 通过蛋白激酶C的活性和一个单独的钙依赖步骤 它们又受多磷肌醇的状态调节 新陈代谢和二十烷类化合物。我们将把我们的动力学数据带入 激活剂对1)多磷肌醇代谢影响的研究 2)花生四烯酸释放与白三烯和前列腺素 综合。我们将把脂类代谢变化的动力学与 Na/H交换和检验这些脂质之间的相互关系 小路。我们将评估特定蛋白质抑制剂的疗效。 激酶C、花生四烯酸释放与白三烯和前列腺素 综合并使用这些数据来确定特定的关系 控制Na/H交换活跃状态的代谢途径。

This project is designed to elucidate the biochemical basis for the activation of amiloride sensitive Na/H exchange in glial cells of the central nervous system. We consider this transport system integral to the ion buffering activity of glial cells in brain and determining the processes controlling its state of activation are important to an understanding of defects associated with epilepsy and brain edema. Our approach is to first analyze the kinetics of activation by monitoring proton efflux using the automatic titration method, i.e. the amount of OH ions required to maintain constant pH. We will use C6 cells grown in suspension; we will take advantage of the fact that Na/H exchange is expressed in these cells but is inactive of the absence of appropriate stimuli. We will stimulate the system with bradykinin, phorbol myristoyl acetate (PMA) and the calcium ionophore A23187. These agents represent a membrane receptor mediated hormone, a direct activator of protein kinase C and an agent which specifically raises intracellular calcium, respectively. We will monitor not only specific kinetic parameters but the reliance on external calcium for activation of transport, the ability to deactivate transport upon removal of activators and the kinetics of restimulation. Since data already indicate that polyphosphoinositide turnover is involved in activation, and, its recycling is affected by LiCl, the effect of this salt on the reactivation process will be studied. Our hypothesis is that the level of activation of Na/H exchange is controlled by the activity of protein kinase C and a separate calcium dependent step which are in turn regulated by the status of polyphosphoinositide metabolism and eicosanoids. We will carry our kinetic data over into the study of the effects of activators on 1) polyphosphoinositide metabolism and 2) arachidonic acid release and leukotriene and prostaglandin synthesis. We will compare the kinetics of changes in lipid metabolism to those in Na/H exchange and examine the interrelationship of these lipid pathways. We will assess the efficacy of specific inhibitors of protein kinase C, arachidonic acid release and leukotriene and prostaglandin synthesis and use these data to determine the relationship of specific metabolic pathways to the control of the active state of Na/H exchange.