ASSEMBLY OF ION TRANSPORT PROTEIN IN GLIAL CELLS

该项目旨在阐明生物化学的基础， 阿米洛利敏感性Na/H交换的激活 中枢神经系统 我们认为这个运输系统是 脑胶质细胞的离子缓冲活性和测定 控制其激活状态的过程对于一个 了解与癫痫和脑水肿相关的缺陷。 我们 方法是首先通过监测来分析激活的动力学 使用自动滴定法的质子流出，即OH的量 我们将使用C6细胞生长在 悬浮液;我们将利用Na/H交换是 在这些细胞中表达，但由于缺乏适当的 刺激。 我们会用缓激肽，佛波肉豆蔻酰 乙酸盐（PMA）和钙离子载体A23187。 这些代理人代表一个 膜受体介导的激素，蛋白激酶C的直接激活剂 和特异性升高细胞内钙的试剂， 分别 我们将不仅监测具体的动力学参数， 依赖外部钙激活运输， 在去除活化剂后使运输失活， 再刺激 因为数据已经表明多磷酸肌醇 周转参与活化，其再循环受LiCl影响， 将研究该盐对再活化过程的影响。 我们 假设Na/H交换的活化水平是受控的 通过蛋白激酶C的活性和单独的钙依赖性步骤 而这又受到多磷酸肌醇的调节 代谢和类花生酸。 我们将把动力学数据 活化剂对多磷酸肌醇代谢影响的研究 花生四烯酸释放和白三烯及前列腺素 合成. 我们将比较脂质代谢变化的动力学， Na/H交换中的脂质，并检查这些脂质之间的相互关系 途径。 我们将评估特异性蛋白质抑制剂的疗效， 激酶C、花生四烯酸释放、白三烯和前列腺素 合成并使用这些数据来确定特定的 代谢途径来控制Na/H交换的活性状态。

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.