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.
