Victor Sapirstein - US grants

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.

The focus of this grant is the assembly of the plasma membrane proteolipid protein (PMPLP) in the synaptic plasma membrane and in myelin. This project will utilize a series of cell biologic techniques to follow this protein from its cite of synthesis in neurons and oligodendrocytes to the respective target membranes, eg. synaptic plasma membrane and myelin. Determination will be made of processing of PMPLP during the membrane assembly focusing on the acylation of the protein. The relative rate of assembly of this protein into myelin will be determined and compared to other myelin proteins such as the major myelin proteolipid and the myelin associated glycoprotein. Coated vesicles will be isolated from white matter and the presence of PMPLP and other myelin proteins determined and studies will be carried out to determine if these proteins have been newly synthesized. We will use immunologic methods to try to select as well as visualize subpopulations of coated vesicles that may transport the PMPLP or other myelin proteins. The final localization of PMPLP in the assembled myelin membrane of rat spinal cord will be examined using cryo- ultramicrotomy and immunoelectron microscopy. Studies will be carried out with anti PMPLP and visualized with colloidal gold conjugated second antibody. The assembly of the plasma membrane proteolipid into synaptic plasma membranes will be studied. Using immunological techniques we will examine if, in grey matter derived coated vesicles, PMPLP defines a specific sub class of vesicles and whether the sub class can be distinguished from those carrying synaptic vesicle proteins. The localization of PMPLP to specific membrane domains at the synapse will be determined using cryo- immunoelectron microscopy on ultra thin frozen sections of the rat dentate gyrus. The presence of PMPLP at the synapse will be analyzed in animals at different ages when a marked synaptogenesis and maturation of synapses is taking place in this brain region. The association of synaptic dysfunction with mental retardation and epilepsy suggests that this major synaptic protein could be involved in both the physiology and pathophysiology of the brain. Moreover, its associated with the myelin sheath suggest that not only may it be involved in the integrity of myelin but the abnormalities in the assembly of this protein could give rise to a constellation brain dysfunction associated with demyelinating and hypomyelinating diseases.

biomedical equipment purchase;

biomedical equipment purchase;

biomedical equipment purchase;