The studies outlined in this proposal are designed to provide new information about age-dependent decreases in the activity of two synaptic membrane systems involved in regulation of intra-synaptosomal Ca++ concentrations. The kinetic characteristics and thermotropic behavior of the Na+ Ca++ exchange activity of synaptic membranes will be studied in membranes from rats at 4 different ages. Similar types of studies will be performed on the (Ca++ Mg++)-ATPase and ATp-dependent Ca++ transport activities in synaptic membranes, in an effort to determine if previously observed de creases were indeed a function of aging. The possibility that altered synaptic membrane lipid composition is responsible for the decreased Na+ Ca++ exchange and (Ca++ Mg++)-ATPase activities will be examined. The cholesterol/phospholipid and unsaturated/saturated fatty acid ratios of synaptic membranes from 6, 18, 24, and 30-month old Fisher 344 rats will be determined by HPLC methods. Studies will be undertaken to manipulate the viscosity of the membranes in vitro since both Ca++ translocating systems appear to be sensitive to the state of their lipid environment. The membrane viscosity can be decreased by incorporation of unsaturatd fatty acids in the cis-configuration and by removal of some of the membrane cholesterol. The viscosity can be increased by addition of exogenous cholesterol through the use of lipid transfer proteins. Effects of the various manipulations on membrane fluidity will be documented by means of electron paramagnetic resonance (EPR) spectroscopy. These in vitro manipulations of the synaptic membrane lipid composition will be used to determine whether age-dependent changes in the ion translocation systems are due to alterations of the lipid environment in which these complexes reside or to some specific change in the macromolecules themselves. Synaptic terminals are constantly faced with significant influxes of Ca++ with each depolarization and must have very efficient means of dealing with this Ca++ accumulation. The evidence that elevations in intracellular free Ca++ are very cytotoxic is quite substantial. Thus, if the aging process leads to a decreased effectiveness in synaptic membrane systems for removing intra-terminal Ca++, the long term result may be a progressive neuronal dysfunction and eventual cell death.
