The general aim of this proposal is a better understanding of the molecular events leading to fusion of synaptic vesicles with the neuronal presynaptic membrane. We have found and partially characterized phospholipase A2 (PLA2) and protein kinase activities in synaptic vesicles and membranes. These enzymatic activities support the concept that the Ca++ dependent PLA2 activity in synaptic vesicles and plasma membrane increases their fluidity, favoring fusion events and that these activities are modulated by lipomodulin, a protein capable of undergoing reversible phosphorylation catalyzed by a kinase/phosphatase system. The combined effects of protein kinase and phospholipase activity upon vesicle-membrane interaction can affect the fusion event. We intend to elucidate how various known neuromodulators (Ca++, calmodulin, several prostaglandins, cAMP) affect the kinetic parameters of these enzymes and if the observed effects are correlated with neurotransmission. We have standardized a technique by which an increase in light scattering yields an in vitro measure of aggregaton and/or fusion of synaptic vesicles and have monitored the effects of both exogenous and endogenous phospholipase activities upon synaptic vesicle preparations. Our initial studies have shown that prostaglandins E2 and F2Alpha inhibit and promote, respectively, the aggregation and apparent fusion of synaptic vesicles. The various neuromodulators affected endogenous PLA2 activity of the synaptic vesicles. We plan to identify the various prostaglandin types produced by vesicle fractions and their precise functions; we plan to purify the endogenous PLA2 and lipomodulin and prepare antibodies to these proteins in order to better understand their role in vesicle-membrane interaction and to quantitate the amount of these proteins present in various subcellular fractions. With the studies proposed we will attempt to explain the complex events that occur just prior to synaptic transmission and thereby gain a better understanding of how neurons carry out their secretory functions.
