The overall objective of this research is the elucidation of subcellular and molecular mechanisms by which lead produces its neurotoxic effects on the central and peripheral neurotransmission. The specific aim of this proposal is to distinguish between the Ca++-mediated and direct intracellular actions of Pb++ on the transmitter release. Specifically, the hypothesis will be tested that Pb++ has two intracellular actions: (a) facilitation of spontaneous transmitter release through direct, Ca++-mimicking, action on the secretory process, including mobilization of synaptic vesicles and synaptic vesicle-plasmalemma fusion (exocytosis), and (b) depression of evoked transmitter release due to impairment of ACh in storage in synaptic vesicles. 1. Fluorescent metallochromic indicators will be employed to measure cytosolic concentrations of Ca++ and Pb++ in lead-exposed synaptosomes, and correlation between these parameters and the lead-induced spontaneous release of ACh will be sought. 2. Permeabilized synaptosomes and chromaffin cells will be employed to assess direct effects of Pb++ on the exocytotic apparatus. 3. Effects of Pb++ on calmodulin- and cAMP-dependent phosphorylation of Synapsin 1 will be studied in intact and lysed synaptosomes, and in isolated synaptic vesicles. 4. Effects of Pb++ on recharging of synaptic vesicles with ACh will be investigated in nervestimulated electric tissue of Torpedo marmorata and with isolated synaptic vesicles in vitro.