Ca2+-independent vesicular catecholarnine release in PC12 cells by nanomolar concentrations of Pb2+

Effects of Pb2+, on vesicular catecholamine release in intact and ionomycin-permeabilized PC12 cells were investigated using carbon fibre microelectrode amperometry. Changes in intracellular Pb2+ and Ca2+, were measured from indo-1 fluorescence by confocal laser scanning microscopy. Depolarization of intact cells and superfusion of permeabilized cells with saline containing greater than or equal to 100 mum Ca2+ rapidly evokes quantal catecholamine release. Superfusion with up to 10 mum Pb2+-containing saline evokes release of similar catecholamine quanta after a concentration-dependent delay. Thresholds to induce exocytosis within 30 min of exposure are between 1 and 10 pm Pb2+ in intact cells and between 10 and 30 nm Pb2+ in permeabilized cells. Additional inhibition of exocytosis occurs in permeabilized cells exposed to 10 mum Pb2+. Using membrane-impermeable and -permeable chelators it is demonstrated that intracellular Ca2+, is not required for Pb 2, -induced exocytosis. In indo-1-loaded cells Pb2+ reduces the fluorescence intensity after a concentration-dependent delay, whereas the fluorescence ratio, indicating intracellular Ca2+ concentration, remains unchanged. The delay to detect an increase in free intracellular Pb2+ (greater than or equal to 30 nm) is much longer than the delay to Pb2+- induced exocytosis, indicating that cytoplasmic components buffer Pb 2, with high affinity. It is concluded that Pb2+ acts as a high-affinity substitute for Ca2+ to trigger essential steps leading to vesicular catecholamine release, which occurs when only similar to20% of the intracellular high-affinity binding capacity (similar to2 attomol/cell) is saturated with Pb2+.