Ca 2+ ‐independent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb 2+

Effects of Pb 2+ on vesicular catecholamine release in intact and ionomycin‐permeabilized PC12 cells were investigated using carbon fibre microelectrode amperometry. Changes in intracellular Pb 2+ and Ca 2+ were measured from indo‐1 fluorescence by confocal laser scanning microscopy. Depolarization of intact cells and superfusion of permeabilized cells with saline containing ≥ 100 µ m Ca 2+ rapidly evokes quantal catecholamine release. Superfusion with up to 10 µ m Pb 2+ ‐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 µ m Pb 2+ in intact cells and between 10 and 30 n m Pb 2+ in permeabilized cells. Additional inhibition of exocytosis occurs in permeabilized cells exposed to 10 µ m Pb 2+ . Using membrane‐impermeable and ‐permeable chelators it is demonstrated that intracellular Ca 2+ is not required for Pb 2+ ‐induced exocytosis. In indo‐ 1‐loaded cells Pb 2+ reduces the fluorescence intensity after a concentration‐dependent delay, whereas the fluorescence ratio, indicating intracellular Ca 2+ concentration, remains unchanged. The delay to detect an increase in free intracellular Pb 2+ (≥ 30 n m ) is much longer than the delay to Pb 2+ ‐induced exocytosis, indicating that cytoplasmic components buffer Pb 2+ with high affinity. It is concluded that Pb 2+ acts as a high‐affinity substitute for Ca 2+ to trigger essential steps leading to vesicular catecholamine release, which occurs when only ∼20% of the intracellular high‐affinity binding capacity (∼2 attomol/cell) is saturated with Pb 2+ .