Ca 2+ -independent vesicular catecholamine release in PC12 cells by nanomolar concentrations of Pb 2+ Remco H. S. Westerink and Henk P. M. Vijverberg Institute for Risk Assessment Sciences, Utrecht University, Utrecht, The Netherlands Abstract Effects of Pb 2+ on vesicular catecholamine release in intact and ionomycin-permeabilized PC12 cells were investigated using carbon fibre microelectrode amperometry. Changes in intra- cellular Pb 2+ and Ca 2+ were measured from indo-1 fluores- cence by confocal laser scanning microscopy. Depolarization of intact cells and superfusion of permeabilized cells with saline containing ‡ 100 lM Ca 2+ rapidly evokes quantal catecholamine release. Superfusion with up to 10 lM Pb 2+ -containing saline evokes release of similar catecholamine quanta after a concentration-dependent delay. Thresholds to induce exocy- tosis within 30 min of exposure are between 1 and 10 lM Pb 2+ in intact cells and between 10 and 30 nM Pb 2+ in permeabilized cells. Additional inhibition of exocytosis occurs in permeabilized cells exposed to 10 lM 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 nM) 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+ . Keywords: Ca 2+ -imaging, carbon fibre microelectrode amperometry, indo-1, ionomycin, Pb 2+ -imaging, rat phaeo- chromocytoma cells. J. Neurochem. (2002) 80, 861–873. Exocytosis is a complex, Ca 2+ -dependent process involving docking and priming of vesicles, intracellular fusion of the vesicle and plasma membranes, the formation of a fusion pore, and the release of vesicle contents. Exocytosis is tightly regulated by a variety of highly conserved proteins (for review see Su ¨ dhof 1995; Martin 1997; Lin and Scheller 2000). Several proteins associated with exocytosis, which require Ca 2+ as a co-factor, are also activated by Pb 2+ . Pb 2+ is far more potent than Ca 2+ as an activator of calmodulin (Habermann et al. 1983; Kern et al. 2000), protein kinase C (PKC; Tomsig and Suszkiw 1995; Sun et al. 1999), calci- neurin (Kern and Audesirk 2000) and synaptotagmin (Bouton et al. 2001). Furthermore, extracellular Pb 2+ inhibits voltage-gated Ca 2+ currents (Reuveny and Narahashi 1991; Audesirk and Audesirk 1993; Oortgiesen et al. 1993; Sun and Suszkiw 1995). Under low Ca 2+ conditions, Pb 2+ may permeate through Ca 2+ channels (Tomsig and Suszkiw 1991) and reduce inactivation of Ca 2+ channels at an intracellular site (Sun and Suszkiw 1995). Two main effects of Pb 2+ on neurotransmitter release have been reported. The first effect observed is inhibition of evoked neurotransmitter release, which has been attributed to a reduced influx of Ca 2+ due to Ca 2+ channel block. The second, delayed effect of Pb 2+ is an enhancement of spontaneous neurotransmitter release, which occurs irrespec- tive of the presence of extracellular Ca 2+ and has been attributed to intracellular effects of Pb 2+ (Manalis and Cooper 1973; Tomsig and Suszkiw 1993; Bressler et al. 1996; Braga et al. 1999). The enhancement of the frequency of miniature endplate potentials at the frog neuromuscular junction (Manalis and Cooper 1973) and of excitatory post-synaptic currents in rat hippocampal neurones (Braga et al. 1999) indicate that Pb 2+ facilitates synaptic vesicle release. Received July 23, 2001; revised manuscript received November 15, 2001; accepted December 10, 2001. Address correspondence and reprint requests to R. H. S. Westerink, Institute for Risk Assessment Sciences, Utrecht University, PO Box 80176, NL-3508 TD Utrecht, The Netherlands. E-mail: R.Westerink@iras.uu.nl Abbreviations used: CLSM, confocal laser scanning microscopy; DMSO, dimethylsulfoxide; PKC, protein kinase C; TPEN, tetrakis- (2-pyridylmethyl)ethylenediamine. Journal of Neurochemistry , 2002, 80, 861–873 Ó 2002 International Society for Neurochemistry, Journal of Neurochemistry , 80, 861–873 861