Free cytoplasmic Ca2+ and neurotransmitter release: Studies on PC12 cells and synaptosomes exposed to α-latrotoxin

The relationship between the free cytoplasmic Ca²⁺ concentration, [Ca²⁺](i), and neurotransmitter release was investigated in guinea pig brain synaptosomes and the neurosecretory cell line PC12. Release was induced by α-latrotoxin, which acts in both Ca²⁺-containing and Ca²⁺-free incubation media, or by the classical depolarizing agents high K⁺ and veratridine, which require extracellular Ca²⁺. Two complementary approaches were used to reveal changes of [Ca²⁺](i): (i) direct measurement by a fluorescent Ca²⁺ indicator (quin2) and (ii) study of the Ca²⁺-dependent phosphorylation of a protein, synapsin I, located at the cytoplasmic surface of synaptic vesicles. Depolarizing agents, when applied in Ca²⁺-containing medium, induced the [Ca²⁺](i) to increase promptly 3- to 6-fold, drastically increased synapsin I phosphorylation, and caused stimulation of transmitter release. With α-latrotoxin, the [Ca²⁺](i) increase was delayed and occurred at a slower rate, the increase of synapsin I phosphorylation was less drastic, and the release response was much more pronounced. In Ca²⁺-free medium, depolarizing agents released no transmitter and had no effect on [Ca²⁺](i) or synapsin I phosphorylation, whereas with α-latrotoxin these processes were dissociated: considerable stimulation of the release without apparent change of [Ca²⁺](i) and synapsin I phosphorylation. We conclude that the relationship between average [Ca²⁺](i) and transmitter release is not straightforward and, in particular, that the release evoked by α-latrotoxin in Ca²⁺-free medium is mediated by a factor(s) other than bulk redistribution of Ca²⁺ from intracellular stores.