RELATION OF EXOCYTOTIC RELEASE OF GAMMA-AMINOBUTYRIC-ACID TO CA-2+ ENTRY THROUGH CA-2+ CHANNELS OR BY REVERSAL OF THE NA(+)/CA-2+ EXCHANGER IN SYNAPTOSOMES

The specific inhibitor of the gamma-aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmeth-ylene)amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridine-carboxylic acid hydrochloride, blocks the Ca2+-independent release of [H-3]GABA from rat brain synaptosomes induced by 50 mM K+ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca2+-dependent release of [H-3]GABA in the total absence of carrier-mediated release. Reversal of the Na+/Ca2+ exchanger was used to increase the intracellular free Ca2+ concentration ([Ca2+]i) to test whether an increase in [Ca2+]i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca2+], may rise to values above 400 nM, as a result of Na+/Ca2+ exchange, without inducing release of [H-3]GABA, but subsequent K+ depolarization immediately induced [H-3]GABA release. Thus, a rise of only a few nanomolar Ca2+ in the cytoplasm induced by 50 mM K+ depolarization, after loading the synaptosomes with Ca2+ by Na+/Ca2+ exchange, induced exocytotic [H-3]GABA release, whereas the rise in cytoplasmic [Ca2+] caused by reversal of the Na+/Ca2+ exchanger was insufficient to induce exocytosis, although the value for [Ca2+]i attained was higher than that required for exocytosis induced by K+ depolarization. The voltage-dependent Ca2+ entry due to K+ depolarization, after maximal Ca2+ loading of the synaptosomes by Na+/Ca2+ exchange, and the consequent [H-3]GABA release could be blocked by 50 muM verapamil. Although preloading the synaptosomes with Ca2+ by Na+/Ca2+ exchange did not cause [H-3]GABA release under any conditions studied, the rise in cytoplasmic [Ca2+] due to Na+/Ca2+ exchange increased the sensitivity to external Ca2+ of the exocytotic release of [H-3]GABA induced by subsequent K+ depolarization. Thus, our results show that the vesicular release of [H-3]GABA is rather insensitive to bulk cytoplasmic [Ca2+] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca2+ entry through Ca2+ channels near the active zones.