Stimulation of [3H]γ‐Aminobutyric Acid Release by Calcium Chelators in Synaptosomes

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Abstract
The effect of EGTA [ethylene glycol-bis (.beta.-aminoethyl ether)-N,N''-tetraacetic acid] on the release of labeled GABA, glutamate, acetylcholine, and dopamine was studied in superfused synaptosomes from mouse brain. In the absence of both Ca2+ and Mg2+, EGTA and also EDTA at 50 .mu.M or higher concentrations induced a 2.5-5-fold stimulation of [3H]GABA release, similar to that produced by K depolarization, whereas only a slight effect, or no effect at all, was observed on the release of the other transmitters studied. The GABA-releasing action of EGTA was practically abolished in the presence of Mg2+. The effect of EDTA was also observed when the medium contained Mg2+. Studies on the ionic dependence showed that the stimulation of GABA release by EGTA was abolished in a Na+-free medium. Li+ did not substitute Na+ for the EGTA effect, which was also independent of Cl. This Na+ dependence does not seem to involve voltage sensitive channels, since tetrodotoxin did not affect the GABA-releasing action of EGTA, whereas in parallel superfusion chambers it blocked over 80% of the stimulation of GABA release by veratridine. Two Ca channel blockers in synaptosomes, La3+ and the cationic dye ruthenium red, greatly inhibited the GABA-releasing effect of EGTA. L-2,4-Diaminobutyric acid, an inhibitor of the Na+-dependent GABA carrier, did not affect the releasing action of EGTA, whereas in a parallel experiment this drug inhibited by more than 90% the exchange of labeled GABA with unlabeled GABA. Apparently, the Na+-dependent releasing action of EGTA and EDTA on GABA is probably due to a destabilization of the synaptosomal membrane by chelation of endogenous membrane Ca2+, which can be prevented by Mg2+. Such destabilization results in Na+ influx through Ca2+ channels, and the consequent increase in the intraterminal Na+ concentration induces the release of GABA by a mechanism probably not involving the amino acid carrier. The possible participation of mitochondrial Na+-Ca2+ exchange is considered improbable in view of the lack of effect of EGTA on the release of other neurotransmitters.