Characterization of the carrier-mediated [3H]GABA release from isolated synaptic plasma membrane vesicles

Synaptic plasma membrane (SPM) vesicles were isolated under conditions which preserve most of their biochemical properties. Therefore, they appeared particularly useful to study the cytoplasmic GABA release mechanism through its neuronal transporter without interference of the exocytotic mechanism. In this work, we utilized SPM vesicles isolated from sheep brain cortex to investigate the process of [3H]GABA release induced by ouabain, veratridine and Na+ substitution by other monovalent cations (K+, Rb+, Li+, and choline). We observed that ouabain is unable to release [3H]GABA previously accumulated in the vesicles and, in our experimental conditions, it does not act as a depolarizing agent. In contrast, synaptic plasma membrane vesicles release [3H]GABA when veratridine is present in the external medium, and this process is sensitive to extravesicular Na+ and it is inhibited by extravesicular Ca2+ (1mM) under conditions which appear to permit its entry. However, veratridine-induced [3H]GABA release does not require membrane depolarization, since this drug does not induce any significant alteration in the membrane potential, which is determined by the magnitude of the ionic gradients artificially imposed to the vesicles. The substitution of Na+ by other monovalent cations promotes [3H]GABA release by altering the Na+ concentration gradient and the membrane potential of SPM vesicles. In the case of choline and Li+, we observed that the fraction of [3H]GABA released relatively to the total amount of neurotransmitter released by K+ or Rb+ is about 28% and 68%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)