gamma-Aminobutyric acid release from synaptosomes as influenced by Ca2+ and Ca2+ channel blockers.

We studied the correlation between the high affinity binding of Ca2+ channel blockers to purified synaptic plasma membranes (SPM) and the effect of these drugs in blocking the 45Ca2+ uptake and the release of [3H]gamma-aminobutyric acid [( 3H]GABA) by preloaded synaptosomes. The Ca2+ channel blocker binding sites were characterized by studying the binding of the dihydropyridine, [3H]nimodipine, and of the phenylalkylamine, (-)-[3H]desmethoxyverapamil, to purified SPM isolated from sheep brain cortex synaptosomes. The purified SPM had high affinity binding sites for both Ca2+ channel blockers. The binding parameters were similar to those previously reported for whole brain homogenates: KD = 0.64 nM and Bmax = 160 fmol/mg of protein for [3H]nimodipine, and KD = 7.9 nM and Bmax = 1,500 fmol/mg of protein for (-)-[3H]desmethoxyverapamil. The Ca2+ channel blockers inhibited the release of [3H]GABA induced by K+ depolarization in the presence or in the absence of Ca2+. The Ca2+-dependent component of [3H]GABA release was inhibited by verapamil, (-)-D 600, d-cis-diltiazem, nifedipine and PY 108-86 with IC50 values of 2.2 X 10(-5) M, 6.3 X 10(-5) M, 3 X 10(-4) M, greater than 10(-4) M and 3 X 10(-5) M, respectively. Furthermore, the Ca2+ channel blockers also inhibited the Ca2+-independent [3H]GABA release which occurred in the presence, but not in the absence, of external Na+. The Ca2+ channel blockers at concentrations which inhibited [3H]GABA release inhibited the entry of Ca2+ through the Ca2+ channels and also the entry of Ca2+ by Na+/Ca2+ exchange. We conclude that the concentrations of Ca2+ blockers necessary to block Ca2+ uptake through the Ca2+ channels and by Na+/Ca2+ exchange coincide with the concentrations at which they inhibit [3H]GABA release, but that their effect on the relationship between Ca2+ uptake and [3H]GABA release is different for the various blockers. The effects of the drugs on Ca2+ movements and [3H]GABA release are not specifically mediated through the high affinity binding of the drugs since relatively high concentrations were necessary (greater than 10(-5) M) for the effects reported here.