γ-Aminobutyric acid release from synaptosomes as influenced by Ca 2+ and Ca 2+ channel blockers

Abstract

We studied the correlation between the high affinity binding of Ca 2+ channel blockers to purified synaptic plasma membranes (SPM) and the effect of these drugs in blocking the 45Ca 2+ uptake and the release of [ 3H] γ-aminobutyric acid ([ 3H]GABA) by preloaded synaptosomes. The Ca 2+ 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 Ca 2+ channel blockers. The binding parameters were similar to those previously reported for whole brain homogenates: K D = 0.64 nM and B max = 160 fmol/mg of protein for [ 3H]nimodipine, and K D = 7.9 nm and B max = 1500 fmol/mg of pprotein for (−)-[ 3H]desmethoxyverapamil. The Ca 2+ channel blockers inhibited the releaseof [ 3H]GABA induced by K + depolarization in the presence or in the absence of Ca 2+. The Ca 2+-dependentcomponent of [ 3H]GABA release was inhibited by verapamil, (−)-D 600, d-cis-diltiazem, nifedipine and PY 108-86 with IC 50 values of 2.2 × 10 −5, 6.3 × 10 −5 M, 3 × 10 −4 M, > 10 −4 M and 3 × 10 −5 M, respectively. Furthermore, the Ca 2− channel blockers also inhibited the Ca 2+-independent [ 3H]GABA release which occured in the presence, but not in the absence, of external Na +. The Ca 2+ channel blockers at concentrations which inhibited [ 3H]GABA release inhibited the entry of Ca 2+ through the Ca 2+ channels and also the entry of Ca 2+ by Na +/Ca 2+ exchange. We conclude that the concentrations of Ca 2+ blockers necessary to block Ca 2+ uptake through the Ca 2+ channels and by Na +/Ca 2+ exchange coincide with the concentrations at which theyr inhibit [ 3H]GABA release, but that their effect on the relationship between Ca 2+ uptake and [ 3H]GABA release is different for the various blockers. The effects of the drugs on Ca 2+ movements and [ 3H]GGABA release are not specifically mediated through the high affinity bindinng of the drugs since relatively high concentrations were necessary (> 10 −5 M) for the effects reported here.