CHEB, a convulsant barbiturate, evokes calcium-dependent spontaneous glutamate release from rat cerebrocortical synaptosomes

Abstract

CHEB [5-(2-cyclohexylidene-ethyl)-5-ethyl barbituric acid] is a potent convulsant barbiturate that causes direct neuronal excitation by an unknown mechanism. We have analyzed the effects of CHEB on the release of endogenous glutamate from rat Cerebrocortical synaptosomes using an on-line enzyme-coupled fluorimetric assay. CHEB evoked spontaneous Ca 2+ -dependent glutamate release with an EC 50 = 14.2 μ M and an E max = 3.2 μ mol/min/mg. The non-convulsant barbiturates pentobarbital and phenobarbital evoked significantly less glutamate release at high concentrations. CHEB (30 μM) increased intrasynaptosomal [Ca 2+ ] by 58 ± 4 nM ( p < 0.01; n = 4) above baseline compared to an increase of 5 ± 4 nM (NS; n = 4) produced by pentobarbital (30 μM). CHEB-evoked glutamate release was inhibited by pentobarbital, phenobarbital, EGTA, CoCl 2 CdCl 2 and flunarizine, but not by local anesthetics, tetrodotoxin, nitrendipine or ω-conotoxin GVIA. These results demonstrate that CHEB acts as a potent and effective secretogogue for glutamate by a pre-synaptic mechanism that does not require activation of Na + channels or of L-type or N-type Ca 2+ channels. Stimulation of spontaneous glutamate release may contribute to the convulsant properties of CHEB.