Release of endogenous glutamate from rat cerebellar synaptosomes: interactions with adenosine and ethanol

The effects of ethanol and adenosine receptor agonist R-PIA and antagonist theophylline on release of endogenous glutamate were tested in rat cerebellar synaptosomal preparation. Release was carried out for 5 to 60 sec after which time the released glutamate was separated from the synaptosomal membranes by rapid filtration. The amount of released glutamate in the filtrate was measured by an enzyme-linked fluorometric assay. Basal endogenous glutamate release was estimated as 3.7 +/- 0.3 nmol/mg protein/5 sec and was stimulated by high K+. Glutamate release consisted of an initial rapid phase for the first 10 sec that was followed by a relatively slower phase. Both Ca2+-dependent and Ca2+-independent glutamate release were observed which suggested the involvement of both neuronal and glial constituents of the synaptosomal preparation, respectively. Pharmacologically relevant concentrations of ethanol (25-100 mM) caused a trend toward a dose-dependent inhibition of glutamate release. R-PIA and theophylline inhibited and stimulated, respectively, basal release of glutamate and R-PIA-inhibited release was blocked by theophylline. Ethanol (25 mM) blocked the stimulatory effect of theophylline and the results of experiments following the inclusion of adenosine deaminase suggested the involvement of adenosine in this effect of ethanol. The results support our previous findings that suggest an involvement of cerebellar adenosine in the motor disturbing effects of acute ethanol and extend those findings by indicating that ethanol inhibits glutamate release from granule cells of the cerebellar cortex through an adenosine-sensitive mechanism.