Effects of ethanol on NMDA receptor-mediated functions in primary cultures of cerebellar granule cells

Primary cultures of cerebellar granule cells were used to investigate the effects of acute and chronic exposure of ethanol on NMDA receptor functions and to examine the possible sites of interaction between ethanol and the NMDA receptor. Activation of NMDA receptors induced elevation of free cytoplasmic Ca2+, Ca2+ influx, activation of protein kinase C and neurotoxicity. Acutely added ethanol potently inhibited these NMDA receptor-mediated functional responses, although the existence of ethanol-sensitive and -insensitive neurons was observed. Glycine, as a necessary co-agonist, was required for full activation of the NMDA receptors and also to demonstrate the inhibitory actions of ethanol. However, glycine and ethanol did not share the same regulatory site at the NMDA receptor. Spermidine, a polyamine site agonist, dramatically potentiated NMDA-induced neurotoxicity. The potentiating effect of spermidine was not altered by ethanol, indicating that ethanol and spermidine produce their effects acting at different sites within the NMDA receptor complex. In contrast, the neuroprotective action of ethanol on NMDA-induced cell death was significantly reduced by spermidine, suggesting that the spermidine enhancement of NMDA receptor function is so potent that it could mask the inhibitory action of ethanol on other sites within the NMDA receptor. When homoquinolinic acid (HQ), a NR2A-13 subunit-selective agonist, was used as agonist, the NMDA receptor- mediated neurotoxicity was more pronounced. Ethanol antagonized the neurotoxic effects of HQ in a competitive- like manner in contrast to the noncompetitive reduction of NMDA-induced neurotoxicity suggesting the existence of a novel site of interaction between ethanol and the NMDA receptor. Enhancement of several NMDA receptor functions was observed in cell cultures chronically exposed to intoxicating concentrations of ethanol. Thus, NMDA-induced neurotoxicity and elevation of cytoplasmic free Ca2+ were clearly potentiated. However, the NMDA-induced 45Ca2+ influx was not altered by chronic ethanol, suggesting that chronic exposure to ethanol mainly affects the ability of neurons to utilize free cytoplasmic Ca2+. Furthermore, the potentiation of NMDA-mediated functional responses following chronic ethanol treatment was not accompanied by changes in the number or affinity of NMDA receptor antagonist [3H] MK-801 binding sites, indicating that the conductance of the NMDA receptor-controlled cation channel was not affect