Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells.
The effects of ethanol and/or glycine on NMDA-induced enhancement of cytoplasmic free Ca2+ concentrations ([Ca2+]i), 45Ca2+ influx, 4-b-[3H]phorbol-12,13-dibutyrate ([3H]PDBu) binding, and neuronal necrosis in cultured rat cortical and cerebellar granule neurons were examined. Using microfluorimetric techniques in combination with rapid perfusion of single brain neurons, we found that glycine (10 microM) was a necessary co-agonist for NMDA-induced depolarization in cerebellar granule cells. In contrast, depolarization with NMDA in cortical cells was observed even without the addition of exogenous glycine as well as in the absence or presence of 1 mM MgCl2. Ethanol (50 mM) inhibited the effects of NMDA in some, but not all, neurons indicative of the existence of ethanol-sensitive and ethanol-insensitive cortical and cerebellar granule neurons. In studies performed in monolayers of cortical and cerebellar granule cells, we observed that the presence of glycine (10 microM) was a necessary prerequisite to unmask inhibitory actions of ethanol on 45Ca2+ influx induced by NMDA. In another set of experiments, we noted that NMDA-induced stimulation of [3H]PDBu binding to monolayers of intact cerebellar granule cells was inhibited by ethanol (50 mM). Finally, we report that ethanol caused a concentration-dependent inhibition of NMDA-induced necrotic cell death, assessed by measuring the ability of cerebellar granule cells to transform 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) into formazan. In none of the four assays used to demonstrate the inhibitory effects of ethanol on NMDA receptor activity, the ethanol-induced inhibition was reversed by glycine (up to 100 microM). Thus, in contrast to earlier reports, our data suggest that ethanol and glycine produce their effects by acting at different regulatory sites within the NMDA receptor system in brain neurons.
Cebers
G
G
Department of Clinical Neuroscience, Karolinska Institute, Stockholm, Sweden.
Cebere
A
A
Zharkovsky
A
A
Liljequist
S
S
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000494:pharmacology*; D005998:Glycine / Q000494:pharmacology*; D016202:N-Methylaspartate / Q000494:pharmacology; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects* / Q000502:physiology
D016194:Receptors, N-Methyl-D-Aspartate; D015240:Phorbol 12,13-Dibutyrate; D000431:Ethanol; D016202:N-Methylaspartate; D002118:Calcium; D005998:Glycine