Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells

Abstract

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.

Alcohols inhibit N-methyl-d-aspartate receptors via a site exposed to the extracellular environment

“…The possibility that alcohol inhibits NMDA receptors via an open-channel blocking mechanism has been excluded based on a study using single-channel recording (Wright et al, 1996), in which ethanol was observed to decrease mean open time and frequency of opening of the ion channel without producing flickering block or affecting channel amplitude. Studies addressing a possible interaction of alcohols with the glycine coagonist site have in some cases reported such an interaction (Hoffman et al, 1989;Rabe and Tabakoff, 1990;Woodward and Gonzales, 1990;DildyMayfield and Leslie, 1991;Buller et al, 1995;Popp et al, 1999), while in other studies no such interaction has been observed Peoples and Weight, 1992;Woodward, 1994;Chu et al, 1995;Mirshahi and Woodward, 1995;Cebers et al, 1996;Peoples et al, 1997). The reasons for these discrepant results are not yet entirely clear, but may in part involve differences in NMDA receptor subunit composition (Buller et al, 1995), as well as differences in intracellular modulators that depend upon cell type and experi- mental protocol.…”

“…Recent studies have not identified interactions of alcohols with a number of the modulatory sites of the NMDA receptor-channel (Chu et al, 1995;Peoples et al, 1997), and alcohols do not appear to act by binding within the ion channel pore (Wright et al, 1996). Although a number of studies have reported an interaction of alcohols with the glycine coagonist site (Hoffman et al, 1989;Rabe and Tabakoff, 1990;Woodward and Gonzales, 1990;Dildy-Mayfield and Leslie, 1991;Buller et al, 1995), other studies have not observed such an interaction Peoples and Weight, 1992;Woodward, 1994;Chu et al, 1995;Mirshahi and Woodward, 1995;Cebers et al, 1996;Peoples et al, 1997).…”

Inhibition ofN-Methyl-d-aspartate Receptors by Straight-Chain Diols: Implications for the Mechanism of the Alcohol Cutoff Effect

“…Alcohol inhibition of NMDA receptors is noncompetitive with respect to the agonist (Göthert and Fink, 1989;Gonzales and Woodward, 1990;Rabe and Tabakoff, 1990;Peoples et al, 1997). Although a number of studies have reported an interaction of alcohols with the glycine coagonist site (Hoffman et al, 1989;Rabe and Tabakoff, 1990;Woodward and Gonzales, 1990;Dildy-Mayfield and Leslie, 1991;Buller et al, 1995), other studies have not observed such an interaction Peoples and Weight, 1992;Woodward, 1994;Chu et al, 1995;Mirshahi and Woodward, 1995;Cebers et al, 1996;Peoples et al, 1997), and results from a study in rat cerebellar granule neurons suggest that the apparent interaction of ethanol with the glycine site results instead from an action of alcohol on an unidentified intracellular modulator (Popp et al, 1999). It thus seems most probable that the glycine site may regulate or influence alcohol sensitivity under certain condi- …”

Enhancement of NMDA-induced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells

“…Results from biochemical, electrophysiologial and behavioural studies indicate that acute administration of ethanol partially inhibits various aspects of glutamate receptor activity, in particular NMDA receptor-mediated functional responses such as Ca 2+ influx, transmitter release and neurotoxicity (Dildy and Leslie 1989;Göthert and Fink 1989;Hoffman et al 1989;Lovinger et al 1989;Fink and Göthert 1991;Peoples and Weight 1995;Cebers et al 1996a). In contrast to inhibitory actions of acute ethanol, chronic treatment with intoxicating concentrations of ethanol has been shown to enhance NMDA-induced functional responses both in vitro and in vivo (Iorio et al 1992(Iorio et al , 1993Chandler et al 1993Chandler et al , 1997Ahern et al 1994;Hu and Ticku 1995;Cebers et al 1996b).…”

“…We have previously used rat cerebellar granule cell cultures to describe the effects of acute and chronic ethanol treatments on various NMDA receptor functions (Cebers et al 1996a(Cebers et al , 1996b. In the present study, cerebellar granule cells were used to investigate the effects of chronic ethanol exposure on NMDA-induced Ca 2+ fluxes and NMDA-induced neurotoxicity.…”