The 71 kDa glutamate-binding protein is increased in cerebellar granule cells after chronic ethanol treatment.

Besides the N-methyl-D-aspartate (NMDA) receptor proteins NR1 and NR2, another complex of proteins which has been shown to contain ligand-binding sites characteristic of NMDA receptors is expressed in cerebellar granule cells. One of the proteins in the latter complex is the 71 kDa glutamate-binding protein (GBP). To determine the role of the GBP in the response to NMDA, primary cultures of cerebellar granule cells were treated with an antisense oligonucleotide complementary to mRNA for this protein. This treatment substantially reduced both mRNA and protein levels of the GBP, as well as the response of the cells to NMDA, measured as an increase in intracellular Ca2+ with fura-2 fluorescence. The antisense oligonucleotide treatment did not alter the Ca2+ responses to KC1 or kainate. Chronic ethanol exposure has previously been shown to increase NMDA receptor function and the density of binding sites for the NMDA receptor channel blocker, dizocilpine, in cerebellar granule cells. Chronic exposure of the cells to 100mM ethanol is now shown to result in significant increases in mRNA and protein levels for the GBP (45% and 100%, respectively). Ethanol treatment did not affect mRNA levels for NR1 or NR2A, caused only a small increase (20%) in protein levels for NR1, and resulted in a decrease (30%) in NR2A protein. Although a role of the NMDA receptor NR1/NR2 subunits cannot be ruled out, these results are compatible with the hypothesis of involvement of the GBP in the chronic ethanol-induced increase in NMDA receptor function in cerebellar granule cells.

Attenuation of glutamate-induced neurotoxicity in chronically ethanol-exposed cerebellar granule cells by NMDA receptor antagonists and ganglioside GM1.

Ethanol, acutely, is a potent inhibitor of the function of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor. After chronic exposure of animals to ethanol, however, the NMDA receptor in brain is upregulated. This upregulation is associated with the occurrence of ethanol withdrawal seizures. When cultured cerebellar granule neurons are exposed chronically to ethanol, the resulting upregulation of NMDA receptor function renders the cells more susceptible to glutamate-induced neurotoxicity. The present studies show that chronic ethanol exposure produces an increase in NMDA receptor number in the cells, measured by ligand binding to intact cells. Glutamate-induced excitotoxicity, both in control and ethanol-exposed cells, is blocked by the same NMDA receptor antagonists previously shown to block ethanol withdrawal seizures in animals. In addition, glutamate neurotoxicity is blocked by acute (2-hr) pretreatment of cells with ganglioside GM1 or by chronic (3 days) treatment with the ganglioside. Acute ganglioside treatment does not interfere with the initial rise in intracellular calcium caused by glutamate, whereas this response is downregulated after chronic ganglioside treatment. These results suggest that therapeutic agents can be developed to block both ethanol withdrawal signs and the neuronal damage that accompanies ethanol withdrawal. Furthermore, chronic ganglioside treatment during ethanol exposure has the potential to prevent changes in the NMDA receptor that lead to withdrawal seizures and enhanced susceptibility to excitotoxicity.

Protein kinase C activation attenuates N-methyl-D-aspartate-induced increases in intracellular calcium in cerebellar granule cells.

Activation of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor increases levels of intracellular calcium and can lead to stimulation of protein kinase C activity. Several reports have demonstrated that stimulation of protein kinase C can, in turn, increase electrophysiological responses to NMDA in certain cells or in oocytes expressing certain NMDA receptor subunits. In the present study, the effects of protein kinase C activation on NMDA receptor-mediated increases in intracellular Ca2+ level were investigated in primary cultures of rat cerebellar granule cells using fura-2 fluorescence spectroscopy. Pretreatment of the cells with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not the inactive analogue 4 alpha-phorbol 12-myristate 13-acetate, inhibited NMDA-induced increases in intracellular Ca2+ levels. Coincubation of cells with PMA and the kinase inhibitor staurosporine or calphostin C blocked the PMA effect. The potency of NMDA was reduced twofold, and the potency of the NMDA receptor co-agonist, glycine, to enhance the response to NMDA was decreased fourfold by pretreatment of cells with PMA. The effect on glycine was mimicked by pretreatment with okadaic acid, a protein phosphatase inhibitor. PMA treatment did not significantly alter Mg2+ inhibition of the NMDA response but decreased the potency of the competitive antagonist CGS-19755. These data suggest that, in cerebellar granule cells, the function of the NMDA receptor may be subject to feed-back inhibition by protein kinase C stimulation. Under physiological conditions, this inhibition may result from a decreased effectiveness of the endogenous co-agonists, glutamate and glycine.

Glutamate-induced neurotoxicity is increased in cerebellar granule cells exposed chronically to ethanol.

Chronic exposure of primary cultures of cerebellar granule cells to ethanol has previously been shown to result in an enhanced response of the cells to N-methyl-D-aspartate (NMDA). To determine if this increase in NMDA receptor function alters glutamate-induced cytotoxicity, cells were incubated in the presence or absence of 100 mM ethanol for 3 days, the ethanol was removed, the cells were treated with glutamate, and cell survival was assessed with fluorescein diacetate fluorescence. The ethanol-treated cells showed a significantly increased cytotoxic response to glutamate. Treatment with receptor-selective antagonists demonstrated that the cytotoxicity was mediated by NMDA receptors. The increased vulnerability to glutamate-induced cytotoxicity in ethanol-exposed cells may underlie the neuronal degeneration observed in animals and humans after chronic ethanol intake and withdrawal.

Chronic exposure of cerebellar granule cells to ethanol results in increased N-methyl-D-aspartate receptor function.

In primary cultures of cerebellar granule cells, activation of the N-methyl-D-aspartate (NMDA) receptor leads to Ca2+ influx. Previous work showed that this response is selectively inhibited by acute exposure to low concentrations of ethanol. The present results demonstrate that the response to NMDA (measured as an increase in intracellular Ca2+ concentration, using fura-2 fluorescence) is significantly enhanced after chronic in vitro exposure of the cells to ethanol (100 mM for 2-4 days; 20 mM for 3 or more days). This enhancement is consistent with an increased number of NMDA receptors, with no change in receptor properties. Specifically, there was no change in the EC50 values for NMDA and glycine or in the magnitude of inhibition of the NMDA response by competitive or uncompetitive antagonists. There was also no change in the ability of acute ethanol to inhibit the NMDA response after chronic exposure of the cells to ethanol. Furthermore, chronic ethanol exposure did not alter depolarization-dependent increases in intracellular Ca2+ observed after exposure of the cells to 30 mM KCl. The data suggest that chronic ethanol exposure produces a selective up-regulation of NMDA receptor function. In the intact animal, such a change may be associated with particular symptoms of ethanol withdrawal, i.e., withdrawal seizures.