Effects of Chronic Ethanol Treatment on the Expression of Calcium Transport Carriers and NMDA/Glutamate Receptor Proteins in Brain Synaptic Membranes

Abstract

Acute exposure to ethanol inhibits both the NMDA receptors and the Na/Ca-exchange carriers in neuronal membranes. This alters intraneuronal signaling pathways activated by Ca 2~.Neurons exposed chronically to ethanol exhibit enhanced density and activity of NMDA receptors and increased maximal activity of the exchangers. In the present study, the expression of brain synaptic membrane proteins with ligand binding sites characteristic of NMDA receptors and of exchange carriers were determined after chronic ethanol administration (15 days) to rats. Such treatment caused an increase in the expression of the NMDAR1 receptor subunit, 15% above the levels in the pair-fed controls, as well as of three subunits of a complex that has properties characteristic of NMDA receptors, the glutamate, carboxypiperazinylphosphonate, and glycine binding proteins. Increases for the three binding proteins were 49, 50, and 62%, respectively. The expression of the 1 20-kDa exchanger proteins was increased by 14% and that of a 36-kDa exchanger-associated protein by 33%. Both the binding proteins and the exchangers returned to basal levels within 36-72 h after withdrawal from ethanol. No changes were detected in synaptic membrane Ca2~, Mg2~-ATPases.The enhanced expression of receptor and exchanger-associated proteins may explain the increases in the density and activity of NMDA receptors and exchange carriers after chronic ethanol treatment. Key Words: NMDA receptors-Receptor subunits-Na/ Ca-exchange carriers. J. Neurochem. 69, 1559-1569 (1997).The importance of changes in the concentration of free intracellular calcium (~Ca2~1 1) for normal synaptic transmission as well as for events that lead to prolonged changes in neuronal excitability, such as longterm potentiation (LTP), neuronal development, and neuronal survival, is well documented (e.g., Choi, 1987;Mattson et al., 1988;Ito, 1989;Madison et al., 1991;Ghosh and Greenberg, 1995). Stimuli that disrupt the regulation of intraneuronal Ca 2~may have profound effects on neurotransmission, synaptic plasticity, such as the establishment of LTP and memory formation, synaptogenesis, and neuronal survival. Acute exposure of neurons to ethanol disrupts both the signaling events that are dependent on the transient elevations of intraneuronal Ca2~concentrations and some of the transport processes involved in the maintenance of low intracellular concentrations of this cation (e.g., Harris and Hood, 1980;Messing et al., 1986;Rabe and Weight, 1988;Tabakoff and Hoffman, 1991;Dildy-Mayfield and Leslie, 1991;Carlen et al., 1993).Two main pathways for transient changes in free Ca2~],that are most important in cell signaling are the plasma membrane Ca2-permeant channels and the Ca2-releasing channels in the membranes of intracellular organelles, such as the endoplasmic reticulum. One of the Ca2~-permeantion channels in neuronal plasma membranes is the NMDA receptor (NMDAR), and this has been the focus of many studies related to the inhibitory effects of ethanol on receptor function (e.g., Dild...

Calcium Influx Through NMDA Receptors, Chronic Receptor Inhibition by Ethanol and 2‐Amino‐5‐Phosphonopentanoic Acid, and Receptor Protein Expression

“…The specificity of the antibody immune reactivities was verified by western blot analyses. The ELISAs were performed exactly as described previously (Roy and Michaelis, 1984;Eaton et al, 1990;Wang et al, 1992;Chen et al, 1997~). The primary antibodies used in these studies were an antibody to the Cterminal peptide of NRl (Chemicon) and to the intact GBP purified from brain (Eaton et al, 1990).…”

“…Electrophoresis and western blot analyses were performed exactly as described previously (Chen et al, 1988;Eaton et al, 1990;Kumar et al, 1994;Xia et al, 1995;Babcock et al, 1996;Chen et al, 1997~). The proteins were extracted from the cultured cortical cells and solubilized in sample buffer containing 2% (wtlvol) SDS, 250 mM dithiothreitol, and 500 mM iodoacetamide.…”

“…After separation by electrophoresis, the proteins were transferred to polyvinylfluoride membranes (Millipore Corp.). The intensity of the antibody-labeled bands was estimated by image analysis as described previously (Mattson et al, 1993;Xia et al, 1995;Hoffman et al, 1996;Chen et al, 1997~).…”

“…This model can therefore be used to explore the mechanisms involved in altered NMDA receptor protein expression and to determine whether changes in receptor protein expression are the result of cellular events selectively regulated by NMDA receptor function. The expression of the NR1 protein is modestly (15-20%) and that of the NR2B is prominently (40 -60%) increased following chronic treatment of either experimental animals or neurons in culture with ethanol (Trevisan et al, 1994;Follesa and Ticku, 1995, 1 9 9 6~;Hu and Ticku, 1995;Hoffman et al, 1996;Chen et al, 1997~).…”

“…Synaptic membranes isolated from the brains of animals treated chronically with ethanol exhibit increases in the expression of three subunits of this protein complex (Chen et al, 1997~). The three subunits of this complex whose expression is increased following chronic ethanol treatment are the glutamatebinding protein (GBP), the (+-)-3-(2-carboxypiperazin-4-y1)propyl-1 -phosphonic acid (CPP)-binding protein (CPP-BP), and the glycine/TCP-binding protein (Gly/ TCP-BP).…”