Differential alterations in the expression of NMDA receptor subunits following chronic ethanol treatment in primary cultures of rat cortical and hippocampal neurones.
In our previous experiments, severe cellular damages and neuronal cell loss were observed following 24h of alcohol withdrawal in primary cultures of rat cortical neurones pre-treated with ethanol (50-200 mM) repeatedly for 3 days. Increased NMDA induced cytosolic calcium responses and excitotoxicity were also demonstrated in the ethanol pre-treated cultures. Thus, the enhancement in functions of NMDA receptors was supposed to be involved in the adaptive changes leading to the neurotoxic effect of alcohol-withdrawal. In this study, we investigated the effect of the 3-day repeated ethanol (100 mM) treatment on the function and subunit composition of the NMDA receptors. Here, we demonstrate that the maximal inhibitory effect of ethanol was significantly increased after ethanol pre-treatment. Similarly, the inhibitory activity of the NR2B subunit selective antagonists threo-ifenprodil, CP-101,606 and CI-1041 was also enhanced. On the contrary, the efficiency of the channel blocker agent MK-801 and the glycine-site selective antagonist 5,7-dichlorokynurenic acid was the same as in control cultures. According to these observations, a shift in subunit expression in favour for the NR2B subunit was suggested. Indeed, we provided evidence for increased expression of the NR2B and the C1 and C2' cassette containing splice variant forms of the NR1 subunit proteins in ethanol pre-treated cultures in further experiments using a flow cytometry based immunocytochemical method. These changes may constitute the basis of the increased NMDA receptor functions and subsequently the enhanced sensitivity of ethanol pre-treated cortical neurones to excitotoxic insults resulting in increased neuronal cell loss after ethanol withdrawal. Such alterations may play a role in the neuronal adaptation to ethanol as well as in the development of alcohol dependence, and might cause neuronal cell loss in certain areas of the brain during alcohol withdrawal.
Nagy
József
J
Pharmacological and Drug Safety Research, Gedeon Richter Ltd., P.O.B. 27, H-1475 Budapest 10, Hungary. joszef.nagy@richter.hu
Kolok
Sándor
S
Dezso
Péter
P
Boros
András
A
Szombathelyi
Zsolt
Z
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002540:Cerebral Cortex / Q000166:cytology / Q000196:embryology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D000431:Ethanol / Q000008:administration & dosage / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects*; D006624:Hippocampus / Q000166:cytology; D007736:Kynurenic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D010880:Piperidines / Q000494:pharmacology; D021122:Protein Subunits; D012326:RNA Splicing / Q000187:drug effects; D051381:Rats; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics
D016895:Culture Media, Serum-Free; D018691:Excitatory Amino Acid Antagonists; C409466:NR1 NMDA receptor; C120997:NR2A NMDA receptor; C121001:NR2B NMDA receptor; C432823:NR2C NMDA receptor; C464436:NR2D NMDA receptor; D009419:Nerve Tissue Proteins; D010880:Piperidines; D021122:Protein Subunits; D016194:Receptors, N-Methyl-D-Aspartate; D000431:Ethanol; D016202:N-Methylaspartate; D016291:Dizocilpine Maleate; C095106:traxoprodil mesylate; D007736:Kynurenic Acid; C010739:ifenprodil; C066192:5,7-dichlorokynurenic acid