The NR2B subtype of NMDA receptor: a potential target for the treatment of alcohol dependence

Ethanol is a small molecule acting on several neurotransmitter systems in the brain. Accumulating evidences suggest that the primary excitatory--i.e. the glutamatergic--neurotransmitter system is a particularly important site of ethanol's action. Several studies showed that ethanol is a potent and selective inhibitor of the N-methyl-D-aspartate (NMDA) receptors and prolonged ethanol exposition leads to a compensatory "up-regulation" of these receptors resulting in enhanced NMDA receptor-mediated functions after removal of ethanol. These alterations are supposed to contribute to the development of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal. In recent papers, alterations in subunit composition of NMDA receptors were reported after long term ethanol exposure. mRNA and/or protein levels of NR2A and NR2B types of subunits were found elevated both by in vivo and in vitro experiments. Our results showed that especially the NR2B subunit expression is increased in cultured hippocampal and cortical neurones after 3 days of intermittent ethanol treatment. According to the high calcium permeability, the increased agonist sensitivity and the relatively slow closing kinetics of NMDA ion channels composed of NR2B subunits, the above mentioned changes may underlie the enhanced NMDA receptor activation observed after long term ethanol exposure. Accordingly, we have tested NR2B subunit selective NMDA receptor antagonists in primary cultures of rat cortical neurones pre-treated with ethanol intermittently for 3 days and found that these compounds potently inhibited the neurotoxic effect of ethanol withdrawal. Hypothesising the involvement of enhanced NR2B subunit expression in development of alcohol dependence and withdrawal symptoms and considering the tolerable side effect profile of the NR2B subunit selective NMDA receptor antagonists, the NR2B type of NMDA receptor subunit may serve as a possible drug target in pharmacological interventions for alcoholism. The aim of this review is to give an update on the role of altered structure and function of NMDA receptors after ethanol exposure and to summarise the recent data about the activity of NR2B subunit selective NMDA receptor antagonists in model systems related to alcoholism.     

Renaissance of NMDA receptor antagonists: do they have a role in the pharmacotherapy for alcoholism?

Long-term alcohol exposure leads to the development of alcohol dependence, which is possibly induced by changes in specific neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of ionotropic glutamate receptor is a particularly important site of action for ethanol. Ethanol potently and selectively inhibits NMDA receptors (NMDARs) and prolonged ethanol exposure produces a compensatory 'upregulation' of NMDAR functions. These changes are believed to underlie the development of ethanol tolerance and dependence as well as acute and delayed signs of withdrawal. Therefore, negative modulators of NMDARs may be useful agents for the pharmacotherapy of alcoholism. NMDAR antagonists attenuate not only the physical symptoms but also some affective and motivational components of alcohol withdrawal. Encouraging experimental results have been obtained with novel uncompetitive (memantine and neramexane (Merz & Co GmbH/Forest Laboratories Inc)), glycine site and NR2B subunit-selective NMDA antagonists (SSNAs). Recently emerged NR2B SSNAs (CP-101606 (Pfizer Inc), Co-101244 (Pfizer Inc/Purdue Neuroscience Corp/Senju Pharmaceutical Co Ltd), CI-1041 (Purdue Neuroscience Corp/Pfizer Inc) and indole-2-carboxamide derivatives) have demonstrated excellent in vitro potency against withdrawal-induced cytotoxicity. Although in vivo data are few, according to their in vitro efficacy and good tolerability, novel NMDA antagonists, especially the NR2B-selective antagonists, may offer a preferable alternative to the presently available pharmacotherapies for treating alcoholism.     

Alcohol Related Changes in Regulation of NMDA Receptor Functions

Long-term alcohol exposure may lead to development of alcohol dependence in consequence of altered neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action. Several studies showed that ethanol potently inhibits NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “up-regulation” of NMDAR mediated functions. Therefore, alterations in NMDAR function are supposed to contribute to the development of ethanol tolerance, dependence as well as to the acute and late signs of ethanol withdrawal.     

Chronic intermittent ethanol treatment selectively alters N-methyl-D-aspartate receptor subunit surface expression in cultured cortical neurons

A chronic intermittent ethanol (CIE) exposure regimen consists of repeated episodes of ethanol intoxication and withdrawal. CIE treatment has been reported to result in a significant enhancement of N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in vivo, and trafficking of NMDA receptors is emerging a key regulatory mechanism that underlies the channel function. Therefore, in the present study, we examined the effects of CIE on NMDA receptor subunit surface expression. Cultured cortical neurons were exposed to 75 mM ethanol for 14 h followed by 10 h of withdrawal, repeated this cycle five times, and followed by 2 or 5 days of withdrawal. Surface-expressed NMDA receptor subunits and their endocytosis were measured by biotinylation and Western blots. CIE significantly increased NMDA receptor (NR) 1 and NR2B but not NR2A subunit surface expression after 5 days of treatment. However, CIE treatment did not reduce the NMDA receptor endocytosis. Quantification of immunocytochemistry confirmed CIE-induced increase in both the total number of NR1 and NR2B subunit clusters and their targeting to synaptic sites. It is noteworthy that this effect persisted even after ethanol withdrawal with a peak expression occurring between 0 and 2 days after withdrawal, and the expression on the plasma membrane was still at high levels after 5 days of withdrawal. In addition, this was accompanied by significant increases in postsynaptic density protein 95 clusters. Protein kinase A inhibitor completely reversed CIE-induced increase in NR1 and partially in NR2B surface level and a long-lasting effect. These changes may contribute to the development of ethanol-induced neurotoxicity and ethanol dependence.     

Chronic ethanol exposure and protracted abstinence alter NMDA receptors in central amygdala.

We recently reported that chronic ethanol treatment (CET) and early withdrawal (2-8 h) altered glutamatergic transmission at both pre- and postsynaptic sites in central nucleus of the amygdala (CeA). Acute ethanol (44 mM) inhibited the NMDA receptor (NMDAR)-mediated EPSCs (NMDA-EPSCs) more in CeA neurons from CET rats than from na?ve rats and also decreased paired-pulse facilitation (PPF) of NMDA-EPSCs only in CET rats. To determine whether these CET effects persisted after prolonged withdrawal, we recorded intracellularly in rat CeA slices and measured mRNA and protein expression of CeA NMDAR subunits from CET rats and those withdrawn from ethanol for 1 or 2 weeks. At 1 week withdrawal, acute ethanol decreased evoked NMDA-EPSC amplitudes and NMDA currents induced by exogenous NMDA ( approximately 20%) equally to that in na?ve rats, indicating that CET effects on postsynaptic mechanisms reversed 1 week after CET cessation. However, acute ethanol still decreased PPF of NMDA-EPSCs, indicating that the acute ethanol-induced increase in glutamate release in CeA seen in CET rats was still present at this time. CET also significantly increased mRNA levels of NR1 and NR2B NMDAR subunits compared to control rats. At 1 week withdrawal, mRNA levels for NR1 and NR2B subunits were significantly decreased. These changes reversed at 2 weeks withdrawal. In Western blots, a significant increase in protein for all three subunits occurred in CeA from CET rats, but not after 1 and 2 weeks of withdrawal. These data indicate that CET induces reversible neuroadaptations in synaptic function, gene expression, and protein composition of NMDAR at CeA synapses.     

Implications of the NR2B subunit-containing NMDA receptor localized in mouse limbic forebrain in ethanol dependence

The present study was designed to further investigate the direct involvement of the NR2B-containing NMDA receptor in ethanol dependence. Using the liquid diet method, mice were chronically treated with skimmed milk containing 5% ethanol for 5 days. After the discontinuation of ethanol, mice revealed tremor, handling-elicited convulsion and death. Treatment with a selective NR2B-containing NMDA receptor antagonist, ifenprodil, significantly suppressed the expression of ethanol withdrawal signs. The protein level of NR2B subunits in the limbic forebrain, but not the cerebral cortex, during chronic ethanol treatment was markedly increased with respect to the levels in control mice. The significant up-regulation of NR2B subunits lasted for at least 9 h after the discontinuation of ethanol and returned to the basal level by 48 h after the withdrawal. These findings suggest that the up-regulation of NR2B subunits during chronic ethanol exposure may be implicated in the initial development of physical dependence on ethanol.     

Opiate physical dependence and N-methyl-D-aspartate receptors

The present review focused the involvement of N-methyl-D-aspartate (NMDA) receptors in morphine physical dependence. The increased levels of extracellular glutamate, NMDA receptor zeta subunit (NR1) mRNA, NMDA receptor epsilon 1 subunit (NR2A) protein, phosphorylated Ca(2+)/calmodulin kinase II (p-CaMKII) protein, c-fos mRNA, c-Fos protein, are observed in the specific brain areas of mice and/or rats showing signs of naloxone-precipitated withdrawal. In preclinical and clinical studies, a variety of NMDA receptor antagonists and pretreatment with an antisense oligonucleotide of the NR1 have been reported to inhibit the development, expression and/or maintenance of opiate physical dependence. In contrast to data obtained in adult animals, NMDA receptor antagonists are neither effective in blocking the development of opiate dependence nor the expression of opiate withdrawal in neonatal rats. In the NMDA receptor-deficient mice, the NR2A knockout mice show the marked loss of typical withdrawal abstinence behaviors precipitated by naloxone. The rescue of NR2A protein by electroporation into the nucleus accumbens of NR2A knockout mice reverses the loss of abstinence behaviors. The activation of CaMKII and increased expression of c-Fos protein in the brain of animals with naloxone-precipitated withdrawal syndrome are prevented by NMDA receptor antagonists, whereas the increased levels of extracellular glutamate are not prevented by them. These findings indicate that glutamatergic neurotransmission at the NMDA receptor site contributes to the development, expression and maintenance of opiate dependence, and suggest that NMDA receptor antagonists may be a useful adjunct in the treatment of opiate dependence.     

Opiate physical dependence and N-methyl-d-aspartate receptors

The present review focused the involvement of N-methyl-d-aspartate (NMDA) receptors in morphine physical dependence. The increased levels of extracellular glutamate, NMDA receptor ζ subunit (NR1) mRNA, NMDA receptor 1 subunit (NR2A) protein, phosphorylated Ca²⁺/calmodulin kinase II (p-CaMKII) protein, c-fos mRNA, c-Fos protein, are observed in the specific brain areas of mice and/or rats showing signs of naloxone-precipitated withdrawal. In preclinical and clinical studies, a variety of NMDA receptor antagonists and pretreatment with an antisense oligonucleotide of the NR1 have been reported to inhibit the development, expression and/or maintenance of opiate physical dependence. In contrast to data obtained in adult animals, NMDA receptor antagonists are neither effective in blocking the development of opiate dependence nor the expression of opiate withdrawal in neonatal rats. In the NMDA receptor-deficient mice, the NR2A knockout mice show the marked loss of typical withdrawal abstinence behaviors precipitated by naloxone. The rescue of NR2A protein by electroporation into the nucleus accumbens of NR2A knockout mice reverses the loss of abstinence behaviors. The activation of CaMKII and increased expression of c-Fos protein in the brain of animals with naloxone-precipitated withdrawal syndrome are prevented by NMDA receptor antagonists, whereas the increased levels of extracellular glutamate are not prevented by them. These findings indicate that glutamatergic neurotransmission at the NMDA receptor site contributes to the development, expression and maintenance of opiate dependence, and suggest that NMDA receptor antagonists may be a useful adjunct in the treatment of opiate dependence.     

Heterozygous deletion of NR1 subunit of the NMDA receptor alters ethanol-related behaviors and regional expression of NR2 subunits in the brain

NMDA receptors have been hypothesized to play a role in various aspects of ethanol-related phenotypes, notably in ethanol withdrawal. However, the role of each of the specific subunits remains unclear. To address this issue, mice that are heterozygous for the NR1 deletion, and thus have a reduction in functional NMDA receptors, were examined for ethanol consumption and acute ethanol withdrawal. Additionally, mice were examined for the level of vocalization following footshock, and behavior in an elevated plus maze, to determine their responses to stress. In these behavioral tests, NR1 heterozygous mice were shown to consume significantly higher levels of ethanol in the two bottle-choice test showing a possible role for this receptor in ethanol consumption. Analysis of acute withdrawal found that the heterozygous mice exhibit lower levels of handling-induced convulsions consistent with a role in ethanol sensitivity or withdrawal. In contrast, no effects on stress-related phenotypes were detected. Levels of NR2A-NR2D subunits of the NMDA receptor in specific brain regions were compared between NR1+/- mice and wild-type controls to assess whether the behavioral responses were specific to the diminution in NR1 expression or whether these changes could be due to secondary changes in expression of other NMDA subunits. Real-time quantitative PCR, Western blot and immunohistochemistry were used to examine expression levels in the hippocampus, neocortex, striatum and cerebellum. For the majority of the subunits, no differences were found between the wild-type and heterozygous mice in any of the brain regions. However, the NR2B subunit exhibited differences in expression of RNA in the hippocampus and protein levels in multiple brain regions, between wild-type and NR1+/- mice. These results show that NR1 plays a role, through mechanisms as yet unknown, in the expression of NR2 subunits in a region and subtype specific manner. This provides evidence of the effects of altered levels of NR1 expression on ethanol withdrawal and consumption, and suggests that concomitant changes in the levels of NR2B may contribute to that effect.     

Mechanisms of morphine-induced rewarding effect: involvement of NMDA receptor subunits

The glutamate receptor contributes to excitatory synaptic transmission in the central nervous system and plays an important role in memory acquisition, learning and neurological disorders. Molecular cloning studies have revealed that NMDA receptors consist of two families, the NR1 and NR2A-NR2D subunits, and NMDA receptors are thought to be pentameric or tetrameric complexes of the NR1 subunit with one or more of the NR2 subunits. It has been proposed that NMDA receptors are implicated in the development of opioid dependence. The non-selective NMDA receptor antagonist dizocilpine has been shown to suppress not only physical but also psychological dependence produced by morphine. An intracerebroventricular (i.c.v.) treatment with a specific antibody against the carboxyl-terminal region of the NR2B subunit abolishes the morphine-induced place preference, whereas antibodies against the NR1 and NR2A subunits do not affect the rewarding effect of morphine, indicating that the blockade of the NR2B subunit suppresses the development of the morphine-induced rewarding effect. Under these conditions, the NR2B subunit protein is up-regulated in the limbic forebrain of morphine-conditioned mice. These findings suggest that the NMDA receptor, especially NR2B subunit, is an important modulator of the development and/or expression of psychological dependence on morphine.     

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

Primary cultures of rat cerebellar granule cells were used to investigate the effects of chronic ethanol exposure (50-100 mM for 3 days) on NMDA receptor functions (Ca2+ fluxes and neurotoxicity), binding parameters of the non-competitive NMDA receptor antagonist [3H]MK-801, relative abundance of mRNAs coding for NMDA receptor subunits, and expression of NMDA receptor subunit proteins. Ethanol exposure caused a marked increase in NMDA-produced neurotoxicity but produced a differential pattern of effects on NMDA-induced Ca2+ fluxes with a marked enhancement of NMDA-stimulated free cytoplasmic Ca2+ concentrations ([Ca2+]i), but no changes in NMDA-induced 45Ca2+ uptake. As shown by [3H]MK-801 binding experiments, chronic ethanol had no effect on affinity or number of the NMDA receptors. Furthermore, ethanol exposure had no effect on the relative abundance of the mRNAs for any of the NMDA receptor subunits (four splice variants of NR1, or NR2A-C), or on the expression of NMDA receptor subunit proteins. Our data confirm previous observations that chronic ethanol exposure enhances NMDA receptor-mediated neurotoxicity and elevation of [Ca2+]i, but also suggest that the increased responsiveness of NMDA receptors is not necessarily associated with alterations in the subunit composition or the ligand binding properties of NMDA receptors.     

Inhibition by 2-Methoxy-4-ethylphenol of Ca2+ Influx Through Acquired and Native N-Methyl-d-aspartate–Receptor Channels

Pharmacological properties were evaluated for the antidiarrheic wood creosote ingredient 2-methoxy-4-ethylphenol (2M4EP), which was shown to be protective against neurotoxicity of N-methyl-d-aspartate (NMDA), to modulate Ca²⁺ influx across acquired and native NMDA receptor (NMDAR) channels. NMDA markedly increased intracellular free Ca²⁺ levels in HEK293 cells transfected with the expression vector of either NR2A or NR2B subunit together with the essential NR1 subunit vector. Further addition of dizocilpine inhibited the increase by NMDA in intracellular Ca²⁺ levels in both types of acquired NMDAR channels, while 2M4EP and the NR2B-subunit–selective antagonist ifenprodil were more effective in inhibiting the increase by NMDA in HEK293 cells expressing NR1/NR2B subunits than in those with NR1/NR2A subunits. 2M4EP significantly prevented the increased intracellular Ca²⁺ levels by NMDA in cultured rat hippocampal neurons. Brief exposure to NMDA led to a drastic decrease in cellular viability 24 h later in cultured hippocampal neurons, while 2M4EP significantly prevented the loss of cellular vitality by NMDA. Similarly, 2M4EP more efficiently protected HEK293 cells with NR1/NR2B subunits than those with NR1/NR2A subunits. These results suggest that 2M4EP may protect neurons from excitotoxicity through inhibition of Ca²⁺ influx across NMDAR channels composed of NR1/ NR2B, rather than NR1/NR2A, subunits.     

Cytotoxic effect of alcohol-withdrawal on primary cultures of cortical neurones

Physical dependence on alcohol was observed previously at the cellular level in cultured IM-9 human lymphoblast cells. To answer the question whether physical dependence can also develop in neurones and to investigate the neuronal processes involved in the development of alcohol dependence and withdrawal symptoms, cultures of cortical neurones were adapted to alcohol. Morphological characteristics of neurones were not altered during the chronic (3-day) repeated (once per day) ethanol (50-100 mM) treatment, whereas obvious signs of neuronal damage were seen after the following 24 h of alcohol-withdrawal. The extent of the damage, quantitated by measuring the release of lactate dehydrogenase (LDH) into the culture media, was dependent on the concentration of ethanol in the medium during adaptation. LDH-release induced by alcohol-withdrawal was significantly reduced by re-addition of ethanol, as well as by administration of non-competitive (MK-801) or NR2B selective (threo-ifenprodil) N-methyl-D-aspartate (NMDA) receptor antagonists. The sigma ligand haloperidol and the L-type voltage sensitive calcium channel blocker nimodipine were also effective, whereas the effect of the gamma-aminobutyric acid type A (GABA(A)) receptor agonist muscimol was not significant. Furthermore, chronic ethanol treatment potentiated the NMDA induced neurotoxicity and the ability of acute alcohol to inhibit LDH-release in response to NMDA. According to these results, (i) the phenomenon of alcohol-dependence can be observed at the level of neurones and (ii) NMDA receptors seem to play a central role in the development of ethanol dependence and in neurotoxicity induced by alcohol-withdrawal.     

Gender-selective effects of ethanol dependence on NMDA receptor subunit expression in cerebral cortex, hippocampus and hypothalamus

Previous investigations have shown subunit-selective alterations in NMDA receptors in ethanol dependent male rats. In the present study, we found pronounced gender differences in the effects of ethanol dependence on NMDA receptor subunit expression in all brain regions investigated. Ethanol dependent female rats exhibited increased NR1 subunit levels in cerebral cortex and hypothalamus, whereas males displayed increased NR1 levels only in hippocampus. NR2A subunit levels were significantly increased only in hippocampus from ethanol dependent male rats, whereas NR2B subunit levels significantly increased in cerebral cortex of both female and male rats. These findings suggest that gender influences neuroadaptations elicited by ethanol dependence at the level of NMDA receptor subunit expression.     

Oral administration of glycine and polyamine receptor antagonists blocks ethanol withdrawal seizures

Cessation of chronic administration of orally administered large amounts of ethanol for 7 days resulted in a markedly increased frequency of audiogenic seizures in Sprague-Dawley rats. Oral administration of the novel glycine receptor antagonist, L-701,324, produced a dose-dependent (2.5 and 5.0 mg/kg; − 30 min) inhibition of ethanol withdrawal signs when measured about 12 h after withdrawal of the ethanol treatment. Similarly, using the same experimental paradigm, oral administration of the specific polyamine receptor antagonist, eliprodil, caused a dose-related (2.0 and 5.0 mg/kg; − 30 min) inhibition of ethanol withdrawal-induced audiogenic seizure activity. The inhibition of ethanol withdrawal seizures produced by L-701,324 and eliprodil, respectively, was obtained at doses which by themselves did not change the locomotor activity in naive Sprague-Dawley rats. The findings that L-701,324 and eliprodil are potent inhibitors of seizure activity induced by cessation of chronic ethanol administration and the fact that they, in contrast to currently available NMDA receptor antagonists, do not produce psychotomimetic and/or sedative effects, suggest that these drugs may represent a new class of therapeutically useful pharmacological agents for the treatment of ethanol withdrawal seizures. Furthermore, since there is evidence that eliprodil produces its pharmacological actions through a specific inhibition of NMDAR1 and/or NMDAR2B subunits, these data may indicate that certain NMDA receptor subunits may be of particular importance for the mediation of seizure activity following the discontinuation of chronic ethanol exposure.     

Oral administration of glycine and polyamine receptor antagonists blocks ethanol withdrawal seizures

Cessation of chronic administration of orally administered large amounts of ethanol for 7 days resulted in a markedly increased frequency of audiogenic seizures in Sprague-Dawley rats. Oral administration of the novel glycine receptor antagonist, L-701,324, produced a dose-dependent (2.5 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal signs when measured about 12 h after withdrawal of the ethanol treatment. Similarly, using the same experimental paradigm, oral administration of the specific polyamine receptor antagonist, eliprodil, caused a dose-related (2.0 and 5.0 mg/kg; –30 min) inhibition of ethanol withdrawal-induced audiogenic seizure activity. The inhibition of ethanol withdrawal seizures produced by L-701,324 and eliprodil, respectively, was obtained at doses which by themselves did not change the locomotor activity in naive Sprague-Dawley rats. The findings that L-701,324 and eliprodil are potent inhibitors of seizure activity induced by cessation of chronic ethanol administration and the fact that they, in contrast to currently available NMDA receptor antagonists, do not produce psychotomimetic and/or sedative effects, suggest that these drugs may represent a new class of therapeutically useful pharmacological agents for the treatment of ethanol withdrawal seizures. Furthermore, since there is evidence that eliprodil produces its pharmacological actions through a specific inhibition of NMDAR1 and/or NMDAR2B subunits, these data may indicate that certain NMDA receptor subunits may be of particular importance for the mediation of seizure activity following the discontinuation of chronic ethanol exposure.     

Alterations of striatal NMDA receptor subunits associated with the development of dyskinesia in the MPTP-lesioned primate model of Parkinson's disease

The development of dyskinesias and other motor complications greatly limits the use of levodopa therapy in Parkinson's disease (PD). Studies in rodent models of PD suggest that an important mechanism underlying the development of levodopa-related motor complications is alterations in striatal NMDA receptor function. We examined striatal NMDA receptors in the MPTP-lesioned primate model of PD. Quantitative immunoblotting was used to determine the subcellular abundance of NR1, NR2A and NR2B subunits in striata from unlesioned, MPTP-lesioned (parkinsonian) and MPTP-lesioned, levodopa-treated (dyskinetic) macaques. In parkinsonian macaques, NR1 and NR2B subunits in synaptosomal membranes were decreased to 66 +/- 11% and 51.2 +/- 5% of unlesioned levels respectively, while the abundance of NR2A was unaltered. Levodopa treatment eliciting dyskinesia normalized NR1 and NR2B and increased NR2A subunits to 150 +/- 12% of unlesioned levels. No alterations in receptor subunit tyrosine phosphorylation were detected. These results demonstrate that altered synaptic abundance of NMDA receptors with relative enhancement in the abundance of NR2A occurs in primate as well as rodent models of parkinsonism, and that in the macaque model, NR2A subunit abundance is further increased in dyskinesia. These data support the view that alterations in striatal NMDA receptor systems are responsible for adaptive and maladaptive responses to dopamine depletion and replacement in parkinsonism, and highlight the value of subtype selective NMDA antagonists as novel therapeutic approaches for PD.     

胍丁胺对吗啡长期处理引起的NMDA受体蛋白表达改变的影响

目的:观察胍丁胺对吗啡长期处理引起的NMDA受体蛋白改变的影响。方法:采用吗啡递增给药制备大鼠慢性依赖模型,并观察依赖状态下大鼠海马和伏隔核NMDA受体NR1和NR2B亚基蛋白表达量的变化,以及胍丁胺对吗啡作用的影响。结果:与对照组相比,吗啡慢性处理大鼠在纳洛酮催促下能出现典型的戒断综合征,提示依赖模型建立成功。用免疫印记(Western blotting)技术发现,海马部位的NR2B亚基明显下调,而NR1亚基未见显著性变化;吗啡慢性处理不引起伏隔核NR2B亚基的明显变化,但NR1亚基却显著上调。胍丁胺与吗啡伴随给药能逆转吗啡对两脑区NMDA受体蛋白表达的调节作用。结论:胍丁胺调节阿片依赖可能与其逆转吗啡对NMDA受体亚基数量和构成的调节有关。     

Development of a three-dimensional model for the N-methyl-D-aspartate NR2A subunit

NR2 subunits of N-methyl-d-aspartic acid (NMDA) receptors are known to bind the neurotransmitter glutamate, competitive agonists, and antagonists. Since crystallographic data of these proteins are not available, we built a homology model of the ligand binding domain of the NR2A subunit. A consensus binding mode of selected AP5-like NMDA antagonists has been ascertained using molecular docking. The present 3D model gives insights for the design of new NMDA subtype selective compounds.     

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

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.