Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.     

亚慢性地卓西平诱导的精神分裂样小鼠前额叶和海马脑区MIF蛋白表达的变化

目的:探讨亚慢性地卓西平(MK-801)诱导的精神分裂样小鼠模型中前额叶和海马脑区巨噬细胞迁移抑制因子(Macrophage migration inhibitory factor,MIF)蛋白表达的变化。方法:将24只7周龄小鼠随机分为对照组、MK-801组和MK-801+奥氮平(olanzapine,olz)组(n=8),三组小鼠分别接受0.9%生理盐水、MK-801(0.6 mg/kg)和MK-801(0.6 mg/kg)+奥氮平(2.5 mg/kg)给药,持续4周。小鼠行为学通过旷场试验、社交实验进行评价,免疫印迹法检测小鼠前额叶和海马组织中MIF蛋白的表达。结果:MK-801处理后,小鼠活动量增加,社交功能受损,且都能被抗精神分裂症药物奥氮平显著改善。MK-801组小鼠前额叶皮层中MIF蛋白表达与对照组比较无明显统计学差异(P0.05),而海马脑区中MIF蛋白表达较对照组明显升高(P0.05);MK-801+奥氮平组小鼠前额叶皮层中MIF蛋白表达较MK-801组无显著变化,而海马脑区中MIF蛋白表达较MK-801组明显降低(P0.05)。结论:亚慢性给予MK-801诱导的精神分裂样小鼠海马脑区中MIF蛋白水平升高,提示MIF蛋白可能参与MK-801诱导的精神分裂样行为。     

NMDA-NR2B subtype selectivity of stereoisomeric 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives

Enantiopure 2-(1,2,3,4-tetrahydro-1-isoquinolyl)ethanol derivatives were tested for their affinity to the ifenprodil binding site of the NMDA receptor, their potency to inhibit [3H]MK801 binding and their NMDA-NR2B subtype selectivity. The (1S,1'S)-configurated series displayed the highest affinity to the ifenprodil binding site. A reasonable potency and NMDA-NR2B subtype selectivity was found for (1S,1'S)-4c (R1=Me, R2=OMe). A high affinity to HERG K+ channels, however, suggests that (1S,1'S)-4c may involve an increased risk of cardiovascular side effects.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of the N-Methyl-d-Aspartate Receptor Channel Complex from Rat and Porcine Brain

The N-methyl-D-aspartate (NMDA) receptor complex as defined by the binding of [3H]MK-801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK-801, could be stored for up to 1 week at 4 degrees C with less than 25% loss in activity. Receptor preparations from both species exhibited [3H]MK-801 binding properties in solution very similar to those observed in membranes (Bmax = 485 +/- 67 fmol/mg of protein, KD = 11.5 +/- 2.9 nM in rat; Bmax = 728 +/- 108 fmol/mg of protein, KD = 7.1 +/- 1.6 nM in pig, n = 3). The pharmacological profile of the solubilized [3H]MK-801 binding site was virtually identical to that observed in membranes. The rank order of potency of: MK-801 greater than (-)-MK-801 = thienylcyclohexylpiperidine greater than dexoxadrol greater than SKF 10,047 greater than ketamine, for inhibition of [3H]MK-801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of [3H]MK801 Binding to Rat Brain NMDA Receptors after the Administration of a Convulsant Drug and an Adenosine Analogue: A Quantitative Autoradiographic Study

Specific [³H]MK801 binding to rat brain NMDA receptors after the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied by means of a quantitative autoradiographic method. MP administration (150 mg/kg, i.p.) caused significant decreases in [³H]MK801 binding in several hippocampus subareas and layers, mainly in CA1 and CA3 at seizure (11–27%) and postseizure (8–16%) and in cerebral occipital cortex at seizure (18–22%). In nucleus accumbens, a rise was observed at postseizure (44%) and a tendency to increase at seizure (24%). CPA (2mg/kg, i.p.) decreased ligand binding in hippocampus (CA1, CA2, CA3) (17–22%) and in occipital cerebral cortex (18–24%). When CPA was administered 30 minutes before MP (which delayed seizure onset) and rats were sacrified at seizure, decreases in [³H]MK801 binding in several layers of CA1 and CA3 of hippocampus (11–27%) and in CA1, CA2, CA3 (24–35%) after CPA+MP postseizure, and an increase in CA2 after CPA and CPA+MP postseizure (20–34%), were observed. A drop was found in the occipital subarea (18–24%) after CPA and in the frontal and occipital subarea after CPA+MP postseizure (24–34%) while no changes were observed in any treatment involving the other cerebral cortex regions, thalamic nuclei, caudate putamen and olfactory tubercle. These results show that [³H]MK801 binding changes according to drug treatment and the area being studied, thus indicating a different role in seizure activity.     

A Comparison of [3H]MK-801 and N-[1-(2-Thienyl)cyclohexyl]-3,4-[3H]Piperidine Binding to the N-Methyl-D-Aspartate Receptor Complex in Human Brain

The binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate ([3H]MK-801) and N-[1-(2-thienyl)cyclohexyl]-3,4-[3H]piperidine ([3H]TCP) to the N-methyl-D-aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK-801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high-affinity sites for [3H]MK-801 (Bmax, 1,500 +/- 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 +/- 170 fmol/mg of protein). In addition, a low-affinity site was detected for [3H]MK-801 binding but not [3H]TCP binding. The pharmacology of the high-affinity [3H]MK-801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 greater than TCP greater than phencyclidine greater than N-allylnormetazocine (SKF 10047). 2-Amino-5-phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7-chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK-801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK-801 (Bmax, 1,403 +/- 394 fmol/mg) and [3H]TCP (Bmax, 1,292 +/- 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of febrile seizures in epileptic chicks by N-methyl-D-aspartate receptor antagonists

Experimental febrile seizures can be evoked in epileptic chicks by elevation of their body temperature. Both competitive N-methyl-D-aspartate (NMDA) receptor antagonists [(3-(+/- )2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), DL-2-amino-7-phosphosphonoheptanoic acid (APH), DL-2-amino-5-phosphonovaleric acid (APV), D-alpha-aminoadipic acid (AAA), and DL-alpha, epsilon-diaminopimelic acid (DAP)] and the noncompetitive NMDA antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5, 10-imine maleate (MK-801) produced dose-dependent increases in latency to the onset of seizures. Of the drugs tested, MK-801 had the highest potency followed in order by CPP = APH greater than APV much greater than AAA greater than DAP. There was a high correlation (r = 0.995) between the dose capable of doubling seizure latency and the affinity of the competitive NMDA antagonists for the NMDA receptor as determined by in vitro binding assays. These data suggest that NMDA receptor mediated mechanisms may be involved in the production of seizures in response to hyperthermia.     

Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of dithiol chelating agents on [3H]MK-801 and [3H]glutamate binding to synaptic plasma membranes

2,3-Dimercaptopropanol (BAL- British Anti-Lewesite) is a dithiol chelating agent used for the treatment of heavy metal poisoning, however, BAL can produce neurotoxic effects in a variety of situations. Based on the low therapeutic efficiency of BAL other dithiols were developed and DMSA (meso-2,3-dimercaptosuccinic acid) and DMPS (2,3-dimercaptopropane-1-sulfonic acid) are becoming used for treatments of humans exposed to heavy metals. In the present investigation the effect of dithiols in the glutamatergic system was examined. The results showed that BAL inhibited [³H]MK-801 and [³H]glutamate binding in a concentration-dependent manner. At 100 M BAL and DMSA caused a significantly inhibition of [³H]MK-801 binding to brain membranes (p < 0.05 by Duncan's multiple range test). BAL at 100 M caused an inhibition of 40% on [³H]glutamate binding. DMPS and DMSA had no significant effect on [³H]glutamate binding. Dithiotreitol (DTT), abolished the inhibitory effect of BAL on [³H]MK-801 binding. The protection exerted by DTT suggests that BAL inhibit [³H]MK-801 binding by interacting with cysteinyl residues that are important for redox modulation of receptor responses. ZnCl₂ inhibited [³H]glutamate and [³H]MK-801 binding to brain synaptic membrane; nevertheless, the inhibitory effect was slight more accentuated for [³H]MK-801 than [³H]glutamate binding (p < 0.05). The inhibition caused by 10 M ZnCl₂ on [³H]MK-801 binding was attenuated by BAL. The findings present in this study may provide the evidence that BAL affect the glutamatergic system and these effects can contributed to explain, at least in part, why BAL, in contrast to DMPS and DMSA is neurotoxic.     

Lactococcus lactis produces short-chain quinones that cross-feed Group B Streptococcus to activate respiration growth

Quinones are essential components of the respiration chain that shuttle electrons between oxidoreductases. We characterized the quinones synthesized by Lactococcus lactis , a fermenting bacterium that activates aerobic respiration when a haem source is provided. Two distinct subgroups were characterized: Menaquinones (MK) MK-8 to MK-10, considered as hallmarks of L. lactis , are produced throughout growth. MK-3 and demethylMK-3 [(D)MK-3] are newly identified and are present only late in growth. Production of (D)MK-3 was conditional on the carbon sugar and on the presence of carbon catabolite regulator gene ccpA . Electron flux driven by both (D)MK fractions was shared between the quinol oxidase and extracellular acceptors O₂, iron and, with remarkable efficiency, copper. Purified (D)MK-3, but not MK-8–10, complemented a menB defect in L. lactis . We previously showed that a respiratory metabolism is activated in Group B Streptococcus (GBS) by exogenous haem and MK, and that this activity is implicated in virulence. Here we show that growing lactococci donate (D)MK to GBS to activate respiration and stimulate growth of this opportunist pathogen. We propose that conditions favouring (D)MK production in dense microbial ecosystems, as present in the intestinal tract, could favour implantation of (D)MK-scavengers like GBS within the complex.     

8-OH-DPAT and MK-801 affect epileptic activity independently of vigilance

Vigilance and parallel occurrence of epileptic activity after administration of the 5-HT_1A agonist 8-OH-DPAT and the NMDA receptor antagonist MK-801 were studied in the genetic absence epilepsy model WAG/Rij rats. Spike-wave discharges (SWD) were present predominantly in passive awake and light slow wave sleep (SWS1) either in control animals or after treatments. Injection of 8-OH-DPAT (20.0 μg/rat i.c.v.) caused marked increase and MK-801 (10.0 μg/rat i.c.v.) decrease in SWD densities, thus the ratios of SWD in passive awake and in SWS1. SWD densities of MK-801 plus 8-OH-DPAT in combination were similar to those of CSF+CSF treated control rats. Both 8-OH-DPAT and MK-801 transiently increased the duration of active awake, increased latency and decreased duration of rapid eye movement (REM) sleep. 8-OH-DPAT increased the amount of SWD despite the decrease in the duration of SWS1. MK-801 decreased the amount of SWD despite the lack of significant change in duration of passive awake or SWS1. Pre-treatment with MK-801 reversed 8-OH-DPAT- induced increase in duration of SWD without any effect on 8-OH-DPAT-induced changes in sleep parameters. Our studies provide evidence that 8-OH-DPAT-induced epileptic activity is independent of its effect on sleep, and that interaction of serotonergic and glutamatergic systems plays a role in the generation of SWD, but not in the regulation of vigilance and sleep.     

Behavioural motor effects of MK-801 and DNQX parenteral administration in adult cats: dose-response analysis. Modulatory role of dopaminergic D1 and D2 antagonists on MK-801 induced motor behaviours

1. Administration of MK-801 a selective antagonist of the NMDA receptors (50, 100 and 150 micrograms/kg, s.c.) elicited in adult cats ataxia and loss of equilibrium. A dose-response effect was observed. 2. Administration of DNQX, a selective antagonist of the non-NMDA receptors, even with doses 20 times higher than those employed with MK-801, did not produce any behavioural disturbances. 3. Previous injection of SCH 23390, a selective parenteral antagonist of dopamine D1 receptor, reduced significantly the intense ataxic effects of MK-801, while sulpiride only increased the latency of the symptoms. 4. The results are discussed considering the reported interactions between the dopaminergic and glutamatergic systems.     

Intracerebroventricular administration of MK-801 increases food intake through mechanisms independent of gastric emptying

Systemic or hindbrain administration of MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, increases meal size. To examine whether MK-801 enhances intake by increasing gastric emptying, we administered MK-801 (2.0 microg/3.0 microl) into the fourth ventricle [intracerebroventricular (ICV)] and measured feeding and gastric emptying of 5-ml NaCl or 15% sucrose loads. In a parallel experiment, we examined food intake and gastric emptying following intraperitoneal (IP) injection of MK-801 (100 microg/kg). MK-801, either IP or ICV, increased 30-min sucrose intake compared with control (12.3 +/- 0.7 vs. 9.8 +/- 0.5 and 16.6 +/- 2.0 vs. 10.7 +/- 0.7 ml, for IP and ICV administration, respectively). Also, IP MK-801 increased 5-min gastric emptying of NaCl (4.13 +/- 0.1 ml emptied) and sucrose (3.11 +/- 0.1 ml emptied) compared with control (3.75 +/- 0.2 and 2.28 +/- 0.1 ml emptied for NaCl and sucrose loads, respectively). In contrast, ICV MK-801 did not alter NaCl emptying (3.82 +/- 0.1 ml emptied) compared with control (3.82 +/- 0.3 ml emptied) and actually reduced gastric emptying of sucrose (2.1 +/- 0.2 and 2.94 +/- 0.1 ml emptied, for MK and vehicle, respectively). These data confirm previous results that systemic as well as hindbrain injection of MK-801 increases food intake. However, because ICV MK-801 failed to increase gastric emptying, these results indicate that MK-801 increases food intake through mechanisms independent of altered gastric emptying.     

Effect of NMDA-receptor ligands on neocortical and hippocampal EEG activity of rat brain.

Neocortex and hippocampus play important role in motor activity, neuronal plasticity and learning and memory mechanisms. Electroencephalographic (EEG) activity of neocortex and hippocampus of rat following NMDA-receptor agonist, N-methyl-D-aspartate (NMDA), 0.25-2 nmol in 10 microliters, ICV and noncompetitive NMDA-receptor antagonists, MK 801 (0.025-0.1 mg/kg, ip) and ketamine (10-50 mg/kg, ip) at OH, 1/2H, 4H, 8H and 24H was recorded. The electrodes were implanted stereotaxically in hippocampus and neocortex respectively. NMDA (0.25 and 1 nmol) showed longer lasting decrease in amplitude in hippocampus and in frequency in cortical neurons while 2 nmol produced epileptogenic neurotoxicity. Opposite effect i.e. increase in amplitude in both, hippocampus and neocortex was observed with MK 801 and ketamine and these agents also showed longer lasting influence. Administration of MK 801 (0.05 mg/kg) and ketamine (50 mg/kg) prior to NMDA 2 nmol protected 40% animals from NMDA-induced neurotoxicity and blockade of NMDA-induced long term influence. The EEG effect of NMDA agonist and NMDA-induced neurotoxicity at higher dose and its modification by NMDA-antagonist, MK 801 and ketamine suggest that beside NMDA agonists (NMDA), its antagonists may, also affect long lasting changes in hippocampus and cortex. These antagonists reverse NMDA-mediated long term influence in these brain areas.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Modulation of MK-801-elicited mouse popping behavior by galantamine is complex and dose-dependent

The ability of phencyclidine (PCP), a noncompetitive antagonist of NMDA receptor-mediated neurotransmission, to precipitate a schizophreniform psychosis in susceptible individuals is consistent with the hypothesized pathologic occurrence of NMDA receptor hypofunction in this disorder. Because the psychosis caused by PCP resembles schizophrenia in all of the relevant domains of psychopathology, investigators have sought to characterize animal models of NMDA receptor hypofunction. MK-801 (dizocilpine) binds to the same hydrophobic channel domain in the NMDA receptor-associated ionophore as PCP, and has been shown to elicit intense irregular episodes of jumping behavior in mice, termed "popping." MK-801-elicited mouse popping is an animal model of NMDA receptor hypofunction that has been used to screen novel candidate compounds for the treatment of schizophrenia. Recently, a selective abnormality in the transduction of the acetylcholine signal at the level of the alpha 7 nicotinic receptor has been described in schizophrenia. The existence of a nicotinic cholinergic abnormality in schizophrenia has stimulated interest in a potential therapeutic role for positive allosteric modulation of nicotinic receptors. Galantamine is a compound that possesses two interesting properties: inhibition of acetylcholinesterase and positive allosteric modulation of nicotinic neurotransmission. Theoretically, galantamine would be expected to increase the efficiency or likelihood that acetylcholine will promote channel opening and ionic conductance at nicotinic receptors. As expected, in the current investigation statistically significant popping behavior was elicited by MK-801 in mice (T(22) = 2.16, P < 0.05). This MK-801-elicited popping was significantly attenuated by 100 mg/kg of galantamine (T(22) = 2.24, P < 0.05). The data show that nicotinic interventions can influence NMDA receptor-mediated neurotransmission in the intact mouse.     

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.     

Coexpression of postsynaptic density-95 protein with NMDA receptors results in enhanced receptor expression together with a decreased sensitivity to L-glutamate

Coexpression in human embryonic kidney (HEK) 293 cells of the postsynaptic density-95 protein (PSD-95) with NMDA receptor NR2A or NR2B single subunits or NR1-1a/NR2A and NR1-1a/NR2B subunit combinations induced an approximately threefold increase in NR2A and NR2B subunit expression. Deletion of the NR2 C-terminal ESDV motifs resulted in the loss of this increase following coexpression of NR1-1a/NR2A(Trunc) and NR1-1a/NR2B(Trunc) with PSD-95. Characterisation of the radioligand binding properties of [(3)H]MK-801 to NR1-1a/NR2A receptors with or without PSD-95 showed that PSD-95 induced a threefold increase in B:(max) values and an apparent approximately fivefold decrease in affinity in the presence of 10 microM: L-glutamate. In the presence of 1 mM: L-glutamate, the K:(i) for MK-801 binding to NR1-1a/NR2A with PSD-95 was not significantly different from that for NR1-1a/NR2A without PSD-95. The EC(50) value for the enhancement of [(3)H]MK-801 binding by L-glutamate to NR1-1a/NR2A was 1.8 +/- 0.4 (n = 4) and 8.9 (mean of n = 2) microM: in the absence and presence of PSD-95, respectively. Thus, coexpression of PSD-95 with NR1-1a/NR2A results in a decreased sensitivity to L-glutamate and an enhanced expression of NR2A and NR2B subunits. Deletion studies show that this effect is mediated via interaction of the C-terminal ESDV motif of the NR2 subunit with PSD-95.