Chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptor polypeptide subunits in mouse cortical neurons in culture

The goal of this study was to determine whether chronic ethanol-mediated up-regulation of the N-methyl-D-aspartate receptors (NMDAR) was associated with an augmentation of the NMDAR polypeptide subunits in the mammalian cortical neurons. The results show that chronic ethanol treatment produced an increase in the R1 and R2B polypeptide subunits. The R2A subunit was not expressed in these neurons. Chronic NMDAR antagonist ((+)-3-2-(carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP)) treatment also increased the R1 and R2B polypeptide subunits. A similar increase was observed when ethanol and CPP were used in combination. Binding studies using [3H]MK-801 ((+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclophenptan-5,10-imine maleate), a noncompetitive NMDAR antagonist, confirmed that concomitant exposure of ethanol and CPP up-regulated the NMDAR. Our results demonstrate for the first time that chronic ethanol treatment increased the NMDA receptor polypeptide subunit synthesis and that it was associated with an increase in [3H]MK-801 binding sites.     

Effects of subacute lead exposure on [3H]MK-801 binding in hippocampus and cerebral cortex in the adult rat

We used the NMDA receptor non-competitive antagonist, [3H]MK-801, as a ligand for an autoradiographic study to determine the effects of lead on NMDA receptor in the rat brain. Adult male rats were administered lead acetate, 100 mg/kg, or sodium acetate, 36 mg/kg (control), by i.p. for 7 days. High lead levels were detected in blood (41.1 microg/dl) and in brain (16.7-29.4 microg/g). Concentrations of lead in brain regions were not significantly different. The [3H]MK-801 binding was heterogeneously distributed throughout the rat brain with the following order of binding densities: hippocampal formation > cortex > caudate-putamen > thalamus > brainstem. Lead exposure produced a significant decrease in [3H]MK-801 binding to the NMDA receptor in the hippocampal formation including CA2 stratum radiatum, CA3 stratum radiatum, hilus dentate gyrus and presubiculum, and in the cerebral cortex including agranular insular, cingulate, entorhinal, orbital, parietal and perirhinal areas. The hippocampal formation is known as a critical neural structure for learning and memory processes, whereas, cortical and subcortical regions have been demonstrated to be involved in the modulation of complex behavioral processes. The NMDA receptor has been demonstrated to play a key role in synaptic plasticity underlying learning and memory. Lead-induced alterations of NMDA receptors in the hippocampal formation and cortical areas may play a role in lead-induced neurotoxicity.     

Characterization of ionotropic glutamate receptor-mediated nitric oxide production in vivo in rats

BACKGROUND AND PURPOSE: Glutamate receptor activation can stimulate nitric oxide (NO) production and possibly play a role in long-term potentiation and excitotoxic-mediated injury. We studied the differential effect of agonist-induced activation of ion channel-linked N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subtypes on NO production in vivo in rat hippocampus. We also studied whether dantrolene, a ryanodine calcium channel inhibitor previously shown to attenuate metabotropic glutamate receptor stimulation of NO production, also attenuated ionotropic glutamate receptor-mediated stimulation of NO production. METHODS: Microdialysis probes were placed bilaterally into the CA3 region of the hippocampus of pentobarbital-anesthetized adult Sprague-Dawley rats and were perfused for 5 hours with artificial cerebrospinal fluid (CSF) containing 3 mumol/L [14C]L-arginine. Recovery of [14C]L-citrulline in the effluent was used as a marker of NO production. In 13 groups of rats, increases in [14C]L-citrulline recovery were compared between right- and left-sided probes perfused with no additional drugs versus combinations of NMDA, AMPA, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), the non-competitive glutamate receptor blocker MK-801, the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and dantrolene. RESULTS: Recovery of [14C]L-citrulline during perfusion with artificial CSF progressively increased to 272 +/- 73 fmol/min (+/-SEM) over 5 hours. Contralateral perfusion with 1 mmol/L L-NAME inhibited [14C]L-citrulline recovery. Perfusion with 1 mmol/L MK-801 or 1 mmol/L CNQX reduced [14C]L-citrulline recovery compared with contralateral perfusion with CSF alone. Perfusion with 1 mmol/L NMDA enhanced [14C]L-citrulline recovery, and this enhancement was attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. Perfusion with 1 mmol/L AMPA enhanced [14C]L-citrulline recovery, and this enhancement was also attenuated by L-NAME, MK-801, and CNQX but not by dantrolene. CONCLUSIONS: Through an indirect method of assessing NO production in vivo, results with MK-801 and CNQX indicate that NMDA and AMPA receptor activation contribute to basal NO production in the rat hippocampus. Enhanced NO production with NMDA and AMPA agonists appears to involve a complex neuronal interaction because the effect of NMDA was attenuated by both MK-801 and CNQX and because the effect of AMPA was attenuated by both CNQX and MK-801. In contrast to metabotropic glutamate receptor activation, release of calcium from intracellular ryanodine calcium channels does not appear to be a prominent mediator of ionotropic glutamate receptor stimulation of NO production.     

Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain

AIM: To investigate age related alterations in glutamate N-methyl-D-aspartate (NMDA) receptor binding produced by the modulatory compounds glutamate, glycine, and magnesium (Mg2+) sulphate. METHODS: The effects produced by glutamate plus glycine, and Mg2+ on the binding of [3H]MK-801, a ligand for the N-methyl-D-aspartate ion channel phencyclidine site, were measured in membrane preparations made from prefrontal cortex from human neonate (n = 5), infant (n = 6), and adult (n = 6) necropsy brains. RESULTS: Neonatal brains had the least [3H]MK-801 binding, suggesting either a low density of NMDA receptors or a more restricted access of [3H]MK-801 to cation channel sites. Infant brains had the most [3H]MK-801 binding which was stimulated to a greater extent by L-glutamate (100 microM) and glycine (10 microM) than in neonatal and adult brains. MG2+ invariably inhibited [3H]MK-801 binding. However, the Mg2+ IC50 value was higher in neonatal brain (3.6 mM) than infant (1.4 mM) and adult (0.87 mM) brains. CONCLUSION: Infant brain may have excess NMDA receptors which are hyper responsive to glutamate and glycine. The lower potency of Mg2+ to inhibit [3H]MK-801 binding in neonatal cortex may be because newborn babies have NMDA receptors without the normal complement of Mg2+ sites. The findings suggest that therapeutic NMDA receptor block in neonates requires higher concentrations of magnesium sulphate in brain tissue.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

Ethanol, stroke, brain damage, and excitotoxicity

The N-methyl-d-aspartate (NMDA)-glutamate receptor could contribute to stroke, trauma, and alcohol-induced brain damage through activation of nitric oxide formation and excitotoxicity. In rat primary cortical cultures NMDA was more potent at activating nitric oxide formation than triggering excitotoxicity. Ethanol dose dependently inhibited both responses. In contrast, treatment of neuronal cultures with ethanol (100 mM) for 4 days significantly increased NMDA stimulated nitric oxide formation and excitotoxicity. These findings suggest that ethanol acutely inhibits but chronically causes supersensitivity to NMDA-induced excitotoxicity in neuronal cultures. To investigate ethanol's interaction with stroke induced damage models of global cerebral ischemia were studied. Transient global ischemia resulted in a loss of hippocampal CA1 pyramidal neurons over a 3- to 5-day period. Determinations of the NMDA receptor ligand binding stoichiometry or postischemic receptor binding changes did not show differences between neurons that undergo delayed neuronal death following ischemia and those that show no toxicity, for example, CA1 and dentate gyrus, respectively. Acute ethanol (3 g/kg) was found to protect against ischemia-induced CA1 hippocampal damage by lowering body temperature, but not under temperature controled conditions. These studies indicate that the factors contributing to stroke-induced brain damage are complex, although they are consistent with chronic ethanol increasing stroke-induced brain damage by increasing NMDA excitotoxicity.     

Activation of PKC reverses apparent NMDA receptor reduction in ALS

The binding of [3H]MK-801 to NMDA receptors was reduced by 40-45% in the dorsal and ventral horns of spinal cords from patients who died with amyotrophic lateral sclerosis (ALS) compared with controls. These results reflect either neurone death with concomitant receptor loss or regulation-related receptor decreases independent of motoneurone degeneration. To distinguish between these possibilities we explored aspects of NMDA receptor regulation using phorbol ester to activate protein kinase C (PKC). Spinal cord sections were exposed to phorbol ester before incubation with [3H]MK-801 to determine levels of NMDA binding. Phorbol ester treatment increased [3H]MK-801 binding in both ALS and control tissue to almost identical levels of specific binding for both groups. The increased [3H]MK-801 binding could be completely blocked by concurrent exposure of spinal cord sections to H-7, a general protein kinase inhibitor. These results suggest that NMDA receptors in ALS spinal cord are decreased as a result of abnormal enzyme activity independent of motoneurone degeneration.     

Differential effects of aging on binding sites of the activated NMDA receptor complex in mice

The NMDA receptor site has been shown to be vulnerable to the effects of aging. Decreases in binding to the receptor site of up to 50% have been reported in aged animals. The present study was designed to quantitate and compare the effects of aging on multiple binding sites of the NMDA receptor complex in various brain regions. Autoradiography with [3H]glutamate, [3H]CPP, [3H]glycine, [3H]MK801 and [3H]TCP was performed on brain sections from 3, 10 and 28-30 month old C57B1/6 mice. The percent declines between 3 and 28-30 months of age in [3H]-glutamate (15-35% declines) and [3H]CPP (20-42% declines) binding were similar within most cortical regions and the caudate nucleus but [3H]glutamate binding showed less change (0-11% declines) than [3H]CPP (13-27% declines) in the occipital/temporal cortex and hippocampal regions. [3H]MK801 and [3H]TCP binding, stimulated by 10 microM glutamate, exhibited intermediate aging changes between the glycine and NMDA sites, both in percent decline (3-28% and 0-26%, respectively) and in the number of brain regions involved. [3H]Glycine binding, stimulated by 10 microM glutamate, showed no significant overall effect of age (declines ranged from 0-34%). [3H]CPP binding was significantly more affected than [3H]glycine binding in many regions. These results suggest that aging has heterogeneous effects on different sites on the NMDA receptor complex throughout the brain and on NMDA receptor agonist versus antagonist binding in selected brain regions.     

Role of NMDA receptors in pentobarbital tolerance/dependence

Effects of continuous pentobarbital administration on binding characteristics of [3H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300 micrograms/10 microliters/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [3H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10 microliters/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [35S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells

The rat N-methyl-D-aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[3H]dichlorokynurenic acid labeled a single high-affinity site (KD = 29.6 +/- 6 nM; Bmax = 19.4 +/- 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca2+ and channel occupancy by MK-801, when expressed in this mammalian cell line.     

Induction of neurosteroid synthesis by NMDA receptors in isolated rat retina: a potential early event in excitotoxicity

Here we investigated the possible regulation of neurosteroidogenesis by N-methyl-D-aspartic acid (NMDA) receptor activation and addressed the hypothesis that neurosteroid synthesis may be involved in acute excitotoxicity. In the isolated retina, exposure to NMDA modified pregnenolone and pregnenolone sulphate formation. This effect was dose and time dependent, the synthesis being increased by relatively moderate NMDA doses (1-100 microM) within 30 min exposure and reduced to its control value by 60 min or by raising drug concentrations. NMDA-stimulated neurosteroid synthesis was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) and 3(2-carboxypiperazine-4-yl)propyl-1-phosphonic acid (CPP), depended on extracellular calcium and reproduced by glutamate. Lactate dehydrogenase (LDH) release and morphological analysis revealed that retinal cell viability was not significantly affected after 30 min exposure to 50 microM NMDA, but severe cell damage occurred by 60 min. When the GABAA (gamma-aminobutyric acid) receptor agonist muscimol (1-1000 microM), known to activate retinal neurosteroidogenesis, was added together with NMDA, no additional increase in neurosteroid synthesis was observed, and NMDA-induced LDH release remained unchanged. However, exposure to a high concentration of muscimol alone (500 microM) provoked a similar degree of toxicity to NMDA. By contrast, bicuculline abolished the increase in neurosteroidogenesis and LDH release. Similarly, pretreatment with R (+)-p-aminoglutethimide (AMG), an inhibitor of cholesterol side-chain cleavage cytochrome P450, attenuated acute retinal cell damage. The inhibitory nature of AMG on NMDA-stimulated neurosteroidogenesis was confirmed in the observation that drug treatment reduced pregnenolone content and did not affect the bindings of [3H] MK-801 and [3H] muscimol. The results demonstrate that NMDA receptors regulate neurosteroidogenesis through a transneuronal mechanism, which implies GABAA receptor activation. The early NMDA-mediated stimulation of neurosteroid synthesis seems to play a critical role in acute excitotoxicity; consequently, its inhibition is likely to delay neuronal cell death.     

Dual actions of nitric oxide in N-methyl-D-aspartate receptor-mediated neurotoxicity in cultured retinal neurons

This study was performed to elucidate the role of nitric oxide (NO) in N-methyl-D-aspartate (NMDA) receptor-mediated glutamate neurotoxicity in the retina. The experiments were done with primary retinal cultures obtained from 17- to 19-day-old rat fetuses. The NOS activity measured by monitoring the conversion of [3H]arginine to [3H]citrulline was approximately 5 pmol/min/mg protein. A 10-min exposure of the cultured cells to glutamate (1 mM) or NMDA (1 mM) followed by a 1-h incubation in a normal medium consistently resulted in 60% cell death. The concomitant addition of an inhibitor of NOS, Nomega-nitro-L-arginine (300 microM), with glutamate or NMDA reduced cell death by 70%. A brief exposure of the cells to sodium nitroprusside (SNP, 500 microM) or S-nitrosocysteine (SNOC, 500 microM), NO-generating agents, caused 60% cell death. Depletion of NO by reduced hemoglobin prevented the cell death induced by either glutamate, NMDA, or NO generating agents. Fifty microM SNOC alone had no effect on the cell viability. However, pretreatment with 50 microM SNOC as well as simultaneous application of 50 microM SNOC with NMDA inhibited cell death induced by NMDA. These findings indicate that a low concentration of NO plays a protective role in glutamate neurotoxicity via closing the NMDA receptor gated ion channel. However, elevated concentrations of NO, interacting with oxygen radicals, become toxic and mediate glutamate-induced neurotoxicity in the cultured retinal neurons.     

Chronic ethanol treatment decreases [3H]epibatidine and [3H]nicotine binding and differentially regulates mRNA levels of nicotinic acetylcholine receptor subunits expressed in M10 and SH-SY5Y neuroblastoma cells

For a study of the underlying mechanisms of a possible interaction between ethanol and nicotinic receptors during ethanol dependence, the aim of this work was to investigate the effect of chronic ethanol exposure on nicotinic receptor subtypes in a transfected fibroblast cell line (M10 cells) stably expressing alpha4beta2 nicotinic receptor subtype and an SH-SY5Y neuroblastoma cell line expressing alpha3, alpha5, alpha7, beta2, and beta4 nicotinic acetylcholine receptor (nAChR) subunits. A significant dose-related decrease (-30-80%) in number of [3H]nicotine binding sites was observed in ethanol-treated (25-240 mM) compared with untreated M10 cells. Similarly, 4-day treatment with ethanol in concentrations relevant to chronic alcoholism (100 mM) decreased the number of nicotinic receptor binding sites in the SH-SY5Y cells when measured using [3H]epibatidine. When M10 cells were chronically treated with nicotine, ethanol partly inhibited the up-regulation of nicotinic receptors when present in the cells together with nicotine. Chronic treatment for 4 days with 100 mM ethanol significantly decreased the mRNA level for the alpha3 nAChR subunit (-39%), while the mRNA levels for the alpha7 (+30%) and alpha4 (+22%) subunits were significantly increased. Chronic ethanol treatment did not affect the mRNA levels for the beta2 nAChR subunit. Changes in the levels of nAChR protein and mRNA may have adaptive significance and be involved in the development of dependence, tolerance, and addiction to chronic ethanol and nicotine exposure. They also may be targets for therapeutic strategies in the treatment of ethanol and nicotine dependence.     

Genetic nomenclature for Trypanosoma and Leishmania

We investigated the effect of chronic ethanol administration and its withdrawal on the polypeptide levels of NMDA receptor subunits such as NR1, NR2A, and NR2B in the rat cerebral cortex and hippocampus using Western blot analysis technique. Our results indicate that chronic ethanol treatment upregulates NMDA receptor subunits NR1, NR2A, and NR2B ( approximately 35%). At 48 h of last dose of ethanol administration, the protein content returned to almost control level, thereby demonstrating the reversibility of the changes.     

Nitric oxide involvement in regulating the dopamine transport in the striatal region of rat brain

Spontaneous [3H]dopamine ([3H]DA) overflow was measured from striatal slices in the presence of different glutamate (Glu) receptor agonists such as N-methyl-D-aspartate (NMDA), kainate (KA) and quisqualate (QA) and their corresponding antagonists, Dizocilpine maleate (MK-801), D-gamma-glutamyl-aminomethanesulfonic acid (GAMS) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. [3H]DA uptake and release in the presence of L-Arginine (L-Arg) and NG-nitro-arginine (L-N-Arg), an inhibitor of nitric oxide (NO) synthesis were also evaluated. L-N-Arg alone or combined with L-Arg significantly reduced [3H]DA uptake at 10 and 100 microM from 33% to 44% from striatal slices. Whereas, in brain synaptosomal fractions L-Arg induced a biphasic effect on that [3H]DA uptake in a dose dependent manner, and L-N-Arg showed an absolute inhibition in 80-90% of this [3H]DA uptake at 1-500 microM. The amino acids, lysine, valine and histidine (100 microM) had a little effect inhibitory on [3H]DA uptake from synaptosomal fractions. Glu agonists, NMDA (10 microM) and KA (10 microM) importantly increased the spontaneous [3H]DA overflow, which was blocked by MK-801 (10 microM) and GAMS (10 microM), respectively. QA had no effect on [3H]DA release. L-Arg (10-200 microM) potentiated the spontaneous [3H]DA overflow in a dose dependent fashion from striatal slices, being reverted by 10 microM L-N-Arg alone or in combination with all other compounds; whereas, lysine, histidine and valine did not modify that spontaneous [3H]DA overflow. Results support the hypothesis related to the participation of NO on DA transport possibly synthesized at the dopaminergic (DAergic) terminals in the striatum; also that L-Arg concentration may determine alternative mechanisms to regulate the DAergic activity at the striatum.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Pharmacological characterization of N-methyl-D-aspartate receptors in spinal cord of rats with a chronic peripheral mononeuropathy

N-Methyl-D-aspartate (NMDA) receptor antagonists, acting in the spinal cord, are analgesic. However, the clinical utility of these antagonists is diminished by their adverse effects on cognition and behavior. To facilitate the development of spinal cord-selective NMDA receptor antagonists, we characterized ligand interactions at NMDA receptors in spinal cord of normal rats and rats with a chronic peripheral neuropathy. NMDA receptors in spinal cord were distinguished from those in cerebral cortex on the basis of differences in the potencies of competitive and noncompetitive antagonists and on the basis of differences in their response to spermidine. D(-)-2-Amino-5-phosphonopentanoic acid (AP-5) and (+)-(1-hydroxy-3-aminopyrrolidine-2-one) (HA-966) were more potent in inhibiting NMDA-dependent [3H]TCP binding in spinal cord while, conversely, MK-801 was more potent in inhibiting [3H]TCP binding to NMDA receptors in cerebral cortex. Spermidine increased [3H]TCP binding to NMDA receptors in cerebral cortex (39+/-8%) but not spinal cord (2+/-1%). Based on these properties, NMDA receptors in spinal cord more closely resembled those in cerebellum than those in cerebral cortex. Generation of a chronic neuropathy had no effect on the density of NMDA receptors in lumbar spinal cord. There were also no major changes in the potencies of competitive antagonists or channel blocking ligands, although there was a trend for kynurenic acid and D-CPP to be more potent in the spinal cords of neuropathic animals. These findings indicate that, in both normal and neuropathic pain states, NMDA receptors in spinal cord can be distinguished pharmacologically from those in cerebral cortex. These findings underscore the feasibility of developing spinal cord-selective NMDA receptor antagonists as novel analgesics.     

Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro

The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.     

Long-term adrenalectomy decreases NMDA receptors in rat hippocampus

The effect of long-term adrenalectomy on NMDA receptors in the rat hippocampus was studied. Hippocampal sections of control and adrenalectomized rats were incubated with [3H]MK-801, a radiolabeled non-competitive inhibitor of the NMDA receptor. Analysis by in vitro autoradiography showed a significant decrease in [3H]MK-801 binding in the dentate gyrus, CA1 and CA4 areas, as well as the temporal cortex. Results of this study suggest that glucocorticoids are vital for the regulation of the NMDA receptors.     

Aromatic analogs of arcaine inhibit MK-801 binding to the NMDA receptor

Aromatic analogs of arcaine were shown to have inhibitory effects on the binding of the channel blocking drug [3H]MK-801 to the NMDA receptor complex. The most potent compound of the series was an N,N'-bis(propyl)guanidinium which inhibited [3H]MK-801 binding with an IC50 of 0.58 microM and an IC50 of 12.17 microM upon addition of 100 microM spermidine. The increase in IC50 upon addition of spermidine suggests competitive antagonism between the inhibitor and spermidine at the arcaine-sensitive polyamine site of the NMDA receptor complex.