MK-801 Binding Sites Research Articles

Regional distribution and properties of [ 3H]MK-801 binding sites determined by quantitative autoradiography in rat brain

[ 3H]MK-801 binding in rat brain was characterized using a quantitative autoradiographic binding assay. [ 3H]MK-801 binding (5 nM) reached equilibrium by 120 min at 23°C. [ 3H]MK-801 appeared to label a single high affinity site with an affinity constant of approximately 11 nM. [ 3H]MK-801 binding was heterogeneously distributed throughout the brain with the following order of binding densities: hippocampal formation > cortical areas > striatum > thalamus. Competitive N-methyl- d-aspartate antagonists, dl-2-amino-5-phosphonopentanoic acid, dl-2-amino-7-phosphonoheptanoic acid, 3-(2-car☐ypiperazin-4-yl)propyl-1-phosphonic acid, and cis-4-phosphonomethyl-2-piperidine car☐ylic acid, inhibited [ 3H]MK-801 binding. Glycine antagonists, 7-chlorokynurenic acid and kynurenic acid, also inhibited [ 3H]MK-801 binding. Furthermore, the inhibition of [ 3H]MK-801 binding by the quinoxalinedione compounds 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2, 3-dione was reversed by glycine. [ 3H]MK-801 binding was also inhibited by zinc ions. [ 3H]MK-801 binding was enhanced by glycine or N-methyl- d-aspartate. These results demonstrate that [ 3H]MK-801 can be used in a quantitative autoradiographic assay as a functional probe for the N-methyl- d-aspartate receptor complex.

NMDA receptors in mice bred to be prone or resistant to ethanol withdrawal seizures

Selective breeding has produced replicate lines of mice that are prone (WSP) or resistant (WSR) to ethanol withdrawal seizures. Ethanol-naive WSP mice inherently have a greater number of hippocampal binding sites for the NMDA receptor-gated ion channel blocker, MK-801, than ethanol-naive WSR mice. After chronic ethanol ingestion, hippocampal (but not cerebral cortical) MK-801 binding sites increase in both lines of mice. However, the number of MK-801 binding sites in the ethanol-treated WSR mice does not exceed the number of MK-801 binding sites in untreated WSP mice. At the time of ethanol withdrawal, the number of hippocampal MK-801 binding sites in each line of WSP mice is 50–70% higher than the number of such sites in WSR mice. Given the past evidence for a role of the NMDA receptor in seizures, the results implicate hippocampal NMDA receptor-gated channels in the generation of ethanol withdrawal seizures.

Nicotine reduces the binding of [ 3H]MK-801 to brain membranes, but not via the stimulation of high-affinity nicotinic receptors

Nicotine (10 and 100 μM) inhibited [ 3H]MK-801 binding to rat cerebral cortical membranes and this effect was not blocked by dihydro-β-erythroidine, (+)-tubocurarine or mecamylamine. Cytisine, muscarine, mecamylamine and (+)-tubocurarine also inhibited [ 3H]MK-801 binding. Neither raising the MK-801 concentration, nor the addition of n-methyl- D-aspartate (NMDA) receptor agonists altered the effects of nicotine. Hence this response is not mediated via high-affinity nicotinic receptor stimulation, competition for MK-801 binding sites or require NMDA receptor activation.

Interaction of Strychnine‐Insensitive Glycine Binding with MK‐801 Binding in Brain Synaptic Membranes

Strychnine-insensitive [3H]glycine binding was detected in brain synaptic membranes treated with Triton X-100 using a filtration assay method. The binding was a time-dependent, inversely temperature-dependent, and reversible process with a relatively high affinity for the neuroactive amino acid. Scatchard analysis revealed that Triton treatment doubled both the affinity and density of the binding sites, which consisted of a single component. The binding was not only displaced by structurally-related amino acid such as D-serine and D-alanine, but also inhibited by some peptides containing glycine, including glycine methylester and N-methylglycine. These ligands invariably potentiated the binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]- cyclohepten-5,10-imine ([3H]MK-801), a noncompetitive antagonist for the N-methyl-D-aspartate-sensitive subclass of the central excitatory amino acid receptors, in a concentration-dependent manner. Among various endogenous tryptophan metabolites, kynurenic acid significantly inhibited the strychnine-insensitive [3H]glycine binding. The Triton treatment did not affect the pharmacological profile of [3H]MK-801 binding sites. These results suggest that brain synaptic membranes treated with Triton X-100 are useful in evaluating the strychnine-insensitive and kynurenate-sensitive binding sites of glycine, which are functionally linked to N-methyl-D-aspartate- sensitive receptor channels.

Autoradiographic evidence that NMDA receptor-coupled channels are located postsynaptically and not presynaptically in the perforant path-dentate granule cell system of the rat hippocampal formation

In vitro quantitative autoradiography with [ 3H]MK-801 was used to determine K d and B max values for the NMDA receptor-coupled channel in subregions of the rat hippocampal formation. A single form of the channel with an apparent K d in the 15–20 nM range was found for [ 3H]MK-801 binding in the presence of both 1μM glutamate and 1μM glycine. Specific binding was highest in the molecular layer of the dentate gyrus, followed by CA1 stratum radiatum and CA1 stratum oriens. Fewer binding sites were observed in the hilus of the dentate gyrus, cerebral cortex, CA1 stratum pyramidale. CA3 subregion (stratum oriens, stratum pyramidale, stratum radiatum), and thalamus. Selective destruction of dentate granule cells by colchicine microinjections reduced the amount of specific [ 3H]MK-801 binding by half in the molecular layer of the dentate, compared to intact tissue. [ 3H]MK-801 binding did not change in other hippocampal subregions as a consequence of colchicine injection. Electrolytic entorhinal cortical lesions produced no changes in regional MK-801 binding site density in any of the regions under study. To address the tissue shrinkage following entorhinal cortex lesions, detailed analysis of the binding site density per fixed (16 μm) length of granule cell dendrite, and of the aggregate density across the entire molecular layer revealed no change in the number of MK-801 binding sites per unit length of dendrite in the molecular layer of the dentate gyrus. These findings indicate that NMDA receptor-coupled channels are confined to a postsynaptic location in the perforant path-dentate granule cell system of the adult rat.

Localization and Quantitative Autoradiography of Glutamatergic Ligand Binding Sites in Chick Brain.

The anatomical localization of glutamate receptor subtype-selective ligand binding sites was investigated in 1-day-old chick brain using quantitative autoradiography. Under the conditions used, the regional distributions of [3H]glutamate, [3H]AMPA (a selective quisqualate receptor ligand) and [3H]kainate binding sites are manifestly different. [3H]l-glutamate binding is densely localized in the telencephalon, particularly in the neostriatum (2.8 pmol/mg protein). In addition, [3H]l-glutamate labels the thalamus, the nucleus mesencephalicus lateralis pars dorsalis, the superficial layers of the optic tectum and the molecular layer of the cerebellum. [3H]AMPA binding sites are most densely localized in the hippocampus (0.90 pmol/mg protein), with an otherwise relatively uniform distribution of binding within the telencephalon. [3H]AMPA also labels the striatum griseum et fibrosum superficiale of the optic tectum and the molecular layer of the cerebellum. [3H]Kainate binding sites are extremely densely packed in the molecular layer of the cerebellum (10 pmol/mg protein). Other regions of [3H]kainate binding include the hyperstriatum and the thalamus. The binding of the NMDA receptor channel blocker [3H]MK-801 is increased in the presence of 1 mM l-glutamate. [3H]MK-801 binding is generally widespread in the telencephalon but is notably absent from the ectostriatum. No evidence of [3H]MK-801 binding sites was detected in the cerebellum, even in the presence of 1 mM l-glutamate. The relatively high densities and the well-defined localizations of the glutamate receptor subtype binding sites suggest that chick brain provides a useful system for the further study of excitatory amino acid receptors.

MK-801 is a potent nematocidal agent. Characterization of MK-801 binding sites in Caenorhabditis elegans.

MK-801, an N-methyl-D-aspartate antagonist in mammalian brain tissue, is a potent nematocidal agent. Specific MK-801 binding sites have been identified and characterized in a membrane fraction prepared from the free-living nematode Caenorhabditis elegans. The high-affinity MK-801 binding site has an apparent dissociation constant, Kd, of 225 nM. Unlike the MK-801 binding site in mammalian tissues, the C. elegans binding site is not effected by glutamate or glycine, and polyamines are potent inhibitors of specific MK-801 binding.

Regional distribution of [ 3H]MK-801 binding sites in the human brain

Specific [ 3H]MK-801 binding was measured under equilibrium conditions in 8 brain regions (frontal cortex, area entorhinalis, hippocampus, amygdala, putamen, thalamus, substantia nigra and nucleus dentatus) in the right hemisphere of the human brain ( n = 4). In addition, binding was assessed in 3 of these regions (frontal cortex, area entorhinalis and putamen) in the left hemisphere. High [ 3H]MK-801 binding levels occurred in the area entorhinalis, frontal cortex, hippocampus and amygdala, medium levels were observed in the putamen and thalamus and low levels were found in the substantia nigra and nucleus dentatus. No evidence for laterality of [ 3H]MK-801 binding sites was observed in the 3 regions which were investigated on both sides of the brain.

(3H]MK-801 binding sites in postmortem brain regions of schizophrenic patients

[3H]MK-801 binding was used as a marker for the NMDA receptorion channel complex in postmortem brain samples from the frontal cortex, hippocampus, putamen, entorhinal region, and amygdala of schizophrenic patients and controls. In schizophrenia [3H]MK-801 binding levels were increased in all brain regions investigated reaching significance in the putamen.

3H]MK-801 binding sites in neonate rat brain

[ 3H]MK-801 binding sites are present in neonate rat brain as early as 3 days after birth. Immature hippocampus and cortex contain approximately one sixth the concentration of binding sites of the adult, while brainstem concentration is twice as high as that of adult. [ 3H]MK-801 binding is stimulated by glutamate and glycine and blocked by phencyclidine and Mg 2+ both in 7-day-old neonate and adult, indicating that as early as 7 days postnatally, the N-methyl- d-asparatate-type glutamate receptor and MK-801 binding site are functionally coupled.

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)

3H]MK-801 binding sites in post-mortem human frontal cortex

The binding of [ 3H]MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate) was investigated in extensively washed homogenates of post-mortem human frontal cortex. The association of [ 3H]MK-801 proceeded slowly ( t 1 2 = 553 min ) and reached equilibrium only after a prolonged incubation (>24 h). The dissociation of [ 3H]MK-801 from the binding site was also slow ( t 1 2 = 244 min ). Glutamate, glycine and magnesium markedly increased the rate of association ( t 1 2 = 14.8 min ) and dissociation ( t 1 2 = 36.5 min ). At equilibrium, the binding was not altered by these substances. Specific binding was linear with protein concentration, was saturable, reversible, stereoselective, heat-labile and was nearly absent in the white matter. Scatchard analysis of the saturation curves obtained at equilibrium indicated that there was a high-affinity (K d1 1.39 ± 0.21 nM, B max1 0.483 ± 0.084 pmol/mg protein) and a low-affinity (K d2 116.25 ± 50.79 nM, B max2 3.251 ± 0.991 pmol/mg protein) binding site. All competition curves obtained with (+)-MK-801, (−)-MK-801, phencyclidine and ketamine had Hill coefficients of less than unity and were best explained by a two-site model. Thus, our results demonstrate the presence of binding sites for MK-801 in post-mortem human brains and provide evidence for binding site heterogeneity. Furthermore, glutamate, glycine and magnesium accelerate the association and dissociation of [ 3H]MK-801 to and from its binding sites. The results add support to the hypothesis that MK-801, glutamate, glycine and magnesium all bind to different sites on the NMDA receptor-ion channel complex.

O-315 - Characterization of [3H]MK-801 binding sites on cell membranes from mammalian central nervous system

O-315 - Characterization of [3H]MK-801 binding sites on cell membranes from mammalian central nervous system

Effect of antemortem and postmortem factors on [ 3H]MK-801 binding in the human brain: Transient elevation during early childhood

The effect of a number of antemortem and postmortem factors on [ 3H]MK-801 binding was investigated under equilibrium conditions in the frontal cortex of human brains of 38 controls. Binding values transiently increased during the early postnatal period reaching a maximum at the age of about 2 years. After age 10 years [ 3H]MK-801 binding sites disappeared at 5.7% per decade. The storage time of brain tissue had a reducing effect on these binding sites. There was no effect of gender, brain weight or postmortem time interval and the binding sites were bilaterally symmetrically distributed in the frontal cortex.

3H]MK-801 binding in Alzheimer's disease

The density of [ 3H]MK-801 binding sites was studied in homogenates prepared from different cortical regions of postmortem brains of Alzheimer patients and age matched controls. Highest number of binding sites for this noncompetitive antagonist of the N- methyl- d-aspartate (NMDA) receptor was in temporal pole (Brodmann area A-38) in both groups. There were no consistent differences between patients and controls in either binding density or affinity constant in any of the areas studied. Involvement of glutamatergic neurotransmission in Alzheimer's disease does not seem to be at the level of the MK-801 recognition site on the NMDA receptor complex.

Inhibition of [formula omitted] evoked sodium flux by MK-801

The inhibition of N- methyl- d-aspartate (NMDA) stimulated 22Na + efflux from rat hippocampal slices was studied using competitive and non-competitive receptor antagonists. There was a good correlation between the abilities of the competitive antagonists to block NMDA evoked 22Na + efflux and their potencies as inhibitors of l-[ 3H]glutamate binding. The recently reported novel NMDA receptor antagonist, (+)-5-methyl-16,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-10-imine (MK-801) was shown to non-competitively inhibit NMDA stimulated 22Na + efflux with an IC 50 value of 0.4 μM. Relatively high (10 μM) concentrations of MK-801 had no effects on quisqualic acid, α-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), or kainic acid stimulated efflux. However, MK-801 was able to block 22Na + efflux induced by ibotenic acid and l-homocysteic acid, amino acids that act as NMDA receptor agonists. MK-801, (−)-MK-801, and non-competitive NMDA receptor antagonists of the arylcyclohexylamine and dioxolane classes inhibited NMDA stimulated 22Na + efflux with potencies that reflected their abilities to compete for [ 3H]MK-801 binding sites in rat cortical membranes. These results indicate the utility of the 22Na + efflux assay in studying the properties of NMDA receptors and confirm the nature and selectivity of the inhibition of NMDA receptor linked ion channel activation by MK-801.

3H-labeled MK-801 binding to the excitatory amino acid receptor complex from rat brain is enhanced by glycine.

We have studied the binding of the excitatory amino acid antagonist 3H-labeled MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to extensively washed rat brain membranes. Binding of 3H-labeled MK-801 was inhibited by phencyclidine, Mg2+, and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid in a manner dependent on the presence of L-glutamate or N-methyl-D-aspartate, suggesting that it labeled a site linked to the N-methyl-D-aspartate subtype of the glutamate receptor. Glycine also regulated 3H-labeled MK-801 binding, enhancing it in the concentration range of 0.01-10 microM. The actions of glutamate and glycine involved increases in binding affinity, without altering the number of 3H-labeled MK-801 binding sites. The effects of glycine in this system were mimicked by L- and D-alanine and L- and D-serine. However, beta-alanine and taurine were much less effective, and strychnine did not block the actions of glycine indicating that they were not mediated by the classical glycine receptors. Glycine also enhanced the ability of N-methyl-D-aspartate to increase Ca2+ influx into primary cultures of mouse striatal neurons measured using the Ca2+-sensitive fluorescent dye fura-2. These results support the suggestion that glycine may be an important regulator of the physiological actions of glutamate in vivo.

The anticonvulsant MK-801 is a potent N-methyl-D-aspartate antagonist.

The compound MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate)] is a potent anticonvulsant that is active after oral administration and whose mechanism of action is unknown. We have detected high-affinity (Kd = 37.2 +/- 2.7 nM) binding sites for [3H]MK-801 in rat brain membranes. These sites are heat-labile, stereoselective, and regionally specific, with the hippocampus showing the highest density of sites, followed by cerebral cortex, corpus striatum, and medulla-pons. There was no detectable binding in the cerebellum. MK-801 binding sites exhibited a novel pharmacological profile, since none of the major neurotransmitter candidates were active at these sites. The only compounds that were able to compete for [3H]MK-801 binding sites were substances known to block the responses of excitatory amino acids mediated by the N-methyl-D-aspartate (N-Me-D-Asp) receptor subtype. These comprised the dissociative anesthetics phencyclidine and ketamine and the sigma-type opioid N-allylnormetazocine (SKF 10,047). Neurophysiological studies in vitro, using a rat cortical-slice preparation, demonstrated a potent, selective, and noncompetitive antagonistic action of MK-801 on depolarizing responses to N-Me-D-Asp but not to kainate or quisqualate. The potencies of phencyclidine, ketamine, SKF 10,047, and the enantiomers of MK-801 as N-Me-D-Asp antagonists correlated closely (r = 0.99) with their potencies as inhibitors of [3H]MK-801 binding. This suggests that the MK-801 binding sites are associated with N-Me-D-Asp receptors and provides an explanation for the mechanism of action of MK-801 as an anticonvulsant.