Glutamate Binding Sites Research Articles

Metabotropic glutamate receptors are differentially regulated during development

The postnatal expression of metabotropic glutamate receptors was studied in rat brain by in situ hybridization and autoradiographic binding techniques. The messenger RNAs encoding five metabotropic glutamate receptor subtypes named mGluR1–5 had distinct regional and temporal expression profiles. mGluR1, mGluR2 and mGluR4 messenger RNA expression was low at birth and increased during postnatal development. In contrast, mGluR3 and mGluR5 were highly expressed at birth and decreased during maturation to adult levels of expression. [ 3H]Glutamate binding competition studies in developing brain disclosed the presence of two types of binding sites with the pharmacological properties of metabotropic glutamate receptors, having high (metabotropic type-1 binding sites; K 1 = 8nM) and low affinity (metabotropic type-2 binding sites; K 1 = 50 μM) for quisqualic acid, as in adult rat brain. The densities of metabotropic binding sites changed during development in a complex, regionally specific fashion. Metabotropic type-1 binding sites were present at low levels at birth and gradually increased during the second postnatal week. In the striatum, globus pallidus and cerebellar granule layer, the increase in density of metabotropic type-1 binding sites was transient but persisted in the cerebellar molecular layer. In contrast, metabotropic type-2 binding sites were present at high densities in most regions in the first postnatal week and decreased during the second and third week, particularly in the thalamic reticular nucleus and globus pallidus. Only in the external cortex did both metabotropic type-1 and metabotropic type-2 binding sites increase during development. A striking correspondence between the temporal pattern of expression of specific metabotropic glutamate receptor transcripts and metabotropic binding sites was observed in the reticular nucleus of the thalamus (mGluR3; metabotropic type-2 binding sites) and cerebellum (mGluR1; metabotropic type-1 binding sites) suggesting early translation of these metabotropic glutamate receptor messenger RNAs into receptor proteins. In other regions the relationship between messenger RNA expression and binding sites was less direct: comparison between expression of metabotropic glutamate receptor messenger RNA and binding sites suggests both a pre- and postsynaptic location of some receptor subtypes. These data imply a functional role of mGluR3 and mGluR5 during synaptogenesis and maintenance of adult synapses and of mGluR1, mGluR2 and mGluR4 in mature synaptic transmission.

Quantitative autoradiographic distribution of glutamate receptors in the cervical segment of the spinal cord of the wobbler mouse

The reduction of glutamate content has been observed in the spinal cord of the wobbler mouse, a purported model of amyotrophic lateral sclerosis (ALS). To elucidate glutamate receptors in the wobbler spinal cord, we measured densities of N-methyl- d-aspartate (NMDA), kainate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and metabotropic glutamate (mGlu) binding sites using in vitro autoradiography. In wobbler mice, NMDA, kainate, and AMPA binding sites were increased in the dorsal horn and kainate binding sites were also increased in the intermediate zone. However, mGlu binding was unchanged. These results disagree with those observed in ALS spinal cords, in which NMDA and kinate binding sites are decreased. The wobbler mouse may have the glutamate dysfunction, but in a different way from ALS.

Hydrodynamic and pharmacological characterization of putative alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate-sensitive L-glutamate receptors solubilized from pig brain.

L-[3H]Glutamate binding sites with characteristics resembling that of membrane-bound alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate-subtype L-glutamate receptors have been solubilized from pig brain synaptic junctions by Triton X-114. Binding of [3H]AMPA to these soluble sites in the presence of KSCN results in a curvilinear Scatchard plot that can be resolved into a high-affinity component and a low-affinity component. These Triton-X-114-solubilized sites can be further separated into two species of binding sites by gel-filtration chromatography or sucrose-density-gradient centrifugation. The pharmacological profiles of these two species of binding site are almost identical, and the rank orders of potency for glutamatergic drugs in displacing L-[3H]glutamate binding to these sites are quisqualate > 6,7-dinitroquinoxaline-2,3-dione > 6-cyano-7-nitroquinoxaline-2,3-dione > AMPA > L-glutamate > kainate >> N-methyl-D-aspartate = L-2-amino-4-phosphonobutyrate. Both sites are found to bind [3H]AMPA, and in the presence of KSCN the binding activities are significantly enhanced. Analysis of the hydrodynamic behaviour of these binding sites by sucrose-density-gradient centrifugation in H2O- and 2H2O-based solvents and gel-filtration chromatography has revealed that one of these sites (Stokes radius 8.3 nm, sedimentation coefficient 18.5 S) consists of 562 kDa protein and 281 kDa detergent, and the other site (Stokes radius 9.6 nm, sedimentation coefficient 13.4 S) consists of 352 kDa protein and 569 kDa detergent. Frictional coefficients of these sites indicate that these receptor-detergent complexes are asymmetrical in structure, consistent with large transmembrane proteins.

Glutamate receptor binding in juvenile and adult Rana pipiens CNS.

Autoradiographic methods were used to map NMDA- and quisqualate-sensitive glutamate binding sites in the brain of mature and juvenile Rana pipiens frogs. NMDA- and quisqualate-sensitive sites were consistently co-localized in the CNS. The highest glutamate binding occurred in the telencephalon, hypothalamus, and cerebellum. Glutamate binding sites were also specifically localized in visual pathways, including the superficial neuropil of the optic tectum, consistent with glutamate being the retinal ganglion cell neurotransmitter. The distribution of glutamate binding sites in the brain of juvenile postmetamorphic frogs was similar to that in adults. In general, Quis binding increased about twofold in adults compared to juveniles, whereas NMDA binding did not show a comparable developmental increase. To test whether glutamate binding sites are located on retinal axon terminals or on tectal cell dendrites in the optic tectum, juvenile postmetamorphic frogs were enucleated unilaterally, and receptor binding was performed following 1, 3, 7, and 14 days survival. The denervated tectal neuropil showed a delayed decrease in NMDA- and quisqualate-sensitive binding, consistent with the receptors being located on postsynaptic tectal cell dendrites.

Glutamate receptors in striatum and substantia nigra: Effects of medial forebrain bundle lesions

We examined NMDA-sensitive [ 3H]glutamate, [ 3H]AMPA, [ 3H]kainate and metabotropic-sensitive [ 3H]glutamate binding sites in neostriatum and substantia nigra pars reticulata (SNr) in rats after unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. One week after the lesion, NMDA, AMPA, kainate and metabotropic receptors were decreased in the ipsilateral neostriatum, whereas at three months NMDA receptors were increased while AMPA, kainate and metabotropic receptors were not changed. In the SNr at one week, only AMPA and metabotropic receptors were significantly decreased whereas three months after the lesion NMDA, AMPA and kainate binding sites were decreased. The early decrease of excitatory amino acid receptors in the striatum is likely to reflect degeneration of dopaminergic fibers, suggesting that specific subpopulations of excitatory amino acid binding sites are located on dopaminergic terminals.

Effect of chemical modification of arginyl and histidyl residues on [ 3H]MK-801 binding to brain synaptic membranes

Chemical modification of the N- methyl- d-aspartate (NMDA) receptor was investigated in a thoroughly washed Triton-treated pig forebrain membrane preparation. Modification of arginyl residues using phenylglyoxal significantly reduced activation of the NMDA receptor as measured by specific binding [ 3H]MK-801 [(+)-5-methyl-10,11-dihydro-dibenzo[ a, d]cyclohepten-5,10-imine]. The reduction was due to a decrease in the affinity of MK-801 for its binding site from 3.2 ± 0.5 nM to 22 ± 8 nM. Protection studies indicated that alteration of the glutamate or glycine binding sites is not directly responsible for the effect. Diethyl pyrocarbonate treatment also reduced [ 3H]MK-801 binding, by modification of histidyl residues. The binding affinity was reduced to 8.3 ± 1.4 nM whereas the B max was unchanged. Protection studies indicated that the modified histidine is unlikely to be a component of the glutamate, glycine or MK-801 binding sites. However, the accessibility of the modified histidine seems to be partly dependent on the activation state of the receptor.

Pharmacological characterization of glutamate binding sites in cultured cerebellar granule cells and cortical astrocytes.

Membranes prepared from cerebellar granule cells and cortical astrocytes exhibited specific, saturable binding of L-[3H]glutamate. The apparent binding constant KD was 135 nM and 393 nM and the maximal binding capacity Bmax 42 and 34 mumol/kg in granule cells and astrocytes, respectively. In granule cells the binding was strongly inhibited by the glutamate receptor agonists kainate, quisqualate, N-methyl-D-aspartate (NMDA), L-homocysteate and ibotenate, and the antagonist DL-5-aminophosphonovalerate. In astrocytes, only quisqualate among these was effective. L-Aspartate, L-cysteate, L-cysteinesulphinate and gamma-D-glutamylglycine were inhibitors in both cell types. The binding was totally displaced in both cell types by L-cysteinesulphinate with IC50 in the micromolar range. In astrocytes the binding was also totally displaced by quisqualate, but in granule cells only partially by NMDA, kainate and quisqualate in turn. It is concluded from the relative potencies of agonists and antagonists in [3H]glutamate binding that cerebellar granule cells express the NMDA, kainate and quisqualate types of the glutamate receptor, while only the quisqualate-sensitive binding seems to be present in cortical astrocytes.

Glutamine synthetase immunoreactivity in the human hippocampus is lamina-specific

Vibratome sections of immersion-fixed (4% paraformaldehyde) hippocampi from four autoptic cases without neuropathological findings were immunostained with a polyclonal anti-glutamine synthetase antibody. Immunoreactivity is restricted to astrocytes and has a layer-specific distribution: The highest levels are observed in the outer molecular layer of the fascia dentata, in the pyramidal cell layer and in the upper stratum oriens. The stratum radiatum displays particularly low immunoreaction. A conspicuous drop in immunoreactivity from the heavily stained outer molecular layer to its inner portion is marked by a clear-cut dividing line. The layer-specific staining pattern found corresponds to the laminated distribution of glutamate binding sites described by others.

A cluster analysis study of acetyl-l-carnitine effect on NMDA receptors in aging

Aging is associated with a reduction in the maximum density of n-methyl-d-aspartate (NMDA)-sensitive glutamate binding sites in the hippocampus of Fischer 344 rats. This study was designed to investigate the effect of acetyl-l-carnitine (ALCAR) on NMDA receptors in the old rat (24 months) after chronic or single-dose treatments. The number of NMDA receptors was significantly decreased in the old rat hippocampus by 19.5% compared with the young rat. A six-month treatment with ALCAR in the old rat attenuated the loss of NMDA binding sites in the hippocampus. A single-dose treatment with ALCAR in the old rat increased the B max value by 35%, while no change was observed in the young group. We conclude that ALCAR can exert two actions: a trophic/neuro-preserving one when chronically administered during aging, and a stimulatory one when given at a single dose in the aged rat.

Autoradiographic localization of [ 3H]-l-glutamate binding sites in a model of cerebellar granule cell ectopia generated by methylazoxymethanol treatment

The distribution of [ 3H]glutamate binding sites was studied in a model of altered cerebellar development obtained by injecting methylazoxymethanol (MAM) in 5-day-old mice. In these mice, at the 25th postnatal day, cerebella were smaller than normal, stratification was normal except for the presence in some lobes of a thin ectopic granule cell layer in the middle of the molecular layer, the proportion of the distribution of [ 3H]glutamate binding sites between molecular and internal granule cell layers was maintained but site density of both quisqualate- and NMDA-sensitive types was increased in the two layers. In the molecular layer, this increase was uniform in spite of the presence of the ectopic cell layer. In the internal granular layer, the increase of quisqualate-sensitive and NMDA-sensitive [ 3H]glutamate binding sites is topographically segregated and the first corresponds to areas of lesser cellular density. These results show that MAM treatment induces persistent alterations of the cerebellar glutamatergic system, which consist of receptor over-expression, possibly due to deficit of innervation, reactive gliosis and immaturity of surviving granule cells.

Characterization of the calcium and chloride [ 3H]glutamate binding site in crude synaptic membranes from human brain tissue

1. The specific binding properties of [ 3H]glutamate to crude synaptic membranes (CSM) from postmortem human brain were studied. 2. Equilibrium binding analysis of [ 3H]glutamate binding to CSM from human brain cortex revealed a K D = 110 ± 12 nM and a B max = 27 ± 4 pmol/mg protein). 3. Calcium increased the number of binding sites, B max = 44 ± 6 pmol/mg protein, without a significant change in the affinity constant, K D = 95 ± 10 nM. 4. The dissociation constant of the [ 3H]glutamate bound to human CSM was 4.0 ± 0.4 min −1 ( n = 3). 5. The relative potencies of glutamate analogs and 2-amino-4-phosphonobutyric acid (APB) to compete for the glutamate binding sites, in human CSM, were glutamate > quisqualate = ibotenic acid > APB ⪢ α-amino-3-hydroxy-5-methyl-4-isoxozolepropionate acid. 6. The glutamate specific binding in CSM from postmortem human brain was particularly rich in the gyrus hippocampus, nucleus accumbens, thalamus and frontal cortex. 7. This glutamate binding protein is related, probably, to a presynaptic neurosecretory pathway.

Cloning and sequence analysis of cDNAs encoding human hippocampus [formula omitted] receptor subunits: evidence for alternative RNA splicing

Several cDNA clones encoding human N- methyl- d-aspartate receptor (hNRl) subunit polypeptides were isolated from a human hippocampus library. Degenerate oligodeoxyribonucleotide (oligo) primers based on the published rat NR1 (rNR1) amino acid (aa) sequence [K. Moriyoshi et al. Nature 354 (1991) 31–37] amplified a 0.7-kb fragment from a human hippocampus cDNA library, via the polymerase chain reaction (PCR). This fragment was used as a probe for subsequent hybridization screening. DNA sequence analysis of 28 plaque-purified clones indicated three distinct classes, designated hNR1-1, hNR1-2 and hNR1-3, presumably generated by alternative RNA splicing. One of these clones, hNR1-1(5A), was isolated as a full-length cDNA. The hNR1-2 and hNR1-3 cDNAs represented 66.8 and 98.9%, respectively, of the total aa coding information predicted for the polypeptides. The hNR1 cDNAs demonstrated an 84–90.8% nucleotide (nt) identity with the corresponding rodent cDNAs. The nt sequences of hNR1-1, hNR1-2 and hNR1-3 would encode 885-, 901- and 938-aa proteins, respectively, that have 99.1–99.8% identity with the corresponding rodent NR1 (roNRl) subunits. The changes between the predicted aa sequences of hNRl and the corresponding roNRl subunits are confined to the extracellular N-terminal regions. We have also identified two possible allelic variations of the hNR1-3 cDNA that result in aa substitutions in the extracellular N- and C-terminal regions. One of these naturally occurring aa variations is situated within a potential glutamate-binding site.

Development of dopamine and [formula omitted] systems in rat brain: the effect of prenatal phencyclidine exposure

Phencyclidine (PCP) inhibits the uptake of the neurotransmitter dopamine (DA), and blocks N- methyl- d-aspartate (NMDA) receptor-regulated ion channels. PCP also binds to sigma receptors in vivo and in vitro in rat brain. Prolonged exposure to PCP in adults has been observed to reduce the number of PCP binding sites in brain. We designed these experiments to evaluate whether prolonged prenatal exposure to PCP produces alterations in the development of DA and NMDA systems in brain. To do so, we characterized the normal course of development of basal and stimulated DA release in striatal slices, the ontogeny of striatal DA concentrations, and the development of NMDA receptor channels and associated glutamate binding sites in frontal cortex. We compared these developmental profiles to those in rats exposed to prenatal PCP, in an attempt to characterize the effect of prenatal PCP exposure on the pattern of brain development. Pregnant CD rats were injected s.c. with either 0, 10 or 20 mg/kg PCP daily on gestational days 8 through 20. On postnatal days (PND) 8, 21, 45, or 100, rats were sacrificed and brain tissues isolated for in vitro assessment. In vitro [ 3H]DA release from striatal slices evoked by either 40 μM glutamate or 15 mM K + increased over 250% from PND 8 to PND 45, and glutamate-stimulated release was still significantly below adult levels at PND 45. In contrast, d-methamphetamine ( d-METH)-evoked [ 3H]DA release, frontal cortical glutamate binding sites and NMDA channels developed early, reaching adult levels on or before PND 21. At PND 8 but not thereafter prenatal PCP (10 or 20 mg/kg) appeared to increase glutamate-evoked [ 3H]DA release but not K +- and METH-evoked release in striatal slices. There were no significant effects of prenatal PCP exposure on the in vivo levels of DA or serotonin or their metabolites in striatum at any PND. PCP exposure also had no effect on the NMDA receptor ion channel or the glutamate binding site in frontal cortex at any age. These data indicate that prenatal exposure to PCP may selectively but transiently and moderately change glutamate evoked DA release early in development without affecting the in vivo levels of DA and serotonin or NMDA receptor complex levels in developing brain.

Glutamate Dehydrogenase Deficiency in Cerebellar Degenerations: Clinical, Biochemical and Molecular Genetic Aspects

Glutamate dehydrogenase (GDH), an enzyme central to glutamate metabolism, is significantly reduced in patients with heterogenous neurological disorders characterized by multiple system atrophy (MSA) and predominant involvement of the cerebellum and its connections. In human brain, GDH exists in multiple isoforms differing in their isoelectric point and molecular mass. These are differentially reduced in quantity and altered in catalytic activity in patients with clinically distinct forms of MSA, thus suggesting that these GDH isoproteins are under different genetic control. Dysregulation of glutamate metabolism occurs in patients with GDH deficiency and is thought to mediate the disease's neurodegeneration via neuroexcitotoxic mechanisms. This possibility is supported by additional data showing that glutamate binding sites are significantly decreased in cerebellar tissue obtained at autopsy from MSA patients. At the molecular biological level, several cDNAs specific for human GDH have been isolated recently and cloned. Northern blot analysis of various human tissues, including brain, has revealed the presence of multiple GDH-specific mRNAs. In addition, multiple GDH-specific genes are present in humans and these data are consistent with the possibility that the various GDH isoproteins are encoded by different genes. These advances have laid the groundwork for characterizing the human GDH genes and their products in health and disease.

Interactions between the glycine and glutamate binding sites of the NMDA receptor

The interactions between the glycine and glutamate binding sites of the NMDA receptor have been studied in outside-out patches and synapses from hippocampal neurons in culture using rapid drug application techniques. Desensitization of NMDA receptor-mediated currents elicited by glutamate in newly excised outside-out patches was reduced in the presence of saturating concentrations of glycine. This suggests that the glutamate and glycine binding sites of the NMDA receptor are allosterically coupled as has been reported in whole-cell preparations. A glycine-insensitive form of desensitization increased rapidly over the first few minutes of recording and largely occluded the glycine concentration-sensitive desensitization in outside-out patches. However, even in old patches that displayed no glycine-sensitive desensitization, the unbinding rate of glycine was increased fourfold by the presence of glutamate, suggesting that the two binding sites were still allosterically coupled. These data suggest the existence of two forms of NMDA receptor desensitization in outside-out patches, only one of which is dependent on the concentration of glycine. In the presence of saturating levels of glycine, activation of NMDA receptors by synaptic stimulation or by exogenous glutamate resulted in currents that relaxed biexponentially. Addition of the partial glycine-site agonist 1-hydroxy-3-aminopyrrolid-2-one (HA-966) increased the rate of decay of both synaptic and patch currents. This suggests that HA-966 increases the dissociation rate of glutamate from NMDA receptors. These results support the hypothesis that the glutamate and glycine binding sites of NMDA receptors interact allosterically; ligand binding at both types of sites can affect the affinity of the other type for its agonist.

Quisqualate resolves two distinct metabotropic [3H]glutamate binding sites.

Metabotropic receptor subtypes have been proposed based on pharmacological, signal transduction and cDNA sequence data. We assessed potential metabotropic binding site subtypes with in vitro quantitative [3H]glutamate autoradiography in adult rat brains in the presence of saturating concentrations of N-methyl-D-aspartate (NMDA) and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionate (AMPA). Quisqualate (QUIS) competition curves resolved two differentially distributed binding sites (KIhigh = 17 nM; KIlow = 62 microM). Trans-1-amino-cyclopentane- 1,3-dicarboxylic acid (t-ACPD) and 1S,3R-ACPD displaced [3H]glutamate binding both in the absence and presence of a quisqualate concentration (2.5 microM) that saturates the high affinity sites, suggesting that both sites are linked to metabotropic receptors. We conclude that two metabotropic binding sites with different distributions and pharmacological profiles can be detected with selective [3H]glutamate binding assays.

Enflurane inhibits NMDA, AMPA, and kainate‐induced currents in Xenopus oocytes expressing mouse and human brain mRNA

Effects of enflurane, an inhalational anesthetic, on NMDA, AMPA, and kainate-gated currents were examined in Xenopus laevis oocytes expressing mouse or human brain mRNA. In oocytes expressing mouse mRNA, enflurane at an anesthetic concentration (1.8 mM) inhibited the NMDA-, AMPA-, and kainate-induced currents by 29-40%, 30-33%, and 20-27%, respectively, suggesting that all three glutamate ionotropic receptors are susceptible to suppression by inhalational anesthetics. Furthermore, inhibition by enflurane was independent of the concentrations of the agonists (NMDA, AMPA, and kainate) or the NMDA-coagonist (glycine). This suggests that enflurane inhibition does not result from a competitive interaction at glutamate or glycine binding sites. Enflurane also suppressed the oscillation and apparent desensitization of NMDA currents, suggesting an inhibition of Ca2+ influx through the NMDA channel. In oocytes expressing human brain mRNA, only kainate produced observable currents. Kainate currents of human channels were smaller in size than those of the mouse; however, the kainate concentration-response curve and percent inhibition (27-29%) by enflurane were similar for mice and humans. The results suggest that human and mouse kainate receptors have similar pharmacological characteristics.

Characterization of a [3H]glutamate binding site in rat pineal gland: Enhanced affinity following superior cervical ganglionectomy

Glutamate, an excitatory neurotransmitter/neuromodulator involved in cell-to-cell communication within the central nervous system, is now believed to play a role in neuroendocrine function. In this study we describe a single, saturable, stereospecific, and temperature-, time-, and pH-dependent binding site for glutamate in the pineal gland of the rat (Kd = 612 +/- 23 nM, Bmax = 3.17 +/- 0.33 pmol/mg protein). After removal of the sympathetic innervation to the pineal gland, [3H]glutamate binding displayed a higher apparent affinity (Kd = 412 +/- 28 nM) (P < 0.05) without a change in binding site number (Bmax = 3.60 +/- 0.24 pmol/mg protein). No difference in [3H]glutamate binding site number was observed in pineal glands obtained from animals sacrificed during the middle of the light and dark periods. These data suggest a possible modulatory role for a glutamate binding site in pineal gland function.

Phospholipase A2 modulates different subtypes of excitatory amino acid receptors: autoradiographic evidence.

Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A2 (PLA2) on N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA2 pretreatment induced a significant increase in alpha-[3H]amino-3-hydroxy-5-methyl-isoxazole-4-propionate ([3H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [3H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [3H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [3H]glutamate binding site were also increased by PLA2 pretreatment. [3H]Kainate binding and NMDA-sensitive [3H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA2 effect was reversed by EGTA, the PLA2 inhibitor p-bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA2. Arachidonic acid failed to mimic the PLA2 effect on metabotropic binding. These results indicate that PLA2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.

Evidence that [ 3H]glutamate binding sites are masked by biologically relevant endogenous factor on cell membranes of frog spinal cord

We identified the possible endogenous factor effective to modulate the binding of [ 3H]-labeled excitatory amino acid agonists and antagonists in the 100,000 × g supernatant of Triton X-100 (0.01%)-treated cell membranes from frog spinal cords. The factor inhibited the binding of [ 3H]glutamate to Triton X-100-treated cell membranes, to which the binding capacity of [ 3H]glutamate increased much more than that to intact cell membranes. The binding capacities of [ 3H]AMPA (an AMPA type agonist) and [ 3H]CPP (an NMDA type antagonist) to cell membranes remained low by Triton treatment, but they were enhanced significantly by the addition of the factor. The effect of the factor on the [ 3H]kainate binding was hardly observable. The factor may provide key information on receptor structures and the classification of receptor types concerning excitatory amino acids in the mammalian central nervous system.