Glutamate Binding Sites Research Articles

3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3H]CGP 61594 labeled with high affinity ( K D=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors.

3H- l-Glutamate Binding and 3H- d-Aspartate Release From Hippocampal Tissue During the Development of Pentylenetetrazole Kindling in Rats

SCHRÖEDER, H., A. BECKER, U. SCHRÖEDER AND V. HOELLT. 3H- l-Glutamate binding and K +-stimulated 3H- d-aspartate release from hippocampal tissue during the development of pentylenetetrazol-induced kindling in rats . PHARMACOL BIOCHEM BEHAV 62(2) 349–352, 1999.—Previous studies have proposed that there is an increase in the density of glutamate binding sites after pentylenetetrazol (PTZ) kindling, whereas the glutamate release is not altered. Little is known about the time course of these changes. Therefore, we studied 3H- l-glutamate binding to hippocampal membranes and K +-stimulated 3H- d-aspartate release from hippocampal slices of rats given PTZ 3, 7, and 13 times up to a fully kindling state. After three PTZ injections, amino acid release from hippocampal tissue slices was significantly enhanced in comparison to controls, whereas 3H- l-glutamate binding was not altered. After seven injections of PTZ, specific glutamate binding to hippocampal membranes tended to increase, and K +-stimulated 3H- d-aspartate release from rat hippocampal slices was normalized. The kindled state characterized by generalized clonic–tonic seizures was reached after 13 PTZ injections, and it was accompanied by an enhancement in the density of glutamate binding sites, whereas the chemically evoked amino acid release remained unchanged. It can be concluded that the amino acid release is increased in the early phase of PTZ kindling development, whereas after completion of kindling, the density of excitatory amino acid binding sites is enhanced.

3H]CNQX and NMDA-sensitive [3H]glutamate binding sites and AMPA receptor subunit RNA transcripts in the striatum of normal and weaver mutant mice and effects of ventral mesencephalic grafts.

Levels of excitatory amino acid receptors were studied in the weaver mouse model of DA deficiency after unilateral intrastriatal transplantation of E12+/+ mesencephalic cell suspensions. Graft integration was verified by turning behavior tests and from the topographical levels of the DA transporter, tagged autoradiographically with 3 nM [3H]GBR 12935 (average increase in grafted dorsal striatum compared to nongrafted side, 60%). Autoradiography of 80 nM [3H]CNQX and 100 nM NMDA-sensitive [3H]glutamate binding was carried out to visualize the topography of non-NMDA and NMDA receptors, respectively, in +/+ mice and in recipient weaver mutants 3 months after grafting. Increases of 30% or more were found for [3H]CNQX binding in the dorsal nongrafted weaver striatum compared to +/+, and a further 6-9% increase in grafted weaver compared to nongrafted side. The added increase of non-NMDA receptors in the transplanted striatum might be explained by a presence of such receptors on DA presynaptic endings of graft origin. A 20% increase in NMDA-sensitive [3H]glutamate binding was measured in the dorsal nongrafted weaver striatum compared to +/+. NMDA-sensitive [3H]glutamate binding in the transplanted side of weaver mutants tended to be slightly higher in all areas of the striatal complex compared to the nongrafted side, without reaching conventional levels of statistical significance. Using in situ hybridization histochemistry with synthetic 32p labeled oligonucleotide probes, we investigated RNA transcripts encoding the four AMPA receptor subunits. RNA transcripts in the striatum are seen with a decreasing signal intensity in the following order: GluRB > GluRA > GluRC > GluRD. The weaver caudate-putamen shows a 12% increase in GluRA subunit mRNA compared to +/+, whereas mesencephalic neuron transplantation leads to slight increases (3%) in the levels of GluRB mRNA in the nucleus accumbens. The results are placed in the context of the important interaction between the converging glutamatergic corticostriatal and the DAergic nigrostriatal pathways in controlling the functional output of the basal ganglia in Parkinson's disease and in experimental models of DA deficiency.

The effect of pentylenetetrazol kindling on synaptic mechanisms of interacting glutamatergic and opioid system in the hippocampus of rats

Endogenous opioids modulate processes of central excitability such as long-term potentiation and electrical kindling. Little is known about the neurochemical alterations in the interaction of the glutamatergic and opioid system in the development of pentylenetetrazol (PTZ) kindling in rats. Therefore, in the present study we investigated glutamate, DAMGO and naltrindole receptor binding, receptor protein expression by Western blot and ex vivo glutamate transmitter release in PTZ kindled rats. The specific 3 H -DAMGO and -naltrindole binding to hippocampal membranes displayed no significant changes in kindled rats compared to controls. In contrast, the 3 H - l-glutamate binding was significantly enhanced after completion of PTZ kindling. The expression of receptor protein for glutamate as well as the naloxone- and naltrindole-induced 3 H - d-aspartate release from hippocampal slices did not alter in any case as a consequence of PTZ kindling. The PTZ induced enhancement of the glutamate binding sites in the hippocampus was downregulated to control level by natrindole treatment of rats prior to each PTZ application. Furthermore, naltrindole pretreatment of rats significantly inhibited the development of seizure susceptibility. In contrast, naloxone was not able to alter the seizure activity induced by PTZ as well as the transmitter receptor binding. The results are discussed in the light of a modulating role of δ-opioid receptors in PTZ kindling.

A Point Mutation in the Glutamate Binding Site Blocks Desensitization of AMPA Receptors

Desensitization of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is thought to shape the synaptic response and act as a neuroprotective mechanism at central synapses, but the molecular mechanism underlying desensitization is poorly understood. We found that replacing the glutamate binding domain S1 of GluR3 (an AMPA receptor) with S1 of GluR6 (a kainate receptor) resulted in a fully active but completely nondesensitizing receptor. Smaller substitutions within S1 identified, besides two additional modulatory regions, a single exchange, L507Y, as is required and sufficient for the block of desensitization. This phenotype was specific for AMPA receptors and required an aromatic residue at this position. L507 lies between two residues (T504 and R509) that form part of the glutamate binding site. The physical proximity of these residues, which are involved in binding and gating, suggests they may form part of the link between these two events.

Molecular and pharmacological characterization of recombinant rat/mice N-methyl-D-aspartate receptor subtypes in the yeast Saccharomyces cerevisiae.

The genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMBO1 and pMB02. The protease-deficient yeast Saccharomyces cerevisiae c13-ABYS-86 (leu-, ura-, his-) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/ pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes. Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains. By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1-3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88+/-5.38 nM (NR1a), 1365.11+/-76 nM (NR1a/NR2D), 22.97+/-3.37 nM for NR1a/NR2B/NR2D and 7.4+/-1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07+/-0.92 (NRla), 14.63+/-0.50 (NR1a/NR2D), 12.85+/-1.68 (NR1a/NR2B/NR2D) and 8.3+/-0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536+/-43, 688+/-60, and 856+/-48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.

Substituted quinolines as inhibitors of l-glutamate transport into synaptic vesicles

This study investigated the structure–activity relationships and kinetic properties of a library of kynurenate analogues as inhibitors of 3H- l-glutamate transport into rat forebrain synaptic vesicles. The lack of inhibitory activity observed with the majority of the monocyclic pyridine derivatives suggested that the second aromatic ring of the quinoline-based compounds played a significant role in binding to the transporter. A total of two kynurenate derivatives, xanthurenate and 7-chloro-kynurenate, differing only in the carbocyclic ring substituents, were identified as potent competitive inhibitors, exhibiting K i values of 0.19 and 0.59 mM, respectively. The K m value for l-glutamate was found to be 2.46 mM. Parallel experiments demonstrated that while none of the kynurenate analogues tested effectively inhibited the synaptosomal transport of 3H- d-aspartate, some cross-reactivity was observed with the EAA ionotropic receptors. Molecular modeling studies were carried out with the identified inhibitors and glutamate in an attempt to preliminarily define the pharmacophore of the vesicular transporter. It is hypothesized that the ability of the kynurenate analogues to bind to the transporter may be tied to the capacity of the quinoline carbocyclic ring to mimic the negative charge of the γ-carboxylate of glutamate. A total of two low energy solution conformers of glutamate were identified that exhibited marked functional group overlap with the most potent inhibitor, xanthurenate. These results help to further refine the pharmacological specificity of the glutamate binding site on the vesicular transporter and identify a series of inhibitors with which to investigate transporter function.

Spinal and supraspinal antinociceptive action of dipyrone in formalin, capsaicin and glutamate tests. Study of the mechanism of action

Dipyrone injected intraperitoneally (i.p.) or subplantarly into the mouse paw caused dose-related antinociception against the early and the late phases of formalin-induced licking, with mean ID 50 values of 154.5 and 263.7 μmol/kg, and 2.6 and 1.2 μmol/paw, respectively. Given either by intracerebroventricular (i.c.v.) or by intrathecal (i.t.) routes, dipyrone produced a similar inhibition of both phases of the formalin-induced licking, with mean ID 50 values of 0.4 and 1.3 μmol/site, and 0.4 and 0.9 μmol/site against the early and the late phase of the formalin response, respectively. Dipyrone, given by i.p., subplantar, i.t. or i.c.v. routes, caused dose-related antinociception of capsaicin-induced licking. The mean ID 50 values were: 207.6 μmol/kg, 2.2 μmol/paw, 0.4 μmol/site and 0.14 μmol/site, respectively. In addition, dipyrone given i.p. caused a significant increase of the latency both in the hot-plate and the tail-flick assays. Dipyrone, given i.p., i.t. or i.c.v., reversed significantly the hyperalgesia caused by i.t. injection of glutamate, with mean ID 50 values of 9 μmol/kg, 29 nmol/site and 94 nmol/site, respectively. The antinociception caused by dipyrone was not influenced by naloxone, l-arginine, phaclofen, glibenclamide, p-chlorophenylalanine methyl ester, pertussis toxin or by adrenal gland hormones, when assessed against the formalin assay. Dipyrone analgesic action was not secondary to its anti-inflammatory effect, nor was it associated with non-specific effects such as muscle relaxation or sedation actions of animals. Dipyrone at a higher concentration caused significant inhibition of [ 3H]glutamate binding (37%) in cerebral cortical membranes from both mice and rats. However, dipyrone had no significant effect on brain constitutive neuronal nitric oxide synthase activity. It is concluded that dipyrone produces peripheral, spinal and supraspinal antinociception when assessed on formalin and capsaicin-induced pain as well as in glutamate-induced hyperalgesia in mice. Dipyrone antinociception seems unlikely to involve an interaction with the l-arginine-nitric oxide pathway, serotonin system, activation of G i protein sensitive to pertussis toxin, interaction of ATP-sensitive K + channels, GABA B receptors, or the release of endogenous glucocorticoids. However, a modulatory effect on glutamate-induced hyperalgesia and, to a lesser extent, an interaction with glutamate binding sites, seems to account for its analgesic action.

Identification of Amino Acid Residues of the NR2A Subunit That Control Glutamate Potency in Recombinant NR1/NR2A NMDA Receptors

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).

Chapter 12 Molecular determinants of NMDA receptor pharmacological diversity

Publisher Summary This chapter describes the molecular basis of the known pharmacologically distinct N-methyl-D-aspartate (NMDA) receptor subtypes. The potent excitotoxic effect of NMDA receptor overactivation has made this receptor a prime suspect in various neurodegenerative diseases such as Alzheimer's, Parkinson's, and AIDS dementia. Two general approaches have been taken to develop agents with a higher therapeutic index—using compounds that act at different regulatory domains on the NMDA receptor or identifying compounds that act at subtypes of NMDA receptors. NMDA receptors display a rich diversity of sites at which pharmacological agents can modify activity. In addition to the glutamate and glycine agonist binding sites, there are sites for channel blockers, polyamines, redox reagents, ifenprodil, protons, steroids, Zn 2+ , Mg 2+ , and histamine. Two families of NMDA receptor subunits have been cloned, together these consist of 12 different subunits. In specific nuclei of the midline thalamus, for example, the paraventricular and intermediodorsal thalamic nuclei and other diencephalic structures, NMDA receptors display a distinct pharmacological profile at both the glutamate recognition site and the channel blocker-binding site.

12] Design and synthesis of conformationally constrained inhibitors of high-affinity, sodium-dependent glutamate transporters

The value of conformationally constrained analogs rests with the hypothesis that restricting the number of attainable conformations of a flexible, acyclic molecule, such as L-glutamate, should limit the number of glutamate binding sites with which the analog can interact, thereby increasing its selectivity as a pharmacological probe. Pharmacophore models have become tools in the development of the next generation analogs that is used to refine the models and identify more selectively acting agents, such as those that can distinguish among closely related transporter subtypes. This chapter explains the calculation of low energy conformations of acidic amino acids. To compare the structures of the active transport inhibitors, computational libraries of the energetically accessible conformations of each must be generated. From a practical point of view, this task must be carried out using molecular mechanics rather than higher levels of theory.

The effects of age and dietary restriction on metabotropic glutamate receptors in C57B1 mice.

This study examined the effects of aging and dietary restriction on metabotropic type 1 (met1; high quisqualate affinity) and type 2 (met2; low quisqualate affinity) binding sites for glutamate and the relationship between these binding sites and spatial memory. Quantitative autoradiography was performed on 3-, 10-, and 26-month-old mice. Ten- and 26-month-old mice were fed ad libitum or fed restricted diets. Met1 binding sites were not notably affected by age or diet. Binding to met2 sites was maintained during aging in the ad libitum-fed mice, but there were decreases in binding in the diet-restricted mice as compared to 3-month-old mice. Binding to met1 and met2 sites did not correlate significantly with performance in the Morris water maze. These results suggest that dietary restriction induces a decrease in one or more of the functions associated with met2 binding sites during the aging process.

Selective metabotropic receptor agonists distinguish non-ionotropic glutamate binding sites

Metabotropic glutamate receptors (mGluRs) are thought to mediate diverse processes in brain including synaptic plasticity and excitotoxicity. These receptors are often divided into three groups by their pharmacological profiles. [ 3H]Glutamate binding in the presence of compounds selective for ionotropic glutamate receptors can be used as a general assay for these receptors; subtypes of this non-ionotropic [ 3H]glutamate binding differ in both pharmacology and anatomical distribution, and are differentially sensitive to quisqualate. The characteristics of these binding sites are consistent with those of group 1 (high-affinity quisqualate) and group 2 (low-affinity quisqualate) mGluRs. Under our assay conditions, no [ 3H]glutamate binding to group 3-like ( l-AP4 sensitive) sites could be demonstrated. We have attempted to characterize particular agents which may selectively measure [ 3H]glutamate binding to mGluR subtypes. We used two isomers of 2-(carboxycyclopropyl)glycine, l-CCG-I and l-CCG-II, and the (2 S,1′ R,2′ R,3′ R) isomer of 2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV) as competitors of non-ionotropic [ 3H]glutamate binding sites. DCG-IV clearly distinguishes two binding sites. Quantitative levels of DCG-IV binding by anatomic region correlate with quisqualate-defined binding subtypes: high-affinity DCG-IV binding correlates with low-affinity quisqualate binding, whereas low-affinity DCG-IV binding correlates with high-affinity quisqualate binding. l-CCG-II displaces only one type of non-ionotropic [ 3H]glutamate binding, corresponding to high-affinity quisqualate binding. Therefore DCG-IV and l-CCG-II at appropriate concentrations appear to distinguish binding to putative group 2 vs. group 1 mGluRs. l-CCG-I displaces both high- and low-affinity quisqualate binding sites, but unlike the other two compounds, does not clearly distinguish between them.

Effects of methylmalonate and propionate on [3H]glutamate binding, adenylate cyclase activity and lipid synthesis in rat cerebral cortex.

The effects of methylmalonic (MMA) and propionic acid (PPA), metabolites that accumulate in methylmalonic and propionic acidemia respectively, on [3H]glutamate binding, adenylate cyclase activity and [U-14C]acetate incorporation into lipids were investigated in rat cerebral cortex. Neither acid effected [3H]glutamate binding, regardless of the presence of sodium in the incubation medium. Also, the acids had no effect on basal or GMP-PNP-stimulated adenylate cyclase activity. These results suggest that MMA and PPA do not interact with glutamate binding sites and have no effect on basal or guanine nucleotide-stimulated adenylate cyclase activity. In contrast, [U-14C]acetate incorporation into brain lipids was significantly blocked by both acids, the effects being more pronounced with PPA, indicating an inhibition of brain lipid biosynthesis caused by MMA and PPA. These results may explain at least in part the hypomyelinization and/or demyelinization characteristic of patients affected by methylmalonic acidemia and propionic acidemia.

Discriminative stimulus effects of ethanol: Lack of antagonism with N-methyl- d-aspartate and d-cycloserine

Several drug discrimination studies reported that both competitive and uncompetitive NMDA receptor antagonist substituted for ethanol stimulus in rats. In the present study we examined if compounds that act as agonists at the NMDA receptor complex, d-cycloserine (a partial agonist at the glycine positive modulatory site) and N-methyl- d-aspartate (an agonist at the glutamate binding site), could antagonize the discriminative stimulus effects of ethanol. Rats were trained to discriminate between IP administered 1.0 g/kg of ethanol (10% v/v) and saline under a sweetened milk-reinforced fixed ratio 10 (FR10) schedule of reinforcement. When the animals met the discriminative criteria, antagonism tests were conducted with d-cycloserine (0.3–10.0 mg/kg, IP) and N-methyl- d-aspartate (15.0–60.0 mg/kg, IP). Neither d-cycloserine nor N-methyl- d-aspartate antagonized the ethanol-mediated discriminative stimulus effects. In addition, d-cycloserine (3.0–300.0 mg/kg, IP) did not substitute for ethanol. These results indicate that at least certain agonists at the NMDA receptor complex do not attenuate the ethanol interoceptive cue in the rat.

The Somatosensory Cortex of Human: Cytoarchitecture and Regional Distributions of Receptor-Binding Sites

The aim of this study is to characterize the regional and laminar distribution patterns of various neurotransmitter binding sites in areas 3a, 3b, 1, and 2 of the human primary somatosensory cortex, and to compare these receptor-based “maps” with the cytoarchitectonic parcelation. Cryostat sections from a dorsomedial region of the postcentral gyrus close to the interhemispheric fissure and from a ventrolateral region close to the Sylvian fissure were examined. Neurotransmitter-binding sites were analyzed with quantitativein vitroreceptor autoradiography. Different muscarinic-binding sites were labeled with [3H]pirenzepine and [3H]oxotremorine-M, noradrenergic-binding sites with [3H]prazosin, different serotoninergic-binding sites with [3H]5-hydroxytryptamine and [3H]ketanserine, glutamate-binding sites withl-[3H]glutamate, and GABA-binding sites with [3H]muscimol. Adjacent sections were stained with a modified Nissl method for cytoarchitectonic analysis. The binding sites either were preferentially localized in the superficial layers ([3H]5-hydroxytryptamine, [3H]prazosin,l-[3H]glutamate, [3H]muscimol, and [3H]pirenzepine) or were more homogeneously distributed with highest densities in layers III–V ([3H]oxotremorine-M and [3H]ketanserine). Changes in the distribution patterns of [3H]oxotremorine-M- and [3H]ketanserine-binding sites precisely matched the borders between areas 4/3a, 3b/1, and 1/2, as defined cytoarchitectonically. In addition, the autoradiographs showed that area 1 possibly consists of two subregions which cannot be distinguished cytoarchitectonically. The results demonstrate that the regional and laminar distribution patterns of some, but not all, transmitter-binding sites are precisely correlated with the cytoarchitectonic parcelation of the human primary somatosensory cortex. In addition, binding sites may reveal new borders not detectable in Nissl-stained sections. Finally, the human primary somatosensory cortex differs clearly from the primary motor cortex due to higher densities ofl-[3H]glutamate-, [3H]muscimol-, [3H]pirenzepine-, [3H]oxotremorine-M-, and [3H]ketanserine-binding sites.

Development of MK-801, kainate, AMPA, and muscimol binding sites and the effect of dark rearing in rat visual cortex.

We used quantitative autoradiography to determine whether the development of glutamate receptors correlates with the plastic period for monocular deprivation in rat visual cortex. To study glutamate receptors, we incubated sections of rat visual cortex with tritiated (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10imin e maleate (MK-801), tritiated kainate, and tritiated amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA). [3H]MK-801 is a noncompetitive ligand for the N-methyl-D-aspartate (NMDA) receptor. [3H]kainate and [3H]AMPA are competitive ligands for non-NMDA receptors. To compare glutamate binding sites with a nonglutamate binding site, we studied [3H]muscimol, which binds to gamma-aminobutyric acid (GABA)A receptors. [3H]MK-801 binding was maximal at postnatal day 26 (P26) and decreased in adulthood. [3H]AMPA binding was maximal at P18. [3H]kainate binding and [3H]muscimol binding were not age dependent. Dark rearing partially prevented the age-dependent decrease in [3H]MK-801 binding but had no effect on [3H]kainate or [3H]AMPA binding. Dark rearing decreased muscimol binding in adult animals. These results suggest that NMDA receptors, but not other glutamate receptors or GABAA receptors, are likely to be critical for developmental plasticity in rat visual cortex.

Neurotoxicity associated with dual actions of homocysteine at the N-methyl-D-aspartate receptor.

Severely elevated levels of total homocysteine (approximately millimolar) in the blood typify the childhood disease homocystinuria, whereas modest levels (tens of micromolar) are commonly found in adults who are at increased risk for vascular disease and stroke. Activation of the coagulation system and adverse effects of homocysteine on the endothelium and vessel wall are believed to underlie disease pathogenesis. Here we show that homocysteine acts as an agonist at the glutamate binding site of the N-methyl-D-aspartate receptor, but also as a partial antagonist of the glycine coagonist site. With physiological levels of glycine, neurotoxic concentrations of homocysteine are on the order of millimolar. However, under pathological conditions in which glycine levels in the nervous system are elevated, such as stroke and head trauma, homocysteine's neurotoxic (agonist) attributes at 10-100 microM levels outweigh its neuroprotective (antagonist) activity. Under these conditions neuronal damage derives from excessive Ca2+ influx and reactive oxygen generation. Accordingly, homocysteine neurotoxicity through overstimulation of N-methyl-D-aspartate receptors may contribute to the pathogenesis of both homocystinuria and modest hyperhomocysteinemia.

Modification of the Glutamate Binding Site of the NMDA Receptor During Graded Hypoxia in the Cerebral Cortex of Newborn Piglets. † 183

We have previously shown that during hypoxia the glutamate binding site of the N-methyl-D-aspartate (NMDA) receptor is altered in newborn piglet brain. The present study tests the hypothesis that hypoxia-induced alteration of the glutamate site of the NMDA receptor is progressive and correlates with the decrease in cerebral energy metabolism induced by hypoxia. Studies were performed in 8 ventilated piglets, 4 normoxic and 4 hypoxic. Varying degrees of hypoxia were achieved by exposure to decreased oxygen at different concentrations and confirmed by brain tissue ATP and phosphocreatine (PCr) levels. 3[H]glutamate binding was performed in P2 membrane fractions at concentrations of 25-1000 nM. Specific NMDA-displaceable3[H]-glutamate binding was determined with 100μM NMDA. Non-specific binding was determined with 1mM unlabelled glutamate. Bmax (receptor number) and Kd (dissociation constant) were calculated from Scatchard plots. ATP(μmol/g brain), PCr (μmol/g brain) and Bmax (fmol/mg protein) were as follows: (5.86, 2.77, 402), (4.77, 2.96, 388), (4.50, 2.05, 364), (4.03, 2.01, 332), (3.37, 0.96, 276), (2.49, 0.24, 253), (1.96, 0.77, 322), and (0.24, 0.46, 174). Bmax decreased in a linear relationship as both ATP (r=0.92) and PCr (r=0.83) decreased, but Kd did not. The data show that the number of glutamate binding sites decreased in a linear relationship as oxidative phosphorylation decreased during hypoxia. A decline in oxidative phosphorylation may lead to a reduction in glutamate binding sites by several mechanisms. Since phosphorylation regulates NMDA receptor distribution in neuronal membranes, we speculate that a decrease in oxidative phosphorylation as seen during hypoxia may lead to a loss in number of glutamate binding sites through changing the location and density of NMDA receptors in brain cell membranes. Alternatively, we speculate that dephosphorylation of the receptor may cause an allosteric or conformational change in the receptor preventing glutamate binding and may lead to lipid peroxidation and cell death.

Conantokin-T selectively antagonizes N-methyl- d-aspartate-evoked responses in rat hippocampal slice

This study investigated the mode of action of conantokin-T, a 21 amino acid peptide toxin isolated from the venom of the fish-hunting cone snail Conus tulipa, on excitatory synaptic transmission in rat hippocampal slices using intracellular recording techniques. Superfusion of conantokin-T (1–500 nM) specifically and irreversibly decreased the pharmacologically isolated N-methyl- d-aspartate receptor (NMDA)-mediated excitatory postsynaptic potential (EPSP NMDA) in a concentration-dependent manner but had no effect on normal excitatory synaptic transmission (EPSP). The sensitivity of postsynaptic neurons to NMDA but not to α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid was also antagonized by conantokin-T pretreatment. In addition, the conantokin-T-induced depression of EPSP NMDA could be antagonized by prior treatment of hippocampal slices with either dl-2-amino-5-phosphonovaleate (10 μM) or ifenprodil (20 μM). However, 7-chlorokynurenic acid (1 μM) had no effect on the action of conantokin-T. These findings indicated that conantokin-T modulates the NMDA receptor by an interaction with its glutamate binding site and polyamine recognition site.