Thiokynurenates: a new group of antagonists of the glycine modulatory site of the NMDA receptor.

Several substituted derivatives of kynurenic acid were tested on the N-methyl-D-aspartate (NMDA) receptor/ion channel complex present in the guinea pig myenteric plexus, on the binding of [3H]glycine and of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) to rat cortical membranes and on the depolarization of mice cortical wedges induced by NMDA or quisqualic acid (QA). Kynurenic acid derivatives, having a chlorine (CI) or a fluorine atom in position 5 or 7 but not in position 6 or 8 had significantly lower IC50s than the parent compound when tested on the antagonism of glutamate-induced ileal contraction and in the glycine binding assay. A further significant increase in potency was obtained by substituting a thio group for the hydroxy group in position 4 of kynurenic acid: the IC50 was 160 +/- 20 microM of kynurenic acid and 70 +/- 15 microM of thiokynurenic acid in the myenteric plexus whereas these IC50s for glycine binding were 25 +/- 3 and 9 +/- 2 microM respectively. Several thiokynurenic acid derivatives were synthetized and showed an increased affinity for the glycine recognition site over the corresponding kynurenic acid derivatives. Glycine competitively antagonized the actions of the thiokynurenates in the ileum, in cortical wedges and on [3H]TCP binding. In this preparation, 7-Cl-thiokynurenic acid had an IC50 of 8 microM for antagonizing 10 microM NMDA-induced depolarization while 50% of the 10 microM QA depolarization was antagonized at 300 microM. Thus thiokynurenic acid derivatives seem to be a new group of potent and selective antagonists of strychnine-insensitive glycine receptors.     

Kynurenic acid analogues with improved affinity and selectivity for the glycine site on the N-methyl-D-aspartate receptor from rat brain.

The glycine site on the N-methyl-D-aspartate (NMDA) subtype of receptors for the excitatory neurotransmitter glutamate is a potential target for the development of neuroprotective drugs. We report here two chemical series of glycine site antagonists derived from kynurenic acid (KYNA), with greatly improved potency and selectivity. Disubstitution with chlorine or bromine in the 5- and 7-positions of KYNA increased affinity for [3H]glycine binding sites in rat cortex/hippocampus P2 membranes, with a parallel increase of potency for antagonism of NMDA-evoked responses in the rat cortical wedge preparation. The optimal compound was 5-I,7-Cl-KYNA, with an IC50 for [3H]glycine binding of 29 nM and an apparent Kb in the cortical wedge preparation of 0.41 microM. Reduction of the right-hand ring of 5,7-diCl-KYNA reduced affinity by 10-fold, but this was restored by substitution in the 4-position with the trans-phenylamide and further improved in the trans-benzylamide. The optimal compound was the transphenylurea (L-689,560), with an IC50 of 7.4 nM and an apparent Kb of 0.13 microM. Both series of compounds displayed a high degree of selectivity for the glycine site, having IC50 values of greater than 10 microM versus radioligand binding to the glutamate recognition sites of NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and kainate receptors and the strychnine-sensitive glycine receptor. Selectivity versus AMPA receptor-mediated responses was also apparent in the rat cortical wedge and in patch-clamp recordings of cortical neurons in culture. Experiments using [3H]dizocilpine (MK-801) binding indicated that 5,7-diBr-KYNA, 5,7-diCl-KYNA, 5-I,7-Cl-KYNA, and L-689,560 all behaved as full antagonists and were competitive with glycine. Patch-clamp recordings of cortical neurons in culture also indicated that NMDA-induced currents were antagonized by competition for the glycine site, and gave no evidence for partial agonist activity. pKi values for 5,7-diBr-KYNA and L-689,560 in these experiments were 7.2 and 7.98, respectively, similar to the affinities of these compounds in the glycine binding assay. The high affinity and selectivity of these new derivatives make them useful tools to investigate the function of the glycine site on the NMDA receptor.     

Activity of 5,7-dichlorokynurenic acid, a potent antagonist at the N-methyl-D-aspartate receptor-associated glycine binding site

5,7-Dichlorokynurenic acid (5,7-DCKA), one of the most potent excitatory amino acid receptor antagonists yet described, binds to a strychnine-insensitive glycine binding site located on the N-methyl-D-aspartate (NMDA) receptor complex (Ki = 79 nM versus [3H]glycine). 5,7-DCKA (10 microM) antagonized the ability of NMDA to stimulate the binding of the radiolabeled ion channel blocker N-[3H][1-(2-thienyl)cyclohexyl]-piperidine ([3]TCP). Glycine was able to overcome this effect and in the presence of 5,7-DCKA enhanced [3H]TCP binding to antagonist-free levels. 5,7-DCKA completely and noncompetitively antagonized several NMDA receptor-mediated biochemical and electrophysiological responses. Thus, micromolar concentrations of 5,7-DCKA inhibited NMDA-stimulated elevation of cytosolic calcium in cultured hippocampal neurons, cGMP accumulation in cerebellar slices, and norepinephrine release from hippocampal slices. The glycine antagonist could also block the action of synaptically released agonist, as shown by its ability to inhibit the increase in the magnitude of the population spike that follows tetanic stimulation of the hippocampus in vitro (long term potentiation). Inclusion of glycine or D-serine prevented all these effects of the antagonist. 5,7-DCKA was a potent anticonvulsant when administered intracerebroventricularly to mice. As in the in vitro experiments, the dose-response curve for the antagonist was shifted rightward in a parallel fashion when D-serine was coinjected. This spectrum of activity displayed by a compound acting at the glycine binding site suggests that the therapeutic utility of glycine antagonists will be similar to those proposed for other types of glutamate receptor antagonists.     

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.     

Cis-2,4-methanoglutamate is a potent and selective N-methyl-D-aspartate receptor agonist

Cis- and trans-2,4-methanoglutamate were compared with L-glutamate as acidic amino acid ligands. Cis-2,4-methanoglutamate had a Ki of 0.052 microM against N-methyl-D-aspartate (NMDA)-specific L-[3H]glutamate binding compared with 0.050 microM for L-glutamate. Cis-2,4-methanoglutamate exhibited no significant affinity against [3H]kainate or [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate ([3H]AMPA) binding. Trans-2,4-methanoglutamate had no significant affinity for any of these sites. Cis-2,4-methanoglutamate increased [3H]N-1[2-thienyl]cyclohexyl-3,4-piperidine [( 3H]TCP) binding with EC50 of 0.35 +/- 0.14 microM. It produced an inward current in rat brain mRNA-injected Xenopus oocytes which was blocked by the NMDA antagonist, D-2-amino-7-phosphonoheptanoate (D-AP7). Cis-2,4-methanoglutamate (EC50 = 15.9 microM) was 100-fold more potent than L-glutamate (EC50 = 1,584 microM) in reducing the excitatory postsynaptic potential in CA1 of hippocampal slices. Cis-2,4-methanoglutamate is the most potent, selective NMDA agonist known.     

A potent antagonist of the strychnine insensitive glycine receptor has anticonvulsant properties

5.7-Dinitro-quinoxaline-2.3-dione (MNQX) displaced [3H]glycine binding to cortical membranes but had no effect n [3H]3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid ([3H]CPP) binding. MNQX potently antagonized N-methyl-D-aspartate (NMDA)-evoked release of [3H]GABA from cultured cortical neurones, NMDA evoked spreading depression and NMDA depolarizations in the rat neo-cortex. All of these responses were reversed by addition of glycine to the perfusion media. These results suggested that MNQX is an antagonist at the strychnine-insensitive glycine receptor associated with the NMDA receptor/ionophore complex. Furthermore the compound was found to antagonise audiogenic seizures in DBA-2 mice indicating the potential of glycine antagonists of this type in anticonvulsant therapy.     

Glycine does not reverse the inhibitory actions of ethanol on NMDA receptor functions in cerebellar granule cells

The effects of ethanol and/or glycine on NMDA-induced enhancement of cytoplasmic free Ca²⁺ concentrations ([Ca²⁺]_i), ⁴⁵Ca²⁺ influx, 4-b-[³H]phorbol-12,13-dibutyrate ([³H]PDBu) binding, and neuronal necrosis in cultured rat cortical and cerebellar granule neurons were examined. Using microfluorimetric techniques in combination with rapid perfusion of single brain neurons, we found that glycine (10 M) was a necessary co-agonist for NMDA-induced depolarization in cerebellar granule cells. In contrast, depolarization with NMDA in cortical cells was observed even without the addition of exogenous glycine as well as in the absence or presence of 1 mM MgCl₂. Ethanol (50 mM) inhibited the effects of NMDA in some, but not all, neurons indicative of the existence of ethanol-sensitive and ethanol-insensitive cortical and cerebellar granule neurons. In studies performed in monolayers of cortical and cerebellar granule cells, we observed that the presence of glycine (10 M) was a necessary prerequisite to unmask inhibitory actions of ethanol on ⁴⁵Ca²⁺ influx induced by NMDA. In another set of experiments, we noted that NMDA-induced stimulation of [³H]PDBu binding to monolayers of intact cerebellar granule cells was inhibited by ethanol (50 mM). Finally, we report that ethanol caused a concentration-dependent inhibition of NMDA-induced necrotic cell death, assessed by measuring the ability of cerebellar granule cells to transform 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MIT) into formazan. In none of the four assays used to demonstrate the inhibitory effects of ethanol on NMDA receptor activity, the ethanol-induced inhibition was reversed by glycine (up to 100 M). Thus, in contrast to earlier reports, our data suggest that ethanol and glycine produce their effects by acting at different regulatory sites within the NMDA receptor system in brain neurons.     

The Antidepressant Metapramine is a Low-affinity Antagonist at N-methyl- -aspartic Acid Receptors

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl- -aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [³H]TCP in the presence of both NMDA and glycine ( ₅₀ = 1.4 ± 0.2 μM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices ( ₅₀ = 13 μM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

Strychnine-insensitive glycine/NMDA sites are altered in two stress models of depression

Chronic severe stress (CSS) and chronic mild stress (CMS) affect the properties of [3H]5,7-dichlorokynurenic acid (5,7-DCKA) binding to strychnine-insensitive glycine/NMDA sites in the rat cerebral cortex. Specifically, CSS decreases, while CMS increases, the potency of glycine to displace [3H]5,7-DCKA binding to glycine/NMDA sites. Moreover, in both models, a reduction of the specific [3H]5,7-DCKA binding was observed. The present results demonstrate the involvement of the cortical NMDA receptor complex in the animal models of depression.     

Current topics on glutamate receptors]

Glutamate (Glu) receptors are classified into two major categories in the mammalian central nervous system: inotropic receptors linked to ion channels and metabotropic receptors linked to phosphatidylinositol (PI) metabolism. Classification of the inotropic Glu receptors is based on the differential sensitivity to excitement by N-methyl-D-aspartic acid (NMDA), DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and kainic acid (KA). The NMDA-sensitive subclass is supposed to be a receptor ionophore complex consisting of at least four different subcomponents, including an NMDA recognition site, a glycine (Gly) recognition site, a polyamine recognition site and a cation channel. The NMDA site is radiolabeled by both Glu and competitive antagonists, such as (+-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoic acid (CGP 39653). The Gly domain, which is labeled by both [3H]Gly and [3H]5,7-dichlorokynurenic acid, is sensitive to D-serine but insensitive to strychnine, and this domain seems to be absolutely required for an activation of the NMDA channel by agonists. The ionophore domain is identified by radiolabeled non-competitive NMDA antagonists that gain access to the binding sites within the channel only when it is gated by agonists. The opening of an NMDA channel is allosterically potentiated by Gly and several polyamines. In contrast, an activation of the NMDA channel is blocked by both H+ and divalent cations such as Mg2+ and Zn2+. [3H]AMPA binding displays pharmacological profiles of the AMPA-sensitive subclass with a rank order of agonistic potencies of quisqualic acid (QA) greater than or equal to AMPA greater than Glu greater than KA, which is apparently different from that found for the KA-sensitive subclass (domoic acid greater than or equal to KA greater than QA greater than Glu). In contrast, several quinoxaline derivatives competitively antagonize neuronal responses mediated not only by the AMPA receptor but also by the KA receptor. The metabotropic Glu receptors, which stimulate PI metabolism through an activation of the guanosine triphosphate-binding proteins, are activated by Glu, QA and trans-1-amino-cyclopentyl-1,3-di-carboxylic acid (ACPD). Responses mediated by the metabotropic receptors are competitively blocked by 2-amino-3-phosphonopropionic acid. Three or four cloned complementary deoxyribonucleic acids (cDNAs) encoding inotropic Glu receptors are isolated from a rat brain cDNA library. Pharmacological and electrophysiological properties of receptor-ion channels encoded by a transfection of these cDNAs are similar to those observed with the AMPA receptor as well as the KA receptor, but not with the NMDA receptor.     

Heterocyclic excitatory amino acids. Synthesis and biological activity of novel analogues of AMPA.

The novel acidic amino acids 6a-c, 7, and 8 have been synthesized via 1,3-dipolar cycloadditions, using nitrile oxides and alkynes. The prepared compounds are heterocyclic analogues of glutamic acid with differing chain lengths. One of these compounds, (RS)-2-amino-3-(3-carboxy-5-methyl-4- isoxazolyl)propionic acid (ACPA, 8), was shown in [3H]AMPA binding studies to be more active than AMPA itself (IC50 = 20 nM compared to IC50 = 79 nM for AMPA). No affinity for NMDA receptors (NMDA-sensitive [3H]glutamic acid binding) was found, and only weak affinity in [3H]kainic acid binding (IC50 = 6.3 microM) was detected. The excitatory activity in rat cortical wedge also showed that ACPA was more potent than AMPA (EC50 = 1.0 microM compared to EC50 = 3.5 microM for AMPA). The depolarizing effect of ACPA could be fully antagonized by the selective non-NMDA antagonist 6-cyano-7-nitro-quinoxazoline-2,3-dione (CNQX), but was unaffected by the selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (AP5).     

Vinpocetine preferentially antagonizes quisqualate/AMPA receptor responses: evidence from release and ligand binding studies.

The effect of vinpocetine on excitatory amino acid receptors was examined in the rat brain by two different biochemical approaches. In release experiments with striatal slices, vinpocetine reduced the efflux of dopamine and acetylcholine evoked by glutamate, quisqualate and N-methyl-D-aspartate (NMDA), but not that evoked by kainate. In binding experiments with cortical membranes, vinpocetine reduced the binding of [3H]2-amino-3-3-hydroxy-s-methylisoxasole-4-yl-propionic acid ([3H]AMPA), a quisqualate partial agonist, in an incomplete manner, but failed to influence the binding of [3H]kainate and [3H]3-(2-carboxypyperazine-4-yl)-propyl-1-phosphonic acid ([3H]CPP), an NMDA agonist. These findings suggest that vinpocetine is a quisqualate/AMPA antagonist of some specificity and selectivity.     

Receptor binding characteristics of the novel NMDA receptor glycine site antagonist [3H]GV150526A in rat cerebral cortical membranes

Binding of the glycine site antagonist 3-[2-(Phenylamino-carbonyl)ethenyl]-4,6-dichloro-indole-2-carboxylic acid sodium salt ([3H]GV150526A) was characterised in rat cerebral cortical membranes. Saturation experiments indicated the existence of a high affinity binding site, with a pK(d) value of 9.08 (K(d)=0. 8 nM) and a B(max) of 3.4 pmol/mg of protein. A strong linear correlation was observed between the displacement potencies for [3H]GV150526A and [3H]glycine of 13 glycine site ligands (r=0.991). The association kinetics of [3H]GV150526A binding was monophasic, with a k(on) value of 0.047 (nM)(-1) min(-1). Dissociation was induced by the addition of an excess of glycine, GV150526A, or 5,7-dichlorokynurenic acid (DCKA), another glycine antagonist. With GV150526A and DCKA, the dissociation curves presented similar k(off) values (0.068 and 0.069 min(-1), respectively), as expected from ligands binding to the same site. Conversely, a significantly lower k(off) value (0.027 min(-1)) was found with glycine. Although these data may suggest that glycine agonists and antagonists bind to discrete sites with an allosteric linkage (rather than interacting competitively), the reason for this difference remains to be elucidated. It is concluded that [3H]GV150526A can be considered a new valuable tool to further investigate the properties of the glycine site of the NMDA receptor.     

Effect of 1-aminocyclopropanecarboxylic acid on N-methyl-D-aspartate-stimulated [3H]-noradrenaline release in rat hippocampal synaptosomes.

1. The effect of 1-aminocyclopropanecarboxylic acid (ACPC), a partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor complex that exhibits neuroprotective, anxiolytic and antidepressant-like actions, was investigated in a functional assay for presynaptic NMDA receptors. 2. NMDA (100 microM) produced a 36% increase of tritium efflux above basal efflux in rat hippocampal synaptosomes preincubated with [3H]-noradrenaline ([3H]-NA), reflecting a release of tritiated noradrenaline. This effect was prevented by 10 microM 7-chlorokynurenic acid, an antagonist of the glycine site of the NMDA receptor. 3. Glycine enhanced the effect of NMDA with Emax and EC50 values of 84 +/- 11% and 1.82 +/- 0.04 microM, respectively. ACPC potentiated the effect of NMDA on tritium overflow with a lower EC50 (43 +/- 6 nM) and a lower maximal effect (Emax = 40 +/- 9%) than glycine. Furthermore, ACPC (0.1 microM) shifted the EC50 of glycine from 1.82 microM to > or = 3 mM. 4. These results show that ACPC can reduce the potentiation by glycine of NMDA-evoked [3H]-NA release and hence, may act as an antagonist at the glycine site of presynaptic hippocampal NMDA receptors when the concentration of glycine is high.     

Kynurenic acid derivatives. Structure-activity relationships for excitatory amino acid antagonism and identification of potent and selective antagonists at the glycine site on the N-methyl-D-aspartate receptor

Derivatives of the nonselective excitatory amino acid antagonist kynurenic acid (4-oxo-1,4-dihydroquinoline-2-carboxylic acid, 1) have been synthesized and evaluated for in vitro antagonist activity at the excitatory amino acid receptors sensitive to N-methyl-D-aspartic acid (NMDA), quisqualic acid (QUIS or AMPA), and kainic acid (KA). Introduction of substituents at the 5-, 7-, and 5,7-positions resulted in analogues having selective NMDA antagonist action, as a result of blockade of the glycine modulatory (or coagonist) site on the NMDA receptor. Regression analysis suggested a requirement for optimally sized, hydrophobic 5- and 7-substituents, with bulk tolerance being greater at the 5-position. Optimization led to the 5-iodo-7-chloro derivative (53), which is the most potent and selective glycine/NMDA antagonist to date (IC50 vs [3H]glycine binding, 32 nM; IC50's for other excitatory amino acid receptor sites, greater than 100 microM). Substitution of 1 at the 6-position resulted in compounds having selective non-NMDA antagonism and 8-substituted compounds were inactive at all receptors. The retention of glycine/NMDA antagonist activity in heterocyclic ring modified analogues, such as the oxanilide 69 and the 2-carboxybenzimidazole 70, suggests that the 4-oxo tautomer of 1 and its derivatives is required for activity. Structurally related quinoxaline-2,3-diones are also glycine/NMDA antagonists, but are not selective and are less potent than the 1 derivatives, and additionally show different structure-activity requirements for aromatic ring substitution. On the basis of these results, a model accounting for glycine receptor binding of the 1 derived antagonists is proposed, comprising (a) size-limited, hydrophobic binding of the benzene ring, (b) hydrogen-bond acceptance by the 4-oxo group, (c) hydrogen-bond donation by the 1-amino group, and (d) a Coulombic attraction of the 2-carboxylate. The model can also account for the binding of quinoxaline-2,3-diones, quinoxalic acids, and 2-carboxybenzimidazoles.     

The Antidepressant Metapramine is a Low-affinity Antagonist at N-methyl-d-aspartic Acid Receptors

Metapramine, a pharmacological compound with antidepressant activity in humans, was tested for possible antiglutamatergic activity, in vitro. We investigated the effects of metapramine on the N-methyl-d-aspartic acid (NMDA) receptor complex, by determining whether this compound would interfere with the binding of [³H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) to rat cortical membranes in the presence of either glycine NMDA, or both. Metapramine in the micromolar range inhibited the binding of [³H]TCP in the presence of both NMDA and glycine (ic₅₀ = 1.4 ± 0.2 μM). That very similar affinities were observed when either NMDA or glycine was present suggests that metapramine exerted a direct action at the PCP site. The affinity of metapramine for this site was about 25 and 350 times lower than that of PCP and MK-801, respectively. Metapramine inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (ic₅₀ = 13 μM). These results suggest that metapramine is a low-affinity antagonist of the NMDA receptor complex channel. This paper discusses the potential application of metapramine to the treatment of diseases linked to excessive stimulation of glutamatergic NMDA receptors. © 1997 Elsevier Science Ltd. All rights reserved.     

Differential modulation of [3H]TCP binding to the NMDA receptor by L-glutamate and glycine

At equilibrium (4 h incubation), [3H]TCP (N-(1-[2-thienyl]-cyclohexyl)-3,4-[3H]piperidine) binding to well-washed rat forebrain membranes was enhanced in a concentration-dependent and 2-APV (2-amino-5-phosphonovaleric acid)-sensitive fashion by L-glutamate (EC50 = 0.2 microM; maximal effect +280%). L-glutamate (10 microM) increased the affinity of [3H]TCP from 78 to 28 nM, but was without effect on the maximal binding capacity. The enhancing effect of L-glutamate on [3H]TCP binding was potentiated by glycine in a concentration-dependent manner (EC50 = 50 nM, maximal effect +30% in the presence of 10 microM L-glutamate; EC50 = 2 microM, maximal effect +29% in the presence of 0.1 microM L-glutamate). This effect was strychnine-insensitive. Glycine failed to enhance [3H]TCP binding in the presence of 10 microM 2-APV. The glycine effect was due to an increase in affinity (Kd = 21 nM in the presence of 10 microM glycine and 10 microM L-glutamate); glycine did not affect the maximal binding capacity. The glycine enhancement of L-glutamate-stimulated [3H]TCP binding was not antagonised by 1 microM strychnine and was mimicked by L-serine and L-alanine but not by GABA, taurine or beta-alanine. Kinetic analysis of the glycine and L-glutamate enhancement of [3H]TCP binding indicated that the L-glutamate effect was related to a decrease in the [3H]TCP dissociation rate while the glycine effect was due to an increase in the rate of [3H]TCP association in the presence of L-glutamate.     

Up-regulation of N-methyl-D-aspartate receptors on cultured cortical neurons after exposure to antagonists.

The density of N-methyl-D-aspartate (NMDA) receptors on membranes prepared from cultured cortical neurons was determined using binding assays with [125I]I-MK-801 after exposure of cultures to antagonists of the NMDA receptor complex. The density of binding sites for [125I]I-MK-801 was increased by 40-80% after exposure to D-2-amino-5-phosphonopentanoic acid (D-AP5), with no change in the number or viability of neurons. The effect of D-AP5 was concentration dependent, with an EC50 of 10 microM. Up-regulation of NMDA receptors was observed after 2-7 days but not after 1 day of exposure to 100 microM D-AP5. The density of NMDA receptors was also increased after exposure of cells to CGS 19755 and MK-801 but not after exposure to the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The binding of [3H]AMPA was unaltered after exposure to D-AP5. These results demonstrate that the density of NMDA receptors on cultured neurons can be selectively up-regulated by exposure to NMDA receptor antagonists. Increases in the density of NMDA receptors occurring in vivo could complicate therapeutic approaches to the treatment of neurological disorders.     

Inhibitory and potentiating influences of glycine on N-methyl-D-aspartate-evoked dopamine release from cultured rat mesencephalic cells.

In the presence of 1.2 mM Mg2+, glycine (30-100 microM) inhibited [3H]dopamine ([3H]DA) release stimulated by N-methyl-D-aspartate (NMDA), in fetal rat mesencephalic cell cultures. Strychnine (1 microM) blocked the inhibitory effect of 100 microM glycine, indicating an action via strychnine-sensitive inhibitory glycine receptors. A higher concentration of strychnine (100 microM), by itself, inhibited NMDA-evoked [3H]DA release in the presence or absence of Mg2+. Spontaneous [3H]DA release and [3H]DA release stimulated by kainate and quisqualate were unaffected by glycine (less than or equal to 100 microM) or strychnine (less than or equal to 100 microM), indicating that glycine and strychnine modulatory effects are only associated with the NMDA receptor subtype. [3H]DA release evoked by K+ (56 mM) was unaffected by glycine (less than or equal to 100 microM) but was attenuated by a high concentration of strychnine (100 microM). In the absence of exogenous Mg2+, glycine (30-100 microM) potentiated NMDA-evoked [3H]DA release by a strychnine-insensitive mechanism. A selective antagonist of the NMDA-associated glycine receptor, 7-chlorokynurenate (10 microM), attenuated NMDA-evoked [3H]DA release in the absence of Mg2+. The effect of 10 microM 7-chlorokynurenate was overcome by 1 microM glycine. Also, when tested in the presence of 1.2 nM Mg2+ and 1 microM strychnine, 100 microM 7-chlorokynurenate inhibited NMDA-evoked [3H]DA release, and this antagonism was overcome by 30 to 100 microM glycine. These results indicate that two distinct glycine receptors modulate NMDA-stimulated [3H]DA release from mesencephalic cells in culture. Manipulation of extracellular Mg2+ permits the differentiation of a strychnine-sensitive glycine response (inhibition of NMDA-evoked [3H]DA release) from a strychnine-insensitive glycine response (potentiation of NMDA-evoked [3H]DA release). It is suggested that voltage-dependent Mg2+ blockade of the NMDA response may allow for the expression of these opposing effects of glycine.     

N-Methyl-D-aspartate and quisqualate/DL-alpha-amino-3-hydroxy-5- methylisoxazole-4-propionic acid receptors: differential regulation by phospholipase C treatment

The effect of phospholipase C (PLC) treatment of rat brain membranes on the binding properties of excitatory amino acid receptors was investigated using both a phosphsphatidylcholine-hydrolyzing PLC from Clostridium perfringens and a phosphatidylinositol-specific PLC from Bacillus thuringiensis. PLC from C. perfringens produced an increased affinity of the quisqualate/DL-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor for its ligand, whereas kainate receptor binding was not affected. Both kinetic analysis and equilibrium saturation experiments indicated that PLC treatment produced a decrease in affinity for [3H]N-(1-[thienyl]cyclohexyl)-piperidine [( 3H]TCP), a ligand for the N-methyl-D-aspartate (NMDA) receptor-associated ionic channel, when the channel was fully activated by high concentrations of glutamate and glycine but increased its binding under conditions in which the channel was presumably closed. This latter component of the binding was not due to an interaction of [3H]TCP with non-glutamate receptor sites, such as sigma opioid and histamine H3 receptors. Binding of [3H]glutamate and [3H] glycine to the NMDA receptors was not modified by PLC treatment, but there was a large decrease in the binding of the NMDA antagonist [3H]3-[(+/-)-2-carboxypiperazine-4-yl)propyl-1-phosphonic acid. Stimulation by glycine of [3H]glutamate binding was also abolished following PLC treatment. In contrast to PLC from C. perfringens, phosphatidylinositol-specific PLC treatment did not detectably modify the binding properties of the quisqualate/AMPA receptor or the NMDA receptor channel. These data indicate that alterations in the lipid microenvironment of the glutamate receptors modulate both the conformation and the function of the receptors and suggest a possible role for phospholipases in the regulation of synaptic transmission at excitatory synapses.