3H]5,7-dichlorokynurenic acid, a novel radioligand labels NMDA receptor-associated glycine binding sites.

A strychnine-insensitive glycine binding site is located on the N-methyl-D-aspartate (NMDA)-preferring glutamate receptor complex. Kynurenic acid analogs are antagonists at this binding site. A derivative of kynurenic acid, 5,7-dichlorokynurenic acid (5,7-DCKA) was radiolabeled with 3H and used to study antagonist binding to the glycine recognition site. This ligand ( [3H]5,7-DCKA) showed high affinity (Kd = 69 nM), saturable (Bmax = 14.5 pmol/mg protein) binding to rat brain membranes. A variety of agonists and antagonists inhibited the binding of [3H]5,7-DCKA and [3H]glycine in a similar fashion (r = 0.93). In addition, glutamate site agonists and antagonists exerted opposite allosteric effects on [3H]5,7-DCKA binding suggesting that [3H]5,7-DCKA preferentially binds to the agonist-activated conformation of the receptor.     

[3H]5,7-Dichlorokynurenic acid,a novel radioligand labels NMDA receptor-associated glycine binding sites

A strychnine-insensitive glycine binding site is located on the N-methyl-d-aspartate (NMDA)-preferring glutamate receptor complex. Kynurenic acid analogs are antagonists at this binding site. A derivative of kynurenic acid, 5,7-dichlorokynurenic acid (5,7-DCKA) was radiolabeled with ³H and used to study antagonist binding to the glycine recognition site. This ligand ([³H]5,7-DCKA) showed high affinity (K_d = 69 nM), saturable (B_max = 14.5 pmol/mg protein) binding to rat brain membranes. A variety of agonists and antagonists inhibited the binding of [³H]5,7-DCKA and [³H]glycine in a similar fashion (r = 0.93). In addition, glutamate site agonists and antagonists exerted opposite allosteric effects on [³H]5,7-DCKA binding suggesting that [³H]5,7-DCKA preferentially binds to the agonist-activated conformation of the receptor.     

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.     

Evidence for a functional coupling of the NMDA and glycine recognition sites in synaptic plasma membranes

Activation of the N-methyl-D-aspartate (NMDA) receptor complex is subject to modulation via interactions at a coupled [3H]glycine recognition site in rat brain synaptic plasma membranes (SPM). We examined the effect of the potent and specific glycine site antagonists, 1-hydroxy-3-amino-2-pyrrolidone (HA-966) and 1-aminocyclobutane-1-carboxylate (ACBC), on the NMDA recognition site. These glycine analogs were found to significantly stimulate the binding of the competitive NMDA antagonist, [3H]3-(2-carboxypiperazin-4-y1)propyl-1-phosphonate ([3H]CPP) in a dose-dependent fashion, whereas both compounds inhibited NMDA-specific L-[3H]glutamate (agonist) binding. Additionally, both glycine antagonists reduced the binding of [3H]1-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) to SPM, a functional assessment of activation of the NMDA receptor-channel complex. The glycine site agonists, glycine and serine reversed these effects in a dose-dependent manner, with the serine reversal being stereospecific for D-serine. The relative potencies of these compounds in reversing the glycine antagonist effects on the NMDA recognition site corresponded with their ability to competitively displace strychnine-insensitive [3H]glycine binding. These results provide evidence for a functional coupling between the glycine and NMDA recognition sites and further, may provide a mechanism by which compounds interacting at the glycine recognition site may modulate NMDA receptor activity.     

Neuroprotective effects of RPR 104632, a novel antagonist at the glycine site of the NMDA receptor, in vitro

The NMDA antagonist and neuroprotective effects of RPR 104632 (2H-1,2,4-benzothiadiazine-1-dioxide-3-carboxylic acid), a new benzothiadiazine derivative, with affinity for the glycine site of the NMDA receptor-channel complex are described. RPR 104632 antagonized the binding of [³H]5,7-dichlorokynurenic acid to the rat cerebral cortex, with a K_i of 4.9 nM. This effect was stereospecific, since the (-)-isomer was 500-fold more potent than the (+)-isomer. The potent affinity of RPR 104632 for the glycine site was confirmed by the observation that RPR 104632 inhibited [³H]N-(1-(2-thienyl)cyclohexyl]-3,4-piperidine ([³H]TCP) binding in the presence of N-methyl- -aspartate (NMDA) (IC₅₀ = 55 nM), whereas it had no effect on the competitive NMDA site or on the dissociative anaesthetic site. RPR 104632 inhibited the NMDA-evoked increase in guanosine 3′,5′-cyclic monophosphate (cGMP) levels of neonatal rat cerebellar slices (IC₅₀ = 890 nM) in a non-competitive manner and markedly reduced NMDA-induced neurotoxicity in rat hippocampal slices and in cortical primary cell cultures. These results suggest that RPR 104632 is a high-affinity specific antagonist of the glycine site coupled to the NMDA receptor channel with potent neuroprotective properties in vitro.     

Glycine regulation of the N-methyl-D-aspartate receptor-gated ion channel in hippocampal membranes

The N-methyl-D-aspartate receptor-gated ion channel (NMDA channel) is regulated by glycine. To examine the interaction of glycine and NMDA receptor ligands on NMDA channel function, we used a biochemical marker of channel opening, [3H]N-(1-[thienyl]cyclohexyl)piperidine (TCP). We quantified [3H]glycine,L-[3H]glutamate, and TCP binding in an identical membrane preparation. This allowed direct comparison of NMDA and glycine receptor occupancy and channel activation. Glycine increased the association and dissociation rates of NMDA-dependent TCP binding to hippocampal membranes, without altering the Kd or Bmax for TCP binding. Structurally similar amino acids mimicked the action of glycine, with D-isomers being more potent than L-isomers. The potency of glycine in regulating TCP binding matched that for displacing [3H]glycine. Glycine stimulation of TCP binding required the presence of NMDA agonists and was inhibited by the NMDA antagonist D-2-amino-5-phosphonovaleric acid. Glycine stimulation of NMDA-dependent TCP binding was not associated with an increase in agonist binding to the NMDA receptor. Likewise, NMDA stimulation of glycine-dependent TCP binding was not associated with an increase in the binding of glycine to the glycine receptor. These findings permit the following conclusions: 1) glycine stimulates TCP binding solely by increasing the access of TCP to its site in the NMDA channel; 2) TCP binding can be used to quantify glycine regulation of the NMDA channel; 3) a stereospecific glycine receptor, as part of the NMDA receptor-channel complex, regulates NMDA-evoked channel opening by a mechanism not involving increased agonist binding to the NMDA receptor. Thus, it appears that the mechanism of glycine and NMDA receptor regulation of the NMDA channel is analogous to that of a two-key lock; both receptors, by independent and mutually required mechanisms, alter channel conformation to allow ion passage.     

NMDA-induced hippocampal [3H]norepinephrine release is modulated by glycine

Kynurenic acid (KYN) non-competitively inhibited N-methyl-D-aspartate (NMDA)-induced [3H]norepinephrine ([3H]NE) release from rat hippocampal slices. At 100 microM KYN, the effect on release was primarily on the maximal obtainable response to NMDA. Glycine was able to completely block the inhibitory effects of 100 microM KYN on NMDA-evoked release. This ability to prevent KYN inhibition of release was shared by other amino acids with the following order of potency: glycine greater than D-serine greater than D-alanine much greater than L-serine greater than or equal to L-alanine. Neither isomer of valine or threonine was able to reverse KYN inhibition of NMDA-induced release. These potencies agreed with the relative abilities of these amino acids to displace strychnine-insensitive [3H]glycine binding to rat brain membranes. Glycine and D-serine had no effect on the inhibition of NMDA-stimulated [3H]NE release produced by D-2-amino-5-phosphonovaleric acid, MK-801 or Mg2+. Also, neither amino acid modified KYN inhibition of kainic acid-induced release. These data demonstrate that the glycine regulatory site associated with the NMDA receptor can be demonstrated in whole brain slices by using an antagonist to attenuate the influences of endogenous glycine.     

Evidence for direct interactions between the NMDA and glycine recognition sites in brain

The interaction between glycine and competitive N-methyl-D-aspartate (NMDA) antagonists was investigated. Glycine (IC50 = 170 nM) partially (approximately 60%) inhibited [3H]CGS-19755 ((+/-)-4-phosphonomethyl-2-piperdine carboxylic acid), but not [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) binding. The action of glycine was mimicked by D-serine and antagonized by 7-chlorokynurenate. CGS-19755 (IC50 = 230 nM) partially inhibited [3H]glycine binding from strychnine-insensitive sites; this effect was antagonized by NMDA. CPP and NPC 12626 (2-amino-4,5-(1,2-cyclohexyl)-7-phosphonoheptanoic acid) inhibited [3H]glycine binding, but only at concentrations 100- to 1000-fold greater than required to displace [3H]CGS-19755 or [3H]CPP. These data provide the first evidence for bidirectional interactions between glycine and NMDA recognition sites and suggest pharmacological differences among competitive NMDA antagonists.     

D,L-(tetrazol-5-yl) glycine: a novel and highly potent NMDA receptor agonist.

This paper describes the pharmacological activity of D,L-(tetrazol-5-yl)glycine, a structurally novel and highly potent agonist at the N-methyl-D-aspartate (NMDA) subtype of excitatory amino acid receptor. D,L-(Tetrazol-5-yl)glycine potently displaced NMDA receptor binding to rat brain membranes as measured using [3H]CGS19755 (IC50 = 98 +/- 7 nM) and [3H]glutamate (IC50 = 36 +/- 18 nM) as ligands. D,L-(Tetrazol-5-yl)glycine did not appreciably inhibit the binding of D,L-alpha-[5-methyl-3H] amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), [3H]kainate, or [3H]glycine (IC50s greater than 30,000 nM). D,L-(Tetrazol-5-yl)glycine was more potent than NMDA or cis-methanoglutamate as a depolarizing agent in the rat cortical slice, and unlike these other agents induced rapid receptor-mediated neurotoxicity. Depolarization by D,L-(tetrazol-5-yl)glycine was antagonized by LY233053, a selective NMDA receptor antagonist. D,L-(Tetrazol-5-yl)glycine was a highly potent convulsant when given to neonatal rats (ED50 = 0.071 mg/kg i.p.). Convulsions in neonatal rats or lethality in mice induced by D,L-(tetrazol-5-yl)glycine were selectively antagonized by competitive and non-competitive NMDA receptor antagonists. D,L-(Tetrazol-5-yl)glycine is a structurally novel (tetrazole-substituted) compound that is a highly potent and selective NMDA receptor agonist. D,L-(Tetrazol-5-yl)glycine could be used to probe further NMDA receptor function in vitro and in vivo.     

4-Amido-2-carboxytetrahydroquinolines. Structure-activity relationships for antagonism at the glycine site of the NMDA receptor.

trans-2-Carboxy-5,7-dichloro-4-amidotetrahydroquinolines, evolved from the lead 5,7-dichlorokynurenic acid, have been synthesized and tested for in vitro antagonist activity at the glycine site on the N-methyl-D-aspartate (NMDA) receptor. Optimization of the 4-substituent has provided antagonists having nanomolar affinity, including the urea trans-2-carboxy-5,7-dichloro-4[[(phenylamino)carbonyl]amino]-1,2,3, 4-tetrahydroquinoline (35; IC50 = 7.4 nM vs [3H]glycine binding; Kb = 130 nM for block of NMDA responses in the rat cortical slice), which is one of the most potent NMDA antagonists yet found. The absolute stereochemical requirements for binding were found to be 2S,4R, showing that, in common with other glycine-site NMDA receptor ligands, the unnatural configuration at the alpha-amino acid center is required. The preferred conformation of the trans-2,4-disubstituted tetrahydroquinoline system, as shown by X-ray crystallography and 1H NMR studies, places the 2-carboxyl pseudoequatorial and the 4-substituent pseudoaxial. Modifications of the 4-amide show that bulky substituents are tolerated and reveal the critical importance for activity of correct positioning of the carbonyl group. The high affinity of trans-2-carboxy-5,7-dichloro-4-[1-(3-phenyl-2-oxoimidazolidinyl)]- 1,2,3,4-tetrahydroquinoline (55; IC50 = 6 nM) suggests that the Z,Z conformer of the phenyl urea moiety in 35 is recognized by the receptor. Molecular modeling studies show that the 4-carbonyl groups of the kynurenic acids, the tetrahydroquinolines, and related antagonists based on N-(chlorophenyl)glycine, can interact with a single putative H-bond donor on the receptor. The results allow the establishment of a three-dimensional pharmacophore of the glycine receptor antagonist site, incorporating a newly defined bulk tolerance/hydrophobic region.     

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.     

HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartate-evoked neurotransmitter release

The role of endogenous glycine in supporting N-methyl-D-aspartate (NMDA)-evoked neurotransmitter release was investigated. HA-966 (1-hydroxy-3-aminopyrrolidone-2) inhibited NMDA-evoked release of [3H]norepinephrine from rat hippocampal brain slices, but was much less effective in inhibiting [3H]norepinephrine release evoked by kainic acid (KA). Glycine (1 mM) reversed the HA-966 (1 mM) antagonism of NMDA-evoked release of [3H]norepinephrine. Strychnine (10 microM) had no effect on the ability of glycine to reverse HA-966 antagonism of NMDA-evoked neurotransmitter release. Other amino acids were also capable of reversing the HA-966 antagonism of NMDA-evoked [3H]norepinephrine release with a rank order of potency: D-serine greater than or equal to glycine much greater than L-serine approximately beta-alanine. These same compounds inhibited strychnine-insensitive [3H]glycine binding to rat cortical membrane fragments with a rank order of potency: glycine greater than D-serine much greater than L-serine greater than or equal to beta-alanine. In addition, HA-966 inhibited [3H]glycine binding (IC50 = 8.5 microM). The results suggest that HA-966 antagonism of NMDA-evoked neurotransmitter release is due to the inhibition of endogenous glycine acting at a strychnine-insensitive modulatory glycine site associated with the NMDA receptor/ionophore complex.     

Evidence that zinc inhibits N-methyl-D-aspartate receptor-gated ion channel activation by noncompetitive antagonism of glycine binding.

Zinc noncompetitively antagonizes N-methyl-D-aspartate (NMDA) receptor-mediated responses in cultured neurons. We investigated the mechanism of this inhibition by examining the effect of zinc on ligand binding to three distinct sites on the NMDA receptor in rat hippocampal membranes. Zinc dose-dependently inhibited both the association and dissociation of the NMDA channel blocker [3H]N-(1-[thienyl]cyclohexyl)piperidine ([3H]TCP) but had no effect on steady state levels of [3H]TCP binding. This suggests that zinc inhibits the receptor-gated access of [3H]TCP to its site in the ion channel but has no effect on the binding site itself. Zinc inhibition of [3H]TCP association was not mediated by an action at the NMDA recognition site, because zinc had no effect on NMDA-displaceable L-[3H]glutamate binding. On the other hand, zinc dose-dependently inhibited [3H]glycine binding by a noncompetitive interaction. Stoichiometric analysis of equilibrium binding data indicated the presence of two [3H]glycine binding sites/[3H]TCP binding site. Comparison of the potencies of zinc in inhibiting glycine-dependent [3H]TCP association and [3H]glycine binding suggests that blockade of only one of the two glycine sites is sufficient to prevent [3H]TCP association. We hypothesize that synaptically released zinc inhibits NMDA receptor-mediated responses by binding to a site on the receptor/channel complex, reducing glycine binding, and thereby decreasing what would otherwise be a tonically present action of endogenous extracellular glycine.     

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.     

High affinity [3H]dextrorphan binding in rat brain is localized to a noncompetitive antagonist site of the activated N-methyl-D-aspartate receptor-cation channel.

[3H]Dextrorphan recognition sites were characterized in rat brain membranes. The pharmacological profile and regional distribution of [3H]dextrorphan binding sites appear to distinguish these sites from those labeled either by [3H]dextromethorphan or by putative sigma receptor radioligands. Data from thoroughly washed forebrain membranes suggest that [3H]dextrorphan predominantly labels a high affinity site defined by the activated state of the N-methyl-D-aspartate (NMDA) receptor-channel complex. Regulation of [3H]dextrorphan binding by specific modulators of NMDA receptor function suggests that [3H]dextrorphan binding is predominantly localized to a domain of the receptor-channel complex also recognized by the prototypical noncompetitive antagonist radioligands (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) and [3H]1-[1-(2-thienyl)cyclohexyl]piperidine (TCP). The critical relationship between [3H]dextrorphan binding and activation of the NMDA receptor-complex is suggested by the profound dependence of [3H]dextrorphan binding on glutamate in well washed membranes. Basal specific [3H]dextrorphan binding is nearly totally suppressed by the specific competitive NMDA antagonist D(-)-2-amino-5-phosphonopentanoic acid (D-AP5), in a glutamate- but not glycine-surmountable manner. Glutamate and glycine each stimulate [3H]dextrorphan binding in a concentration-dependent manner, effecting maximal increases from control of up to 30- and 14-fold, respectively. The NMDA receptor specificity of the modulation of [3H]dextrorphan binding by glutamate and glycine is indicated by the sensitivity of their effects to competitive antagonism by D-AP5 and 3-amino-1-hydroxy-2-pyrrolidone (HA-966), respectively, and by the accordant rank orders of potency of glycine analogs as modulators of [3H]dextrorphan binding and as ligands at the strychnine-insensitive glycine site. The divalent cations Mg2+ and Zn2+ and the polyamines spermine and spermidine regulate [3H]dextrorphan binding in a manner consistent with radioligand interaction at the noncompetitive NMDA antagonist domain. Mg2+ and spermidine regulate [3H]dextrorphan binding biphasically in well washed forebrain membranes, whereas Zn2+ monotonically inhibits [3H]dextrorphan binding. Mg2+ and spermidine regulate [3H]dextrorphan binding with qualitative similarity and in a contrasting fashion to their regulation of [3H]MK-801 and [3H]TCP binding. First, spermidine and Mg2+ are significantly more potent modulators of [3H]dextrorphan binding than of [3H]MK-801 and [3H]TCP binding in well washed membranes; second, whereas the potencies of spermidine and Mg2+ as modulators of [3H]MK-801 and [3H]TCP binding are significantly increased by glutamate and glycine in well washed membranes, their potencies as regulators of [3H]dextrorphan binding appear to be unaffected by glutamate and glycine.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.     

CGP 37849 and CGP 39551: novel and potent competitive N-methyl-D-aspartate receptor antagonists with oral activity

1. The pharmacological properties of CGP 37849 (DL-(E)-2-amino-4-methyl-5-phosphono-3-pentenoic acid; 4-methyl-APPA) and its carboxyethylester, CGP 39551, novel unsaturated analogues of the N-methyl-D-aspartate (NMDA) receptor antagonist, 2-amino-5-phosphonopentanoate (AP5), were evaluated in rodent brain in vitro and in vivo. 2. Radioligand binding experiments demonstrated that CGP 37849 potently (Ki 220 nM) and competitively inhibited NMDA-sensitive L-[3H]-glutamate binding to postsynaptic density (PSD) fractions from rat brain. It inhibited the binding of the selective NMDA receptor antagonist, [3H]-((+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonate (CPP), with a Ki of 35 nM, and was 4, 5 and 7 fold more potent than the antagonists [+/-)-cis-4-phosphonomethylpiperidine-2-carboxylic acid) (CGS 19755), CPP and D-AP5, respectively. Inhibitory activity was associated exclusively with the trans configuration of the APPA molecule and with the D-stereoisomer. CGP 39551 showed weaker activity at NMDA receptor recognition sites and both compounds were weak or inactive at 18 other receptor binding sites. 3. CGP 37849 and CGP 39551 were inactive as inhibitors of L-[3H]-glutamate uptake into rat brain synaptosomes and had no effect on the release of endogenous glutamate from rat hippocampal slices evoked by electrical field stimulation. 4. In the hippocampal slice in vitro, CGP 37849 selectively and reversibly antagonized NMDA-evoked increases in CA1 pyramidal cell firing rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of RPR 118723, a novel antagonist at the glycine site of the NMDA receptor, in vitro

RPR 118723 ((8-chloro-5-methyl-2,3-dioxo-1,4-dihydro-5H-indeno[1, 2-b]pyrazin-5-yl) acetic acid) was previously reported to exhibit potent affinity for the glycine site of the N-methyl-D-aspartate (NMDA) receptor-channel complex in the nanomolar range (K(i)=3.1+/-0. 8 nM). We now report on the effects of RPR 118723 in two functional tests reflecting the interaction between the glycine site and the NMDA receptor. First, RPR 118723 potently inhibited [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) binding in the presence of NMDA (IC(50)=3.5+/-0.4 nM). Second, RPR 118723 antagonized the NMDA-induced increase in [3H]dopamine release in mouse striatal slices (IC(50)=8.0+/-1.1 nM). In both experimental models, an excess of glycine reversed the effect of RPR 118723. These results show that RPR 118723 interferes functionally in the nanomolar range with the glycine site coupled to the NMDA receptor in vitro. The blockade of the glycine site with RPR 118723 may be useful for the therapy of the disorders linked to excessive NMDA stimulation.     

Attenuation of morphine dependence and withdrawal by glycine B site antagonists in rats

Numerous data indicate that noncompetitive and competitive N-methyl-D-aspartate (NMDA) receptor antagonists inhibit the development of physical dependence on opioids when these substances are administered together, and NMDA receptor antagonists are used at lower range of doses. Higher doses of these antagonists can enhance some opioid-induced effects. The present study extends these findings to the effects of NMDA/glycine (glycine(B)) site antagonists. Wistar rats were rendered dependent on morphine by implantation of morphine pellets. Both of the glycine(B) site antagonists used, 7-chloro-4-hydroxy-3-(3-phenoxy)-phenyl-2(H)-quinolone (L-701,324; 2.5 and 5.0 mg/kg) and 5,7-dichlorokynurenic acid (5,7-DCKA; 25, 50, and 100 mg/kg), suppressed the expression of morphine withdrawal syndrome estimated as wet dog shakes. Furthermore, L-701,324 (2.5 and 5 mg/kg), given twice a day during the development of morphine dependence, attenuated the development of morphine dependence, and the results were comparable to those obtained after administration of noncompetitive NMDA receptor antagonist - MK801 (0.1 mg/kg). Our data suggest that glycine(B) site antagonists may attenuate wet dog shakes (withdrawal) and the development of dependence, both being induced by chronic morphine administration in rats.     

Differential modulation of the associated glycine recognition site by competitive N-methyl-D-aspartate receptor antagonists

The competitive N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphonopentanoate and two other five-atom linkage (C-5) omega-phosphono-alpha-amino acid analogs reduced [3H]glycine binding, in a dose-dependent manner, to a maximum of 45-55%, whereas seven-atom linkage (C-7) analogs had significantly less effect. The IC50 of the C-5 antagonists for the inhibition of [3H]glycine binding closely paralleled their potency both in displacing NMDA-selective L-[3H]glutamate binding and in negatively modulating (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) binding. Additionally, reduction of glycine binding by the C-5 antagonists was reversed by both NMDA receptor agonists and C-7 competitive NMDA antagonists, providing evidence that the site of action of these C-5 antagonists is the NMDA recognition site, resulting in indirect modulation of the glycine site. These data imply a functional coupling between the NMDA and associated glycine recognition sites and, furthermore, suggest a differential interaction of C-5 and C-7 competitive NMDA antagonists with the NMDA receptor complex.