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.     

Light microscopic autoradiographic localisation of [3H]glycine and [3H]strychnine binding sites in rat brain

Receptor autoradiography has been employed to determine the distribution of strychnine-insensitive glycine binding sites in rat brain using [3H]glycine as a ligand. The location was significantly different from and more widespread than glycine sensitive [3H]strychnine binding sites. Highest binding densities were observed in hippocampus, cortex, subiculum and amygdala followed by striatum, cerebellum and olfactory areas. Characterisation of the binding indicated that it was saturable, of high affinity, stereoselective and displaced by structurally related amino acids. The results support the existence of two glycine receptor subtypes: strychnine-sensitive and strychnine-insensitive.     

Selective potentiation by L-cysteine of apparent binding activity of [3H]glutathione in synaptic membranes of rat brain

Significant apparent binding activity of [3H]glutathione was detected in synaptic membranous preparations of the rat brain. In vitro addition of sucrose (50-1000 mM) and Triton X-100 (0.02-0.1%) significantly diminished the apparent binding activity, whereas pretreatment of the membranes with Triton X-100 (0.01-0.4%) did not affect the activity. A slight but statistically significant reduction of the apparent binding activity was induced by the in vitro addition (1 mM) of two constituent amino acids, L-glutamic acid and glycine. In contrast, another constituent amino acid, L-cysteine, potently enhanced the binding activity at a concentration higher than 0.1 mM. No prominent alteration of the activity occurred following the inclusion of structurally-related amino acids, dithiothreitol, dithioerythritol and numerous other amino acids. Scatchard analysis revealed that the apparent binding consisted of two independent separate components with Kd values of 0.76 and 11.0 microM, and Bmax values of 4.00 and 27.0 pmol/mg protein respectively. In vitro addition of 1 mM L-cysteine resulted in a single component with a Kd of 8.5 microM and a Bmax of 105 pmol/mg protein. Pretreatment of the membranes with 1 mM L-cysteine potentiated the apparent binding, with a further addition of L-cysteine having no effect. The retina had the highest activity followed by the hypothalamus, striatum, spinal cord, midbrain, hippocampus, medulla-pons, cerebellum and cerebral cortex, which occurred independently of the incubation temperature. In peripheral organs examined, the pituitary possessed higher activity than the retina, with progressively lower activities in the adrenal, liver, spleen, skeletal muscle and heart. No significant activity was detected in the kidney. Addition of 1 mM L-cysteine significantly potentiated the activities at 30 degrees, but not at 2 degrees, in the hippocampus and cerebral cortex without affecting those in other central structures. In contrast, a profound inhibition of the activity was induced by the addition of L-cysteine in the pituitary, adrenal, intestinal mucosa, skeletal muscle and retina independently of the temperature. These results suggest that L-cysteine may selectively potentiate the apparent binding activity of [3H]glutathione in particular regions of the brain, while eliminating that in the peripheral excitable tissues.     

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.     

Effects of thiol-reagents on [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding to rat telencephalic membranes

The binding of [3H]alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H]AMPA), a ligand for the quisqualate subtype of excitatory amino acid receptors, was measured after chemical modifications of rat brain synaptic membranes. Treatment with oxidizing or thiol-alkylating agents did not modify [3H]AMPA binding, whereas treatment with several sulfhydryl reagents produced marked increases in binding. The involvement of free sulfhydryl groups in the regulation of the properties of [3H]AMPA binding sites was suggested by the specificity of p-chloromercuribenzoic acid (PCMB), its sulfonate analog p-chloromercuriphenyl-sulfonic acid (PCMBS), and HgCl2, plus the reversal of their effects after reduction with dithiothreitol. Pretreatment of synaptic membranes with the oxidizing agent 5,5'-dithiobis(2-nitrobenzoic acid) or the alkylating agent N-ethylmaleimide did not significantly affect [3H]AMPA binding but markedly reduced the enhancing effect of PCMBS. On the other hand, the increase in [3H]AMPA binding produced by PCMBS was not prevented by treatment with agonists such as quisqualate or L-glutamate and was produced equally well in resealed postsynaptic membranes with both lipophilic or nonlipophilic SH-reagents. Using filtration assays, two types of binding sites could be detected with high and low affinity for [3H]AMPA. Treatment with SH-reagents produced an increase in the Bmax for the high affinity component and a decrease in the Bmax for the low affinity component, accompanied by an increase in its affinity for the ligand. Using centrifugation assays, the same two types of sites could be detected under control conditions but treatment with SH-reagents produced an increase in affinity of the large component that prevented the analytical differentiation of the two sites. Treatment with SH-reagents also increased the binding of [3H] glutamate to the N-methyl-D-aspartate receptors but did not modify the binding of [3H]kainate to the kainate receptors or the strychnine-insensitive [3H]glycine binding. These results suggest that free sulfhydryl groups allosterically modulate the affinity of the quisqualate subtype of excitatory amino acid receptors and also indicate that different types of glutamate receptors might be differentially affected by chemical modification.     

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.     

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.     

Melatonin enhancement of [3H]-gamma-aminobutyric acid and [3H]muscimol binding in rat brain

The pineal hormone, melatonin, enhanced the sodium-independent binding of [3H]-gamma-aminobutyric acid ([3H]GABA) and [3H]muscimol in the rat cerebral cortex in vitro. This effect was augmented by preincubation of synaptic membranes with melatonin but was abolished by preincubation with Triton X-100. Saturation binding studies using [3H]GABA (2.5 to 1000 nM) indicated that the melatonin-induced enhancement of binding is due to an increase in low-affinity GABAA binding sites. These findings suggest that the central effects of melatonin involve modulation of GABAergic function.     

Improvement of 5-HT3 receptor binding assay: enhancement of specific [3H]quipazine binding with Triton X-100-treated membranes from rat cortex.

The 5-hydroxytryptamine (5-HT)3 receptor binding assay using [3H]quipazine was examined. It was impossible to obtain specific [3H]quipazine binding with the membrane fractions from rat cortex prepared by the usual procedure. When the membranes were pretreated with detergent Triton X-100, the ratio of specific [3H]quipazine binding markedly increased, depending upon the concentration of Triton X-100 in the range of 0.01-0.1% (w/v). At a concentration of more than 0.05%, the specific binding reached a maximum of 55 to 60% of the total binding. The specific [3H]quipazine binding to the Triton X-100-treated membranes was reversible and was potently inhibited by several 5-HT3 antagonists, while 5-HT1, 5-HT2 receptor antagonists and other receptor-specific ligands had no effect on the binding. Scatchard analysis indicated a single class of binding sites with a Kd of 0.62 nM and Bmax of 97 fmol/mg protein. Thus, the Triton X-100-treated membranes retained the characteristics of 5-HT3 binding sites, making it possible to use [3H]quipazine for a 5-HT3 receptor binding assay with a high ratio of specific binding.     

An endogenous modulator of N-methyl-D-aspartate receptor-coupled glycine receptors

Extensive washing of a membrane preparation from rat brain resulted in a progressive enhancement of strychnine-insensitive [3H]glycine binding, which was due to an increase in the number of binding sites with no changes in the apparent affinity of this radioligand, precluding an explanation based solely on the elimination of endogenous glycine. Moreover, after extensive washing a population of [3H]glycine binding sites with very high affinity for L-serine was observed in addition to the sites with low affinity for L-serine present in less extensively washed tissue. The observed changes in [3H]glycine binding were attributable to the elimination of a low molecular weight, heat-stable compound which was readily detected in the wash supernatant. Extensive washing also altered [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo-[a,d]cyclohept-5,10-i min e maleate ([3H]MK-801) binding to N-methyl-D-aspartate (NMDA) receptor-associated channels, decreasing basal binding at equilibrium and producing slower association rates in the presence of either glycine or L-glutamate. Moreover, in well-washed membranes both glycine and glutamate enhanced [3H]MK-801 binding acting at high- and low-affinity sites. These findings suggest that the NMDA receptor complex can assume interconverting conformational states regulated by an endogenous substance(s).     

DL-tetrazol-5-ylglycine, a highly potent NMDA agonist: its synthesis and NMDA receptor efficacy.

At physiological pH, the spatial arrangement of the three charges of DL-tetrazol-5-ylglycine (5) could be viewed as similar to those found in certain conformations of the two excitatory amino acids (EAAs)--aspartic and glutamic acids. Given significant binding to one or more EAA receptors, 5 would offer unique modeling and perhaps biological opportunities. We have previously shown it to be the most potent NMDA agonist known, with a unique and marked in vitro neutrotoxicity at depolarizing concentrations. Now we report the details required for its synthesis, together with its potency and efficacy in two assays of functional activation of the NMDA receptor, namely agonist-influenced [3H]MK801 binding and agonist-induced release of the neurotransmitter [3H]-norepinephrine from brain slices. In both these assays DL-tetrazol-5-ylglycine proved to be more potent and efficacious than NMDA and cis-methanoglutamate. It was more potent than, and equally efficacious to, L-glutamate in [3H]MK801 binding. The structural features of 5 may well reflect optimal agonist interaction at the NMDA receptor site. (We considered the possibility that some decarboxylation of DL-tetrazol-5-ylglycine may have occurred during testing. This would give 5-(aminomethyl)tetrazole (13), the tetrazole acid analog of glycine; and glycine is involved in NMDA receptor activation. Compound 13 does not affect [3H]glycine binding at the strychnine-insensitive glycine binding site, and [3H]MK801 binding studies showed that the (aminomethyl)-tetrazole, even if is formed, would probably have no effect on the activity of tetrazol-5-ylglycine at the NMDA receptor.     

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.     

Glycine-related amino acids stereoselectively affect N-methyl-D-aspartate receptor-mediated contractions of guinea pig ileum: comparison with the inhibition of strychnine-insensitive [3H]glycine binding to rat cortical membranes

Ten microM glycine, D-serine and D-alanine potentiated L-glutamate (30 microM)-induced contractions of the guinea pig ileum by an average of 35, 53 and 24%, respectively. On the contrary, D-cysteine, at the same concentration, caused a 21% inhibition of the contractile response to L-glutamate. This inhibitory effect of D-cysteine was abolished by 10 microM glycine. The corresponding L-isomers of these amino acids, namely L-serine, L-alanine and L-cysteine and the other amino acids tested, possessed negligible activity or were inactive in this test. The IC50 values of the same compounds for strychnine-insensitive binding of [3H]glycine (20 nM) to cortical membranes from the brain of the rat were: 0.26 microM, glycine; 1.2 microM, D-serine; 2.1 microM, D-alanine; 8.6 microM, D-cysteine; 51 microM, L-serine; 90 microM, L-alanine; greater than 1000 microM, L-cysteine. On the whole, these results point out a strict requirement for stereoselectivity for both of the effects examined. In addition, the results obtained in the ileum preparation suggest that D-cysteine may act as an antagonist, rather than as an agonist at the glycine site which regulates the responses of N-methyl-D-aspartate receptors.     

Characterization of the binding of radioligands to the N-methyl-D-aspartate,phencyclidine, and glycine receptors in buffy coat membranes

Rapid vacuum filtration assays were used to quantitate radioligand binding to phencyclidine (PCP), N-methyl-D-aspartate (NMDA), and strychnine-insensitive glycine receptors in a simple buffy coat preparation of rat cortical membranes. K_D and B_max values for [³H]glycine binding were very similar to those previously reported by workers who used centrifugation for the separation of free and bound [³H]glycine. We also found that this preparation had a high percentage of NMDA-displaceable L-[³H]glutamate binding sites, which demonstrated a pharmacology very similar to that previously observed in more purified synaptic plasma membranes. Hill analysis of the displacement curves indicated that glutamate bound to a single class of sites, but that NMDA and NMDA antagonists may interact with this site in a negatively cooperative fashion. This preparation was also found to be suitable for the study of NMDA and glycine receptor regulation of the associated ion channel, as these effectors, alone and in combination, increased the affinity with which [³H]TCP bound to the PCP receptor believed to be located within the ion channel. Thus, the ability to measure radioligand binding to these three sites in the same simple membrane preparation should greatly facilitate the study of the interaction between them.     

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)     

[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.     

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.     

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.     

Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.     

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.