Glycine Antagonist 7-chlorokynurenic Acid Research Articles

Expression of Glycine-Activated Diheteromeric NR1/NR3 Receptors in Human Embryonic Kidney 293 Cells Is NR1 Splice Variant-Dependent

In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-d-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells.

GlycineB Receptor Antagonists and Partial Agonists Prevent Memory Deficits in Inhibitory Avoidance Learning

Activation of N-methyl-d-aspartate (NMDA) receptors has been hypothesized to mediate certain forms of learning and memory. This hypothesis is based on the ability of competitive and uncompetitive NMDA receptor antagonists to disrupt learning. We investigated the effects of glycine site antagonists and partial agonists on deficits of acquisition (learning) and consolidation (memory) in a single trial inhibitory avoidance learning paradigm. Posttraining administration of either hypoxia (exposure to 7% oxygen) or the convulsant drug pentylenetetrazole (PTZ) (45 mg/kg) to mice impaired consolidation without producing neuronal cell death. Pretreatment with the competitive glycine antagonist 7-chlorokynurenic acid (7KYN) and the glycine partial agonists 1-aminocyclopropanecarboxylic acid (ACPC) and (+)HA-966 prevented memory deficits induced by hypoxia and PTZ, but did not affect scopolamine-induced learning impairment. In addition, ACPC prevented consolidation deficits evoked by a nonexcitotoxic concentration of l-trans-pyrrolidine-2, 4-dicarboxylate, a competitive inhibitor of glutamate transport that increases extracellular levels of glutamate. Moreover, (+)HA-966, 7KYN, and ACPC facilitated both acquisition and consolidation of inhibitory avoidance training, an effect that was dose-dependent and reversed by glycine. These results indicate that memory deficits induced by both hypoxia and PTZ involve NMDA receptor activation. Furthermore, the present findings demonstrate that glycine site antagonists and partial agonists prevent memory deficits of inhibitory avoidance learning by affecting consolidation, but not acquisition processes.

Glutamate regulation of dopamine release in guinea pig striatal slices

The effect of l-glutamic acid ( l-Glu) on basal and electrically evoked [ 3H]-dopamine efflux in guinea pig striatal slices was studied. In the presence of magnesium, l-Glu significantly increased spontaneous [ 3H]-dopamine efflux. This response was unaffected by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the non-competitive NMDA receptor antagonist, d-5-methyl-10,11,dihydro-5-H-dibenzo-[ a,d]-cyclohepten-5,10-imine maleate (MK-801), the glycine antagonist 7-chlorokynurenic acid (7-CL-KYN) and by the metabotropic receptor antagonist (+)-α-methyl-4-carboxyphenyl-glycine (α-M4CPG) and l-2-amino-3-phosphonopropionic acid ( l-AP3). However, the metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid ( t-ACPD), increased spontaneous [ 3H]-dopamine efflux, as did l-Glu, and this response was completely counteracted by α-M4CPG. In the absence of magnesium, l-Glu induced a concentration-dependent increase in basal [ 3H]-dopamine efflux, which was prevented by MK-801. In electrically stimulated striatal slices l-Glu, applied 25 min before the stimulation, concentration-dependently increased the [ 3H]-dopamine efflux both in the presence and in the absence of magnesium. This effect was completely prevented by CNQX, but not by MK-801 or dl-2-amino-5-phosphono-pentanoic acid (AP5). On the contrary, l-Glu, applied during electrical stimulation (2 min) in the absence of Mg 2+, increased the [ 3H]-dopamine efflux to 200%, and this effect was partly counteracted by MK-801. These results provide evidence that different subtypes of excitatory amino acid receptors modulate [ 3H]-dopamine efflux depending on the functional state (rest or activity) of the nerve endings. The spontaneous [ 3H]-dopamine efflux appears to be controlled by metabotropic receptors in the presence of Mg 2+ and by NMDA receptors in its absence. Conversely, the AMPA/kainate receptors facilitate the electrically evoked [ 3H]-dopamine efflux in the presence of Mg 2+, whereas the NMDA receptors appeared to be operative, in the absence of Mg 2+, as long as l-Glu was applied simultaneously with the electrical stimulation.

The glycine binding site of the N-methyl-D-aspartate receptor subunit NR1: identification of novel determinants of co-agonist potentiation in the extracellular M3-M4 loop region.

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors is a heterooligomeric membrane protein composed of homologous subunits. Here, the contribution of the M3-M4 loop of the NR1 subunit to the binding of glutamate and the co-agonist glycine was investigated by site-directed mutagenesis. Substitution of the phenylalanine residues at positions 735 or 736 of the M3-M4 loop produced a 15- to 30-fold reduction in apparent glycine affinity without affecting the binding of glutamate and the competitive glycine antagonist 7-chlorokynurenic acid; mutation of both residues caused a >100-fold decrease in glycine affinity. These residues are found in a C-terminal region of the M3-M4 loop that shows significant sequence similarity to bacterial amino acid-binding proteins. Epitope tagging revealed both the N-terminus and the M3-M4 loop to be exposed extracellularly, whereas a C-terminal epitope was localized intracellularly. These results indicate that the M3-M4 loop is part of the ligand-binding pocket of the NR1 subunit and provide the basis for a refined model of the glycine-binding site of the NMDA receptor.

Ethanol inhibition of recombinant heteromeric NMDA channels in the presence and absence of modulators.

The NMDA receptor/channel has been shown to be inhibited by ethanol in the clinically relevant range 25-100 mM. We studied heteromeric assemblies (NR1b/NR2) of the NMDA receptor, expressed in oocytes, to address three questions regarding this inhibition, and discovered the following: (1) The inhibition was nearly equivalent when ethanol was coapplied with the agonist, and when ethanol was introduced after steady-state current was established, suggesting that ethanol does not act by interfering with the activation process of the NMDA receptor. (2) The degree of inhibition was controlled by the NR2 subunit, with both NR2A and NR2B significantly more sensitive to ethanol than NR2C and NR2D. (3) Manipulation of the NMDA receptor with a number of agents that normally modulate it did not alter the degree of inhibition produced by ethanol. The presence of Mg2+ (3 and 12.5 microM), Zn2+ (1 and 10 microM), or the glycine antagonist 7-chlorokynurenic acid (1.25 or 5 microM), did not alter the ethanol sensitivity of heteromeric (NR1b/NR2A, NR1b/NR2B, NR1b/NR2C) NMDA receptors. Redox modulation of the NMDA receptor by dithiothreitol (2 mM) or 5,5'-dithiobis(2-nitrobenzoic acid) (1 mM) also did not alter the degree to which ethanol inhibits NMDA receptors. Taken together, these results indicate that the ethanol sensitivity of native NMDA receptors, which likely exist in heteromeric form, results from actions at a site different from those of known modulators of the receptor.

Anticonvulsant activity of antagonists and partial agonists for the NMDA receptor-associated glycine site in the kindling model of epilepsy

Several previous studies have shown that competitive and uncompetitive N-methyl- d-aspartate (NMDA) receptor antagonists are ineffective as anticonvulsants in fully amygdala kindled rats, i.e. a widely-used model of complex-partial seizures (limbic epilepsy). In the present experiments, different categories of ligands for the NMDA receptor-associated glycine modulatory site were evaluated in the kindling model in rats. For this purpose, the ‘silent’ glycine antagonist 7-chlorokynurenic acid (7-CKA), the low efficacy glycine partial agonist (+)-HA-966( R(+)-3-amino-1-hydroxypyrrolid-2-one), and the high efficacy glycine partial agonist d-cycloserine (D-CS) were used. In view of the poor brain penetration of some of these compounds after systemic administration, all drugs were injected bilaterally into the lateral ventricles of fully kindled rat. Anticonvulsant activity was evaluated by determination of the threshold for induction of focal afterdischarges in the amygdala (ADT) and by monitoring seizure parameters (seizure severity, seizure duration, afterdischarge duration) at ADT current. In addition to anticonvulsant activity, the behavioural adverse effects of test drugs were determined. All three drugs increased the focal seizure threshold, although at different potencies. In terms of injected dosages and percentage ADT increase, the most potent compound was 7-CKA, followed by (+)-HA-966 and D-CS. None of the drugs induced behavioural adverse effects or motor impairment (measured in the rotarod test) at anticonvulsant doses. However, D-CS produced proconvulsant effects at higher doses. The data demonstrate that pharmacological manipulation of the glycine modulatory site of the NMDA receptor is an effective means of increasing seizure threshold in amygdala-kindled rats. In this respect, glycine antagonists and partial agonists are clearly superior to competitive or uncompetitive NMDA receptor antagonists. Systemically active glycine antagonists or partial agonists may thus provide a novel strategy for treatment of limbic seizures.

Mutational analysis of the glycine-binding site of the NMDA receptor: Structural similarity with bacterial amino acid-binding proteins

Activation of the NMDA subtype of ionotropic glutamate receptors requires binding of both L-glutamate and the coagonist glycine. Site-directed mutagenesis of the NMDAR1 (NR1) subunit revealed that aromatic residues at positions 390, 392, and 466 are crucial determinants of glycine binding. Glutamate efficacy was little affected by mutations at these positions; however, inhibition of channel gating by the glycine antagonist 7-chlorokynurenic acid was drastically reduced. In addition, glutamine (Q387), valine (V666), and serine (S669) substitutions were found to reduce glycine efficacy. Since the mutated residues correspond to positions forming the binding site of homologous bacterial amino acid-binding proteins, a common amino acid-binding fold appears to be conserved from prokaryotic periplasmic proteins to glutamate receptors in the mammalian brain.

MK-801, but not drugs acting at strychnine-insensitive glycine receptors, attenuate methamphetamine nigrostriatal toxicity

Repeated administration of methamphetamine (METH) results in damage to nigrostriatal dopaminergic neurons. Both competitive N-methyl- d-aspartate (NMDA) receptor antagonists and use-dependent cation channel blockers attenuate METH-induced damage. The objectives of the present study were to examine whether comparable reductions in METH-induced damage could be obtained by compounds acting at strychnine-insensitive glycine receptors on the NMDA receptor complex. Four injections of METH (5 mg/kg i.p.) resulted in a ∼70.9% depletion of striatal dopamine (DA) and ∼62.7% depletion of dihydroxyphenylacetic acid (DOPAC) content, respectively. A significant protection against METH-induced DA and DOPAC depletion was afforded by the use-dependent channel blocker, MK-801. The competitive glycine antagonist 7-chlorokynurenic acid (7-Cl-KA), the low efficacy glycine partial agonist (+)-3-amino-1-hydroxy-2-pyrrolidone ((+)-HA-966), and the high efficacy partial glycine agonist 1-aminocyclopropane-car☐ylic acid (ACPC) were ineffective against METH-induced toxicity despite their abilities to attenuate glutamate-induced neurotoxicity under both in vivo and in vitro conditions. These results indicate that glycinergic ligands do not possess the same broad neuroprotective spectrum as other classes of NMDA antagonists.

In vitro and in vivo characterization of the NMDA receptor-linked strychnine-insensitive glycine site

Modulation of the NMDA receptor by the strychnine-insensitive glycine site was studied both in vitro and in vivo. In vitro the glycinergic stimulation of [ 3H]MK801 binding was measured in three different rat forebrain membrane preparations. An increased association rate of [ 3H]MK801 in the presence of glycine was observed. The binding of the radioligand was also enhanced by d-serine, whereas l-serine was less potent. The concentration-effect curves were shifted to the right by the glycine antagonist 7-chlorokynurenic acid (7CKA). In vivo modulation of the N- methyl- d-aspartate (NMDA) receptor was studied using NMDA induced convulsions in 7 day old rats. The NMDA effect was blocked by (+)-5-methyl-10,11-dihydro- 5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801) and d-(-)-2-amino-5-phosphono-pentanoic acid (AP5). The effect of a submaximal dose of NMDA was dose-dependently potentiated by 1–10 mg/kg d-serine, whereas higher doses of l-serine were needed to obtain a similar effect. 7CKA did not affect NMDA-induced convulsions but reduced the d-serine potentiation of NMDA responses. This study illustrates the ability of the strychnine-insensitive glycine site to modulate the NMDA receptor function both in vitro and in vivo.

Potentiation of N-methyl-D-aspartate-stimulated dopamine release from rat brain slices by aluminum fluoride and carbachol.

N-Methyl-D-aspartate (NMDA) stimulated the release of endogenous dopamine from striatal slices prepared from adult Sprague-Dawley rats. A mixture of sodium fluoride and aluminum chloride (AlF4-) added to the slices significantly potentiated the NMDA-stimulated release of dopamine in a concentration- and time-dependent manner. The AlF4- mixture had no effect on the nonstimulated basal efflux of dopamine, and no increases in NMDA-stimulated release were observed when NaF was replaced with NaCl. Similarly, AlCl3 or a mixture of NaCl and AlCl3 had no effect on NMDA-stimulated release. The AlF(4-)-induced increase in NMDA-stimulated dopamine release was totally blocked by magnesium or the selective NMDA glycine antagonist 7-chlorokynurenic acid. Striatal slices depolarized with KCl (15 mM) also released dopamine and this release was similarly potentiated by AlF4-. However, KCl-stimulated dopamine release from striatal synaptosomes was not potentiated by concentrations of AlF4- that greatly increased release from striatal slices. NMDA did not stimulate the release of dopamine from striatal synaptosomes in the absence or presence of aluminum fluoride. Modulators of adenylate cyclase (forskolin) and protein kinase C (phorbol esters) did not enhance NMDA-stimulated dopamine release. The protein kinase C inhibitor H-7 also did not reduce the potentiating effects of AlF4-. The mixed cholinergic agonist carbachol and the calcium ionophore A23187 mimicked the AlF4- effect although the increase in NMDA-stimulated dopamine release produced by these agents was less than that seen with AlF4-.(ABSTRACT TRUNCATED AT 250 WORDS)

The Glycine Antagonist 7‐Chlorokynurenic Acid Blocks the Effects of N‐Methyl‐d‐Aspartate on Agonist‐Stimulated Phosphoinositide Hydrolysis in Guinea‐Pig Brain Slices

Despite having no effect on basal phosphoinositide hydrolysis. N-methyl-D-aspartate (NMDA) inhibited carbachol-stimulated accumulation of 3H-inositol phosphates and enhanced that due to noradrenaline in guinea-pig cerebral cortex slices. The glycine antagonist 7-chlorokynurenic acid inhibited the effects of NMDA and this inhibition was reversed by glycine. The action of 7-chlorokynurenic acid was not mimicked by strychnine or HA 966 (1-hydroxy-3-aminopyrrolid-2-one). L-Glutamate also inhibited carbachol-stimulated accumulation of 3H-inositol phosphates, but this inhibition was not blocked by 7-chlorokynurenic acid. The data are consistent with glycine maintaining tonic control over NMDA receptor activity in guinea-pig brain.

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.

Modulation of N‐methyl‐D‐aspartic acid receptor desensitization by glycine in mouse cultured hippocampal neurones.

1. Responses to N-methyl-D-aspartic acid (NMDA) were recorded from mouse embryonic hippocampal neurones in dissociated culture, using the tight-seal, whole-cell, patch-clamp technique for voltage clamp. A rapid perfusion system, with an exchange time constant of less than 10 ms, was used to apply NMDA under conditions which minimized slow, calcium-sensitive desensitization. With no added glycine, responses to 100 microM-NMDA applied for 1.5 s declined by greater than 90%, due to an additional component of desensitization of time constant 250 ms. 2. Adding glycine to the extracellular solution, over the range 30 nM to 3 microM, both potentiated responses to NMDA and to L-glutamate, and reduced fast desensitization. In the presence of 3 microM-glycine responses to NMDA declined by only 10%. Similar potentiation and reduction of desensitization was obtained with 3 microM concentrations of the glycine analogues D-alanine and D-serine. 3. Analysis of dose-response curves for the action of glycine on responses to 100 microM-NMDA revealed a 3-fold higher potency of glycine for potentiation of peak versus steady-state responses, with concentrations for half-activation of 134 and 382 nM, respectively. The competitive glycine antagonist 7-chlorokynurenic acid produced a similar shift of both the peak and steady-state dose-response curves for glycine, consistent with an equilibrium dissociation constant of 280 nM for interaction of 7-chlorokynurenic acid with the glycine binding site on NMDA receptors. 4. In the presence of 100 nM-glycine, 10 microM-7-chlorokynurenic acid produced nearly complete block of the response to 3 nM-NMDA, suggesting that glycine is absolutely required for activation of the NMDA receptor channel complex. 5. In some neurones responses to NMDA showed essentially no desensitization in the presence of 3 microM-glycine. Under these conditions, 7-chlorokynurenic acid produced a concentration-dependent block of both the initial and equilibrium response to NMDA, with a 4-fold greater sensitivity for block of the steady-state current (IC50 = 2.25 microM) than for block of the peak current (IC50 = 8.96 microM). As a result, in the presence of 7-chlorokynurenic acid, responses to NMDA showed strong desensitization, even in the presence of 3 microM-glycine. 6. Our results show that glycine-evoked potentiation of NMDA receptor activity is accompanied by reduced desensitization.(ABSTRACT TRUNCATED AT 400 WORDS)

Glycine decreases desensitization of N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes and is required for NMDA responses.

In Xenopus oocytes injected with rat brain mRNA, as in neurons, glycine greatly potentiated responses of the N-methyl-D-aspartate (NMDA) type of excitatory amino acid receptor. Injected oocytes generated a partially desensitizing inward current in response to NMDA with 30 nM added glycine. As the added glycine concentration was increased from 30 nM to 1 microM, the NMDA response was increased and exhibited less desensitization. The relationship between the NMDA peak response and added glycine concentration indicated a single component response with apparent affinity of 0.29 microM and a Hill coefficient of 0.77. The desensitized response was also fit by the Hill relation with a lower affinity but similar coefficient. The time course of desensitization at 500 microM NMDA was exponential with a time constant (350 msec) that was independent of glycine concentration between 0.03 and 0.3 microM. At higher glycine concentration a slower component of decay (tau = 1.4 sec) was observed. This component was enhanced by increasing the extracellular Ca2+. NMDA without added glycine evoked a small transient response. However this response was suppressed completely by prewashing with the glycine antagonist 7-chlorokynurenic acid, suggesting that it may have been due to glycine contamination. The dose-response relation for low concentrations of glycine indicated that the measured level of glycine contamination accounted for these responses. These results indicate that glycine has at least two actions at the NMDA receptor: it enables channel opening by the agonist and decreases desensitization.

Novel heteroaryl-spaced APV and APH analogs as ligands for the NMDA receptor : S.J. Hays, C.F. Bigge, J.T. Drummond, G. Johnson, L.J. Brahce, L.L. Coughenour, L.J. Robichaud, A.W. Probert and P.A. Boxer. Parke-Davis Pharmaceutical Research Division, Warner Lambert Company, Ann Arbor, MI. USA

The effect of l-glutamic acid (l-Glu) on basal and electrically evoked [3H]-dopamine efflux in guinea pig striatal slices was studied. In the presence of magnesium, l-Glu significantly increased spontaneous [3H]-dopamine efflux. This response was unaffected by the non-NMDA receptor antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), the non-competitive NMDA receptor antagonist, d-5-methyl-10,11,dihydro-5-H-dibenzo-[a,d]-cyclohepten-5,10-imine maleate (MK-801), the glycine antagonist 7-chlorokynurenic acid (7-CL-KYN) and by the metabotropic receptor antagonist (+)-α-methyl-4-carboxyphenyl-glycine (α-M4CPG) and l-2-amino-3-phosphonopropionic acid (l-AP3). However, the metabotropic glutamate receptor agonist, trans-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD), increased spontaneous [3H]-dopamine efflux, as did l-Glu, and this response was completely counteracted by α-M4CPG.In the absence of magnesium, l-Glu induced a concentration-dependent increase in basal [3H]-dopamine efflux, which was prevented by MK-801.In electrically stimulated striatal slices l-Glu, applied 25 min before the stimulation, concentration-dependently increased the [3H]-dopamine efflux both in the presence and in the absence of magnesium. This effect was completely prevented by CNQX, but not by MK-801 or dl-2-amino-5-phosphono-pentanoic acid (AP5). On the contrary, l-Glu, applied during electrical stimulation (2 min) in the absence of Mg2+, increased the [3H]-dopamine efflux to 200%, and this effect was partly counteracted by MK-801.These results provide evidence that different subtypes of excitatory amino acid receptors modulate [3H]-dopamine efflux depending on the functional state (rest or activity) of the nerve endings. The spontaneous [3H]-dopamine efflux appears to be controlled by metabotropic receptors in the presence of Mg2+ and by NMDA receptors in its absence. Conversely, the AMPA/kainate receptors facilitate the electrically evoked [3H]-dopamine efflux in the presence of Mg2+, whereas the NMDA receptors appeared to be operative, in the absence of Mg2+, as long as l-Glu was applied simultaneously with the electrical stimulation.