Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site

The interaction of newly described antagonist of the non-NMDA glutamate receptor 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) with the glycine site of the NMDA receptor complex has been investigated. In whole-cell patch recordings from hippocampal neurons maintained in culture, currents induced by N-methyl-D-aspartate (NMDA) were dependent on extracellular glycine. Responses to both NMDA (30 microM) and kainate (20 microM) were reduced by CNQX (10-30 microM). The antagonism by CNQX of NMDA, but not kainate, receptor-mediated responses could be reversed by increasing the concentration of glycine in the external medium. Glycine concentration-response curves constructed in the presence of 30 microM NMDA were shifted to the right by CNQX, suggesting that CNQX was competing with glycine for the glycine binding site. However, even at high concentrations of glycine (300 microM) the maximal NMDA current obtained in the presence of CNQX (10-30 microM) was not restored to control levels. Because CNQX had no effect on responses produced by supramaximal concentrations of NMDA (500 microM) and glycine (300 microM), it is suggested that CNQX also interacts with the NMDA recognition site. The antagonism of currents induced by NMDA was not dependent on the membrane potential, and the rapid onset and offset of the block suggested that there was little or no use dependence. Radioligand binding experiments were performed using [3H]glycine to label the strychnine-insensitive glycine regulatory site of the NMDA receptor complex in guinea pig brain frontal cortex membranes. CNQX displaced [3H]glycine binding in a concentration-dependent manner (IC50 = 5.7 microM). Scatchard analysis of the inhibition showed a decrease in the affinity (increase in Kd) of [3H]glycine binding, but no change in the number of binding sites (Bmax) in the presence of 5 microM CNQX, suggesting a competitive interaction. These data provide evidence that CNQX antagonizes NMDA receptor-mediated responses by competing with glycine for a modulatory site associated with the NMDA receptor complex. Furthermore, the results indicate that CNQX may not be as selective an antagonist for non-NMDA receptors as initially described, although its selectivity will depend on the concentration of the NMDA receptor ligand and may be enhanced by increasing the extracellular concentration of glycine.     

6,7-Dinitro-quinoxaline-2,3-dion and 6-nitro,7-cyano-quinoxaline-2,3-dion antagonise responses to NMDA in the rat spinal cord via an action at the strychnine-insensitive glycine receptor

6,7-Dinitro-quinoxaline-2,3-dion (DNQX) and 6-nitro,7-cyano-quinoxaline-2,3-dion (CNQX) produce an unsurmountable antagonism of responses to N-methyl-D-aspartate (NMDA) in the baby rat hemisected spinal cord. These effects of DNQX and CNQX can be prevented in a dose-dependent manner by co-superfusion with D-serine or glycine (in the presence of strychnine). The results suggest that the unsurmountable blockade of NMDA responses by DNQX and CNQX reflects an antagonist effect mediated at the allosterically linked strychnine-insensitive glycine receptor.     

Antagonism of monosynaptic excitations in the mouse olfactory cortex slice by 6,7-dinitroquinoxaline-2,3-dione

The effects of 6,7-dinitroquinoxaline-2,3-dione (DNQX) have been tested in slices of olfactory cortex of the mouse against responses evoked by N-methyl-D-aspartate, kainate and quisqualate and on the surface field potentials evoked on electrical stimulation of the lateral olfactory tract. At a concentration of 5 microM, DNQX competitively antagonized responses evoked by kainate and quisqualate, with only a small reduction in the responses to N-methyl-D-aspartate. In contrast, DL-(+-)-2-amino-5-phosphonopentanoic acid (APP, 50 microM) selectively antagonized depolarizations to N-methyl-D-aspartate. The amplitude of the field potential known as the N-wave was reduced by DNQX in a concentration-dependent reversible manner (IC50 = 2.92 +/- 0.33 microM; mean +/- SE mean, n = 4). DL-(+-)-2-Amino-5-phosphonopentanoic acid (50 microM) did not significantly affect this action of DNQX. It is concluded that DNQX inhibits monosynaptic excitations in the olfactory cortex by selectively blocking kainate and/or quisqualate receptors, although it is unclear whether the receptors are located at pre- and/or postsynaptic sites.     

4-Hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b] pyrimidine derivatives: synthesis and their biological evaluation for the glycine site acting on the N-methyl-D-aspartate (NMDA) receptor

Bioisostere approach has been shown to be useful to augment potency or to modify certain physiological properties of a lead compound. Based upon well documented bioisosterism, an isosteric replacement of benzene ring of 4-hydroxy-2-quinolone compound (L-695902) with a thiophene moiety was carried out to prepare the title compounds, 4-hydroxy-6-oxo-6,7-dihydro-thieno[2,3-b] pyrimidines 15. The resulting bioisosteric compounds 15 were evaluated for their antagonistic activity (binding assay) for NMDA receptor glycine site.     

BMY-14802 antagonizes harmaline- and D-serine-induced increases in mouse cerebellar cyclic GMP: neurochemical evidence for a sigma receptor-mediated functional modulation of responses mediated by the N-methyl-D-aspartate receptor complex in vivo

BMY-14802 [alpha-(4-flurophenyl)-4-(5-fluoro-pyramidinyl)-1-piperazine butanol], a potent sigma ligand with poor affinity for dopamine and phencyclidine receptors in vitro, attenuated parenteral harmaline- and direct intracerebellar D-serine-induced increases in mouse cerebellar cGMP. Intracerebroventricularly injected BMY-14802 also antagonized the effects of intracerebellar D-serine, indicating a central mechanism. However, direct co-injection of BMY-14802 into the cerebellum failed to antagonize the D-serine-induced increases in cGMP, indicating a locus of action outside the cerebellum. In contrast, quisqualate-induced cGMP increases were not attenuated by BMY-14802. These results indicate a functional modulation of the N-methyl-D-aspartate/glycine/phencyclidine/ion channel complex-mediated events by BMY-14802, possibly through a transsynaptic mechanism, thus representing the first in vivo demonstration of a sigma ligand modulation of a response mediated through the N-methyl-D-aspartate receptor complex.     

Identification of a novel structural class of positive modulators of the N-methyl-D-aspartate receptor, with actions mediated through the glycine recognition site

This study describes a new structural class of compounds which interact at the N-methyl-D-aspartate (NMDA) receptor-associated glycine recognition site. These E-gamma-substituted vinylglycine derivatives were active in displacing [3H]glycine binding from the NMDA receptor-associated recognition site in rat forebrain synaptic plasma membranes, with Ki values in the range of 0.24-8.7 microM. Functional analyses of these compounds indicate that they positively modulate basal [3H](+)-5-methyl-10,11-dihydro-5H- [a,d]cyclohepaten-5,10-imine ([3H]MK-801) binding, consistent with their having agonist characteristics. Little stereospecificity is observed with the gamma-substituted methyl and propyl derivatives while the L-isomer of the hexyl analog is significantly more potent than the D-isomer. The D- and L-hydroxyethyl gamma-substituted vinylglycines were the most potent inhibitors of [3H]glycine binding with Ki values of 0.75 +/- 0.06 microM and 0.24 +/- 0.02 microM, respectively. The 3,4-double bond was necessary for activity in that the saturated hexyl derivative (2-aminodecanoate) was inactive. Based on the results reported herein, the hypothesis that there is a distinct size restriction for functional agonists which interact with the glycine recognition site, should be altered to include these larger extensions of vinylglycine.     

Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles.

1. 7-Nitro indazole (7-NI) produces potent inhibition of rat cerebellar nitric oxide synthase (NOS) with an IC50 of 0.9 +/- 0.1 microM (n = 6). NOS activity is dependent on the presence of both exogenous CaCl2 and NADPH. The inhibitory potency of 7-NI remained unaltered in the presence of different concentrations of either CaCl2 (0.75-7.5 mM) or NADPH (0.05-5.0 mM). 2. Kinetic (Lineweaver-Burke) analysis of the effect of 7-NI on rat cerebellar NOS revealed that inhibition was of a competitive nature with a Ki value of 5.6 microM. The Km of of cerebellar NOS with respect to L-arginine was 2.5 microM. 3. The following indazole derivatives (IC50 values shown in parentheses, all n = 6) caused concentration-related inhibition of rat cerebellar NOS in vitro: 6-nitro indazole (31.6 +/- 3.4 microM), 5-nitro indazole (47.3 +/- 2.3 microM), 3-chloro indazole (100.0 +/- 5.5 microM), 3-chloro 5-nitro indazole (158.4 +/- 2.1 microM) and indazole (177.8 +/- 2.1 microM). The IC50 values for 5-amino indazole, 6-amino indazole and 6-sulphanilimido indazole were in excess of 1 mM; 3-indazolinone was inactive. 4. 7-NI (10 mg kg-1) administered i.p. to rats produced 60 min thereafter a significant inhibition of NOS activity in cerebellum (31.1 +/- 3.2%, n = 6), cerebral cortex (38.2 +/- 5.6%, n = 6), hippocampus (37.0 +/- 2.8%, n = 6) and adrenal gland (23.7 +/- 3.0%, n = 6). NOS activity in olfactory bulb and stomach fundus were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Opipramol, a potent sigma ligand, is an anti-ischemic agent: neurochemical evidence for an interaction with the N-methyl-D-aspartate receptor complex in vivo by cerebellar cGMP measurements

Opipramol, a potent sigma ligand and a tricyclic antidepressant compound, provided significant neuronal protection (P less than 0.0001) against ischemia-induced neuronal cell loss in the hippocampus in Mongolian gerbils, at a dose of 50 mg/kg (30 min pretreatment). However, opipramol did not offer protection when given 60 min after the ischemic insult. Opipramol decreased basal levels of cGMP in the cerebellum of the mouse and harmaline-induced increases in levels of cGMP, with approximate ED50 values of 4 and 27 mg/kg. Opipramol antagonized methamphetamine- and pentylenetetrazol-induced increases in levels of cGMP. Parenteral administration of opipramol also antagonized the increases in levels of cGMP in the cerebellum of the mouse after the local administration of D-serine, an agonist at the N-methyl-D-aspartate (NMDA)-associated, strychnine-insensitive glycine receptor. These results indicate that opipramol attenuates responses mediated through the NMDA receptor complex. These results further support the functional modulation of the NMDA receptor complex by sigma ligands and provide a neurochemical correlate for the observed anti-ischemic properties of opipramol.     

Differentiation of L- and D-S-nitrosothiol recognition sites in vivo

The main aim of this study was to determine the effects of the lipophobic electron acceptor, nitroblue tetrazolium (NBT), on the vasodilator responses elicited by femoral vein injections of L- and D-S-nitrosocysteine (L- and D-SNC), L- and D-S-nitroso-beta,beta-dimethylcysteine (L- and D-SNPEN) and the nitric oxide (NO) donor, MAHMA NONOate, in pentobarbital-anesthetized rats. L- and D-SNC, L- and D-SNPEN, and MAHMA NONOate elicited dose-dependent falls in mean arterial blood pressure (MAP), and hindquarter (HQR), renal (RR), and mesenteric (MR) vascular resistances. The L-SNC- and L-SNPEN-induced depressor and vasodilator responses were markedly attenuated after injection of NBT. The D-SNC- and D-SNPEN-induced falls in mean arterial pressure, hindquarter, and mesenteric vascular resistances were also reduced after injection of nitroblue tetrazolium whereas the falls in renal resistances were not affected. However, nitroblue tetrazolium inhibited the L-SNC and L-SNPEN responses much more profoundly than the D-SNC and D-SNPEN responses in each vascular bed. In contrast, the MAHMA NONOate-induced responses were not attenuated by nitroblue tetrazolium. This study demonstrates that nitroblue tetrazolium attenuates L- and D-SNC-and L- and D-SNPEN- mediated but not NO-mediated vasodilation. The lack of effects of NBT on the NO responses suggests that NBT does not interfere with the intracellular mechanisms by which NO relaxes vascular smooth muscle. The more pronounced effects of NBT on the vasodilator effects of L-SNC and L-SNPEN than D-SNC and D-SNPEN suggests that these stereoisomers differentially interact with stereoselective S-nitrosothiol recognition sites in the vasculature and that these sites (or their signaling elements) contain thiol residues that may be susceptible to occupation and/or oxidation (ie, disulfide-bond formation) by nitroblue tetrazolium.     

Polyamines modulate events mediated by the N-methyl-D-aspartate (NMDA) receptor complex through an ifenprodil-insensitive pathway: in vivo measurements of cyclic GMP in the cerebellum.

In the present investigation, the effects of polyamines, spermidine and spermine on events mediated by the N-methyl-D-aspartate (NMDA) receptor complex were examined. Spermine and spermidine did not alter basal levels of cyclic GMP (cGMP) in the cerebellum of the mouse, over a wide range of concentrations. However, exogenously added spermine, spermidine, D- and L-ornithine and putrescine attenuated the increases in cGMP seen after the administration of D-serine, an agonist of the NMDA receptor-associated glycine recognition sites. Spermine and/or spermidine also antagonized harmaline-, methamphetamine- and pentylenetetrazol-induced increases in the levels of cGMP. Spermidine also potentiated (+)-MK-801 (dizocilipine)-induced attenuation of basal levels of cGMP. Intracerebroventricular administration of ifenprodil, a suggested polyamine antagonist, did not antagonize spermine- and spermidine-induced attenuation of the response to D-serine. These data suggest that exogenously added polyamines attenuate events mediated by the NMDA receptor complex, in an ifenprodil-insensitive manner.     

Antagonism of synaptic potentials in ventral horn neurones by 6-cyano-7-nitroquinoxaline-2,3-dione: a study in the rat spinal cord in vitro.

1. The rat spinal cord in vitro has been used to assess the effect of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) on the dorsal root evoked extracellular ventral root reflex (DR-VRR) and the intracellular excitatory postsynaptic potential (e.p.s.p.) in ventral horn neurones and motoneurones. 2. CNQX (1-5 microM) produces a selective and dose-dependent reduction in the amplitude of the monosynaptic component of the DR-VRR recorded from lumbar spinal segments. 3. With low intensity dorsal root stimulation CNQX selectively attenuates the amplitude of the short latency intracellular e.p.s.p. (70% reduction, P < 0.005) and its rise-time (75%, P < 0.01) without affecting the half-time to decay. 4. When high intensity stimulation is used CNQX significantly attenuates the amplitude of the e.p.s.p. (56%, P < 0.005), rise-time (76%, P < 0.01) and abolishes the short latency spike. In addition longer latency synaptic components are attenuated and the half-time to decay significantly reduced (47%, P < 0.005). 5. The results with CNQX are compared to D-aminophosphonovalerate and discussed in relation to the recruitment of low versus high threshold afferents. The data supports an involvement of non-NMDA receptors in transmission through both mono- and polysynaptic pathways in the ventral horn.     

6-Cyano-7-nitroquinoxaline-2,3-dione as an excitatory amino acid antagonist in area CA1 of rat hippocampus.

1. A quantitative pharmacological investigation of the excitatory amino acid antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) has been made in area CA1 of rat hippocampal slices bathed in 1 mM Mg2+ containing medium. 2. At a concentration of 10 microM, CNQX reversibly antagonized responses to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), quisqualate and kainate; it produced a parallel shift in their log dose-response curves. Responses to N-methyl-D-aspartate (NMDA) were not antagonized by 10 microM CNQX (dose-ratio: 1.04 +/- 0.06, n = 3). 3. Schild plots (constructed over the range 1-100 microM) yielded the following estimated pA2 values, AMPA 5.8, quisqualate 5.9, and kainate 5.9. NMDA was antagonized by 100 microM CNQX, giving an apparent log K of 4.44 +/- 0.06. 4. The slopes (+/- s.e. mean) of the Schild plots were for AMPA 0.84 +/- 0.06, quisqualate 0.79 +/- 0.04 and kainate 0.68 +/- 0.07. These were all significantly less than unity. 5. Synaptic responses elicited by low frequency activation of the Schaffer collateral-commissural pathway were blocked completely by CNQX (10 microM) providing that a low stimulus intensity was used. With high intensity stimulation a small component remained that was blocked by the selective NMDA antagonist D-2-amino-5-phosphonovalerate (APV). 6. These results suggest that CNQX does not differentially affect the responses of CA1 neurones to AMPA, quisqualate and kainate. It does, however, depress responses to these agonists to a greater degree than it does responses to NMDA and it is a highly effective synaptic antagonist.     

Localization of sensitive sites to taurine, gamma-aminobutyric acid, glycine and beta-alanine in the molecular layer of guinea-pig cerebellar slices.

1 The localization, in the molecular layer of guinea-pig cerebellar slices, of the sites most sensitive to iontophoretically applied taurine, gamma-aminobutyric acid (GABA), glycine and beta-alanine was investigated. 2 The most sensitive sites were located (expressed as the distance from the Purkinje cell body to the pial surface): 0 micrometers, 60 micrometers and 220 micrometers for taurine; 0 micrometers and 180 micrometers for GABA; 80 micrometers and 200 micrometers for glycine, and 80 micrometers, 180 micrometers and 300 micrometers for beta-alanine. 3 The sensitive site (at 0 micrometers) to GABA was considered to represent the basket synapses on the Purkinje cell soma, while the sites (60 to 80 micrometers and 200 to 300 micrometers) for taurine, glycine and beta-alanine were tentatively assigned to the synapses of the stellate neurones on the Purkinje cell dendrites. 4 Inhibitory actions of all four amino acids tested at the most sensitive sites were antagonized by both picrotoxin and strychnine. 5 The possibility that taurine might be the neurotransmitter of the stellate neurones in guinea-pig cerebellum is discussed.     

Metabolism of 1,4-Dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525) in Rats and Cats

1. The metabolism of 1,4-dihydro-6-trifluoromethylquinoxaline-2,3-dione (Lilly 72525), a sedative hypnotic drug, was studied in rat and cat.

2. Plasma concentrations of Lilly 72525 were measured fluorometrically after oral and intravenous doses of the compound in rats. A comparison of the area under the two curves suggested that 84% of the oral dose was absorbed.

3. Studies with ¹⁴C-labelled material in both species confirmed that the drug was well absorbed after oral administration and revealed that the dione was mainly eliminated unchanged in the urine. Bile duct cannulation experiments suggested that biliary excretion accounted for most or all of the drug present in faeces of rats.

4. Metabolites isolated from urinary extracts by t.l.c. were identified by g.l.c.-mass spectrometry. The only metabolite detected in rat urine or bile extracts was a ring-hydroxylated compound. This metabolite plus two N-hydroxylated metabolites were identified in extracts of cat urine.     

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.     

Clozapine attenuates N-methyl-D-aspartate receptor complex-mediated responses in vivo: tentative evidence for a functional modulation by a noradrenergic mechanism.

Recent studies revealed a role for dopamine and noradrenaline in the etiology of ischemia-induced neuronal cell death. In the present investigation, the modulation by clozapine, an atypical antipsychotic agent that interacts with adrenergic receptors, of N-methyl-D-aspartate (NMDA) receptor complex-mediated events were studied by examining its effects on levels of cGMP in the cerebellum. Clozapine decreased basal levels of cGMP in the cerebellum and antagonized harmaline-, methamphetamine-, pentylenetetrazol- and D-serine-induced increases in levels of cGMP with ED50 values of 3.9, 2.36, 2.13 and 2.1 mg/kg (i.p.). However, clozapine (1.25-25 mg/kg) did not attenuate the quisqualate-induced increases in levels of cGMP, indicating a specific modulation of events modulated by the NMDA receptor complex. Antagonists of dopamine (D2), serotonin (5-HT)-5-HT1, 5-HT2 and 5-HT3 [haloperidol, propranolol, ritanserin, ICS 205-930 [(3-tropanyl-indole-3-carboxylate methiodide)] respectively], did not reverse the response to harmaline. However, WB-4101 [(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane HCl], and alpha 1-adrenergic antagonist, reversed harmaline-, D-serine-, PTZ- and MA-induced increases in levels of cGMP, indicating an adrenergic modulation of the events mediated by the NMDA receptor complex. Intracerebellar and intracerebroventricular administration of clozapine and intracerebellar administration of WB-4101 reversed the D-serine-induced response, indicating a central locus of action. These results indicated that clozapine modulates levels of cGMP predominantly through its interactions with central adrenergic receptors.     

Increased striatal dopamine efflux in vivo following inhibition of cerebral nitric oxide synthase by the novel monosodium salt of 7-nitro indazole.

The role of nitric oxide (NO) in striatal dopamine release has been controversial. Most NO synthase inhibitors affect more than one isoform of the enzyme and exert vasoconstrictor effects which may also affect striatal dopamine function. We now report on the effect of a soluble monosodium salt of the selective brain NO synthase inhibitor 7-nitro indazole (7-NINA). Using 7-NINA the first study of selective inhibition of the brain isoform of NO synthase on dopamine efflux in rat striatum was undertaken by use of in vivo microdialysis. Perfusion with 7-NINA (1 mM) increased striatal dopamine efflux. The effect of 7-NINA was partially antagonized (67%) by co-perfusion with L-arginine (1 mM), the precursor of NO formation in vivo. This suggests that 7-NINA induces a competitive inhibition of NO synthase activity. These data show that endogenous NO has an inhibitory effect on striatal dopamine efflux in vivo.     

Acute effects of phenoxybenzamine on alpha-adrenoceptor responses in vivo and in vitro: relation of in vivo pressor responses to the number of specific adrenoceptor binding sites

Pressor responses to intravenous phenylephrine, an adrenoceptor agonist, and to the alpha 2-adrenoceptor-selective agonist guanabenz were examined in conscious rabbits 30 min after treatment with a range of doses of phenoxybenzamine (10(-5) to 5 mg/kg). The maximum number of specific prazosin- and clonidine-binding sites in the spleen of rabbits sacrificed 30 min after receiving phenoxybenzamine were measured using radioligand binding techniques. Treatment with phenoxybenzamine resulted in a dose-dependent reduction in the maximum pressor response to both phenylephrine and guanabenz, although phenoxybenzamine was a more potent antagonist at postsynaptic alpha 1- than at postsynaptic alpha 2-adrenoceptors. Phenoxybenzamine treatment also caused a dose-dependent reduction in specific [3H]prazosin and [3H]clonidine binding. The maximum in vivo pressor response to guanabenz was observed only when all specific clonidine-binding sites were present. There was a close correlation between in vitro receptor number and in vivo pressor responses for alpha 2-adrenoceptor stimulation but not for alpha 1-adrenoceptor-mediated responses. The maximum pressor response to phenylephrine could be obtained in rabbits in which the number of specific prazosin-binding sites was reduced by 60%. These experiments provide an approach to relating in vitro receptor number to in vivo responses.     

Equilibrium analysis of [3H]TCP binding: effects of glycine, magnesium and N-methyl-D-aspartate agonists

It has been reported that glutamate can increase the binding of [3H]TCP to phencyclidine (PCP) receptors by an action on receptors which are selective for N-methyl-D-aspartate (NMDA). Recently this laboratory has reported that glycine and magnesium can amplify this effect of NMDA agonists in well-washed, lysed cortical membranes. Here we report that maximally effective concentrations of glutamate (10 microM), NMDA (300 microM), MgCl2 (300 microM) and glycine (10 microM) increase the affinity of the PCP receptor for [3H]TCP by approximately 4-fold in the absence of any change in the density of PCP receptors. However, in combination with glutamate, magnesium had the further effect of increasing the Bmax by about 75%. Finally, a synaptosomal P2 preparation, which had not been washed to minimize the concentration of endogenous effectors had a Bmax value similar to the well-washed preparation, but had a KD value 8-fold lower. These data indicate that the primary effect of NMDA agonists, glycine, and low concentrations of magnesium ions is to convert the PCP receptor from a low-affinity to a high-affinity state. These data are discussed in relation to the functional regulation of the NMDA ionophore.