Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

6,7-Dichloroquinoxaline-2,3-Dione is a Competitive Antagonist Specific to Strychnine-Insensitive [3H]Glycine Binding Sites on the N-Methyl-D-Aspartate Receptor Complex

Multiple binding sites on the N-methyl-D-aspartate (NMDA) receptor complex were examined using rat brain synaptic membranes treated with Triton X-100. Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801), a noncompetitive NMDA antagonist, in the presence of 10 microM L-glutamate not only was inhibited by different types of antagonists, such as 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, 7-chlorokynurenate, and 6,7-dichloroquinoxaline-2,3-dione (DCQX), but also was abolished by non-NMDA antagonists, including 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. The inhibition of [3H]MK-801 binding by these compounds was invariably reversed or attenuated by addition of 10 microM glycine. Among these novel antagonists with an inhibitory potency on [3H]MK-801 binding, only DCQX abolished [3H]glycine binding without inhibiting [3H]glutamate and [3H](+-)-3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate bindings. Other antagonists examined were all effective as displacers of the latter two bindings. These results suggest that DCQX is an antagonist highly selective to the strychnine-insensitive glycine binding sites with a relatively high affinity.     

Rats With Different Thresholds to Clonic Convulsions Induced by DMCM Differ in the Binding of [3H]-MK-801 and [3H]-Ouabain in the Membranes of Brain Regions

Considering the putative participation of N-methyl-D-aspartate (NMDA) receptors and the Na(+), K(+)-ATPase enzymes in the susceptibility to convulsions induced by the benzodiazepine inverse agonist methyl 6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate (DMCM), the present study sought to determine if rats with high (HTR) and low (LTR) thresholds to clonic convulsions induced by DMCM differed in the following aspects: the binding of NMDA receptors by [(3)H]-MK-801, Na(+), K(+)-ATPase activity (K(+)-stimulated p-nitrophenylphosphatase) and high-affinity [(3)H]-ouabain binding to membranes from discrete brain regions. Compared to the HTR subgroup, the LTR subgroup presented a lower binding of [(3)H]-MK-801 in the hippocampus, frontal cortex and striatum. The subgroups did not differ in K(+)-p-nitrophenylphosphatase activity, but the LTR subgroup had a lower density of isozymes with a high-affinity to ouabain in the brainstem and in the frontal cortex and a lower affinity to ouabain in the hippocampus than the HTR subgroup. These results suggest that NMDA receptors and ouabain-sensitive Na(+), K(+)-ATPase isozymes may underlie the susceptibility to DMCM-induced convulsions.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

[3H]MK-801 Binding to N-Methyl-d-Aspartate Receptors Solubilized from Rat Brain: Effects of Glycine Site Ligands, Polyamines, Ifenprodil, and Desipramine

The N-methyl-D-aspartate (NMDA) receptor is thought to contain several distinct binding sites that can regulate channel opening. In the present experiments, the effects of ligands for these sites have been examined on [3H]MK-801 binding to a soluble receptor preparation, which had been passed down a gel filtration column to reduce the levels of endogenous small-molecular-weight substances. Glycine site agonists, partial agonists, and antagonists gave effects similar to those observed in membranes [EC50 values (in microM): glycine, 0.31; D-serine, 0.20; D-cycloserine, 1.46; (+)-HA-966, 4.06; and 7-chlorokynurenic acid, 1.81]. Spermine and spermidine enhanced [3H]MK-801 binding to the soluble receptor preparation (EC50, 4.3 and 20.1 microM, respectively), whereas putrescine and cadaverine gave small degrees of inhibitions. When spermine and spermidine were tested under conditions where [3H]MK-801 binding approached equilibrium, their ability to enhance [3H]MK-801 binding was much reduced, a result suggesting that the polyamines increase the rate to equilibrium. Putrescine antagonised the effects of spermine. Ifenprodil reduced [3H]MK-801 binding under both equilibrium and nonequilibrium conditions, although the high-affinity component of inhibition described in membranes was not observed. Ifenprodil antagonised spermine effects in an apparently noncompetitive manner. Desipramine was able to give total inhibition of specific [3H]MK-801 binding under nonequilibrium conditions with an IC50 of 4 microM, and this value was unaltered when [3H]MK-801 binding was allowed to reach equilibrium. These results suggest that the sites mediating the effects of glycine and its analogues, polyamines and desipramine are integral components of the NMDA receptor protein.     

Stimulation of Dopamine Release from Cultured Rat Mesencephalic Cells by Naturally Occurring Excitatory Amino Acids: Involvement of Both N-Methyl-D-Aspartate (NMDA) and Non-NMDA Receptor Subtypes

In rat mesencephalic cell cultures, L-glutamate at concentrations ranging from 100 microM to 1 mM stimulated release of [3H]dopamine that was attenuated by the non-N-methyl-D-aspartate (non-NMDA) receptor antagonist 6,7-dinitroquinoxalinedione, but not by the selective NMDA receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801; 10 microM) and 3-(2-carboxypiperazine-4-yl)propyl-1-phosphonate (300 microM). Even at 1 mM glutamate, this release was Ca2+ dependent. These observations suggest that the release was mediated by a non-NMDA receptor. Only release stimulated by a lower concentration (10 microM) of glutamate was inhibited by MK-801 (10 microM), indicating that glutamate at this concentration activates the NMDA receptor. By contrast, L-aspartate at concentrations of 10 microM to 1 mM evoked [3H]dopamine release that was completely inhibited by MK-801 (10 microM) and was also Ca2+ dependent (tested at 1 and 10 mM aspartate). Thus, effects of aspartate involved activation of the NMDA receptor. Sulfur-containing amino acids (L-homocysteate, L-homocysteine sulfinate, L-cysteate, L-cysteine sulfinate) also evoked [3H]dopamine release. Release evoked by submillimolar concentrations of these amino acids was attenuated by MK-801 (10 microM), indicating involvement of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and characterization of a radiolabelled derivative of the phencyclidine/N-methyl-D-aspartate receptor ligand (+) MK-801 with high specific radioactivity

A [3H]-labelled derivative of the drug (+)MK-801 with a high specific radioactivity was synthesized by first preparing a tribromo derivative of (+)MK-801 followed by catalytic reduction in the presence of [3H]-gas and subsequent purification of the radioactive product by reversed-phase high performance liquid chromatography (RP-HPLC). This resulted in pure (+) [3H]MK-801 with a specific radioactivity of 97 Ci/mmol. The (+) [3H]MK-801 was shown to interact with high affinity and selectivity with the phencyclidine (PCP) receptor in guinea pig brain membrane suspensions. The PCP receptor is associated with a cation channel that is chemically gated by glutamate and N-methyl-D-aspartate (NMDA). Drugs that interact with the PCP receptor block this channel. The (+) [3H]MK-801 described here will be useful to investigate the biochemistry of PCP/NMDA receptors in experiments where a high specific radioactivity is essential.     

Characterization of [3H]MK-801 binding to N-methyl-D-aspartate receptors in cultured rat cerebellar granule neurons and involvement in glutamate-mediated toxicity

The conditions required for growth and survival of cerebellar granule neurons in vitro are known to alter the developmental regulation of NMDA receptor subunit mRNA. In the present report, we have examined the functional and pharmacological characteristics of NMDA receptors on cerebellar granule neurons at 12 days in culture (12 DIC). Under open-channel conditions in extensively washed membranes, [³H]MK-801 labeled a uniform population of sites (K_d = 3.2 ± 0.3 nM) in a saturable manner (B_max = 416 ± 18 fmol/mgl); however, biexponential association and dissociation kinetics indicated the possible existence of at least two NMDA receptor populations that differ in pharmacological properties. The kinetically derived equilibrium dissociation constants for the high- and low-affinity binding components were 0.56 and 771 nM, respectively. The equilibrium competition analysis of MK-801 and other channel-blocking compounds as displacers of [³H]MK-801 revealed the presence of high- and low-affinity binding sites with relative apportionments of 70% and 30%, respectively. The rank-order potency profile of competitor binding at the high-affinity site was (+)-MK-801 > TCP > dextrorphan > dextromethorphan > (+)-ketamine. When tested for the ability to protect 12 DIC cerebellar granule neurons from acute glutamate-induced toxicity, the neuroprotective rank-order potency of these compounds was MK-801 > TCP > dextrorphan > (+)-ketamine > dextromethorphan, which correlated significantly with the high-affinity competition binding profile and thus established the role of NMDA receptors in glutamate toxicity. The findings of these experiments indicate that NMDA receptors on 12 DIC cerebellar granule neurons are a heterogenous population that functionally mediate glutamate-induced neurotoxicity. The heterogenous [³H]MK-801 binding sites may represent NMDA receptor channels composed of different subunits. © 1997 John Wiley & Sons, Inc.     

Solubilization of Spermidine-Sensitive (+)-[3H]5-Methyl-10,11 -Dihydro-5H-Dibenzo[a,d]cyclohepten-5,10-Imine ([3H]MK-801) Binding Activity from Rat Brain

The receptor-ionophore complex of the N-methyl-D-aspartate (NMDA)-sensitive receptor was solubilized by deoxycholic acid from rat brain using (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne ([3H]MK-801) binding as a marker for the receptor. Gel filtration of the solubilized preparations on a Sephadex G-25 column revealed significant [3H]MK-801 binding sensitive to potentiation by glutamate and glutamate/glycine, which was prevented by competitive antagonists for the NMDA and strychnine-insensitive glycine (GlyB) sites. In contrast to NMDA and glycine, spermidine markedly potentiated the amount of [3H]MK-801 binding in solubilized preparations by increasing the apparent affinity of the ligand. In the presence of all three stimulants, the solubilized preparations exhibited pharmacological profiles similar to those in the membrane preparations. These results clearly indicate that the whole macromolecular NMDA receptor-ionophore complex is solubilized under the experimental conditions used.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Regional Variations in [3H]MK801 Binding to Rat Brain N-Methyl-D-Aspartate Receptors

This study examined (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine maleate [( 3H]MK801) binding to the N-methyl-D-aspartate (NMDA) receptor in membranes prepared from six regions of rat brain. Highest levels of binding were found in hippocampus and cortex, whereas much lower densities were found in brainstem and cerebellum. NMDA receptors in cerebellum exhibited a significantly lower affinity for [3H]MK801 than cortical NMDA receptors. To determine whether forebrain and hindbrain NMDA receptors were distinct, the actions of glutamate, NMDA, ibotenate, quinolinate, glycine, and spermine were investigated. These agents increased [3H]MK801 binding in all brain regions examined. However, agonists were uniformly less efficacious in hindbrain compared to forebrain regions. NMDA mimetics and spermine were less potent in cerebellum compared to cortex whereas glycine was equipotent. Antagonists that act at the various modulatory sites on the NMDA receptor were also examined. DL-Amino-phosphonopentanoic acid and 7-chlorokynurenate were approximately equipotent in cortex and cerebellum. However, antagonists that are believed to act inside the NMDA-operated ion channel, including Mg2+ and phencyclidine, were approximately threefold less potent in cerebellum. The diminished regulation of [3H]MK801 binding by glutamate and glycine in the cerebellum was associated with a smaller effect of these agonists on the dissociation of [3H]MK801 from its binding site. The levels of glutamate, aspartate, glycine, serine, and glutamine in the membrane preparations were determined. However, variations in the levels of endogenous amino acids were not sufficient to account for the regional differences in [3H]MK801 binding. These results do not support the hypothesis that a distinct NMDA receptor exists in hindbrian regions of the rat CNS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Abolition of the NMDA-mediated responses by a specific glycine antagonist, 6,7-dichloroquinoxaline-2,3-dione (DCQX)

Among various quinoxaline derivatives examined, only 6,7-dichloroquinoxaline-2,3-dione (DCQX) competitively displaced the strychnine-insensitive binding of [3H]glycine, without affecting the other binding sites on the N-methyl-D-aspartate (NMDA) receptor complex. This novel specific antagonist abolished the ability of L-glutamate to potentiate [3H]MK-801 binding activity in brain synaptic membranes treated with Triton X-100. Inclusion of glycine reversed this preventive action of DCQX on the potentiation induced by glutamate.     

The lack of CB1 receptors prevents neuroadapatations of both NMDA and GABA(A) receptors after chronic ethanol exposure

As the contribution of cannabinoid (CB1) receptors in the neuroadaptations following chronic alcohol exposure is unknown, we investigated the neuroadaptations induced by chronic alcohol exposure on both NMDA and GABA(A) receptors in CB1-/- mice. Our results show that basal levels of hippocampal [(3)H]MK-801 ((1)-5-methyl-10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine) binding sites were decreased in CB1-/- mice and that these mice were also less sensitive to the locomotor effects of MK-801. Basal level of both hippocampal and cerebellar [(3)H]muscimol binding was lower and sensitivity to the hypothermic effects of diazepam and pentobarbital was increased in CB1-/- mice. GABA(A)alpha1, beta2, and gamma2 and NMDA receptor (NR) 1 and 2B subunit mRNA levels were altered in striatum of CB1-/- mice. Our results also showed that [(3)H]MK-801 binding sites were increased in cerebral cortex and hippocampus after chronic ethanol ingestion only in wild-type mice. Chronic ethanol ingestion did not modify the sensitivity to the locomotor effects of MK-801 in both genotypes. Similarly, chronic ethanol ingestion reduced the number of [(3)H]muscimol binding sites in cerebral cortex, but not in cerebellum, only in CB1+/+ mice. We conclude that lifelong deletion of CB1 receptors impairs neuroadaptations of both NMDA and GABA(A) receptors after chronic ethanol exposure and that the endocannabinoid/CB1 receptor system is involved in alcohol dependence.     

Noncompetitive Inhibition of N-Methyl-D-Aspartate by Conantokin-G: Evidence for an Allosteric Interaction at Polyamine Sites

Conantokins T and G are polypeptide toxins present in snails of the genus Conus. These substances were recently reported to act as N-methyl-D-aspartate (NMDA) antagonists. In the present study, we examined the possible mechanisms producing this antagonism. Conantokin-G inhibited spermine- and spermidine-stimulated [3H]MK-801 binding to extensively washed rat forebrain membranes in a noncompetitive manner with IC50 values of approximately 507 and approximately 946 nM, respectively. In contrast, glutamate-enhanced [3H]MK-801 binding was unaffected by conantokin-G concentrations of less than or equal to 20 microM. At concentrations greater than or equal to 5 microM, conantokin-G effected a modest, noncompetitive inhibition of glycine-stimulated [3H]MK-801 binding and also produced a small enhancement of basal [3H]MK-801 binding. Conantokin-G reduced (IC50 approximately 1.08 microM) the NMDA-stimulated accumulation of cyclic GMP in cerebellar granule cell cultures to basal values, but did not affect kainate-mediated increases in cyclic GMP. These findings indicate that conantokin-G acts as a noncompetitive NMDA antagonist through an allosteric inhibition of polyamine responses. The neurochemical profile of this polypeptide is distinct from previously described noncompetitive NMDA antagonists.     

Inhibition by Calmodulin Antagonists of [3H]MK-801 Binding in Brain Synaptic Membranes

In brain synaptic membranes not extensively washed, (+)-5-[3H]methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine ([3H]MK-801) binding was markedly inhibited in a concentration-dependent manner (at concentrations above 1 microM) by several compounds having antagonistic activity at the Ca(2+)-binding protein calmodulin. Scatchard analysis revealed that N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 microM. In membranes extensively washed and treated with a low concentration of Triton X-100, L-glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK-801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W-7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]-MK-801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W-7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in rat brain.     

Characterization of the non-competitive antagonist binding site of the NMDA receptor in dark Agouti rats

The ability of non-competitive NMDA antagonists and other selected compounds to inhibit [3H]MK-801 binding to the NMDA receptor in brain membranes was evaluated in female, dark Agouti rats. In homologous competition binding studies the average apparent affinity (KD) of [3H]MK-801 for its binding site was 5.5 nM and the binding site density (Bmax) was 1.83 pmol/mg protein. Inhibition of [3H]MK-801 binding by non-competitive NMDA antagonists was best described with a one-site competition model and the average Hill coefficients were -1. A series of eight non-competitive NMDA antagonists inhibited [3H]MK-801 binding with the following rank order of affinity (K(i), nM): MK-801 (5.5) > dexoxadrol (21.5) > or = TCP (24.2) > phencyclidine (100.8) > (+)-SKF 10,047 (357.7) > dextrorphan (405.2) > ketamine (922.2) > dextromethorphan (2913). These inhibition binding constants determined in dark Agouti rat brain membranes were significantly correlated (P = 0.0002; r2 = 0.95) with previously reported values determined in Sprague-Dawley rats [Wong et al., 1988, J. Neurochem. 50, 274-281]. Despite significant differences in metabolic capability between these strains, the central nervous system NMDA receptor ion channel shares similar characteristics.     

Differential Modulation by Divalent Cations of [3H]MK-801 Binding in Brain Synaptic Membranes

Endogenous divalent cations, such as Mg2+, Ca2+, and Zn2+, differentially affected the binding of (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne maleate ([3H]MK-801) to an ion channel associated with an N-methyl-D-aspartate-sensitive subclass of excitatory amino acid receptors in different preparations of brain synaptic membranes. Both Mg2+ and Ca2+ were weak inhibitors of the binding in membranes which had not been extensively washed (nonwashed membranes), over a concentration range effective in markedly potentiating the binding in the absence of any added stimulants in membranes which had been extensively washed, but not treated with a detergent (untreated membranes). In membranes extensively washed and treated with Triton X-100 (Triton-treated membranes), both cations significantly potentiated the binding in the presence of added glutamate alone. In contrast, Zn2+ was invariably active as a potent inhibitor of the binding irrespective of the membrane preparations used. In untreated membranes, Ca2+ markedly accelerated the initial association rate of [3H]MK-801 binding without affecting the binding at equilibrium in a manner similar to that found with glycine, as well as with glutamate; Mg2+, however, facilitated the initial association rate with a concomitant reduction of the binding at equilibrium. Zn2+ was effective in accelerating the initial rapid phase of association, with the initial slow phase being delayed, and in markedly reducing the binding at equilibrium. Both Mg2+ and Ca2+ also facilitated dissociation of the bound [3H]MK-801 and Zn2+ slowed the dissociation in untreated membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of ifenprodil on the N-methyl-D-aspartate receptor ionophore complex in rat brain.

The effects of a cerebral anti-ischemic drug ifenprodil on the receptor ionophore complex of an N-methyl-D-aspartate (NMDA)-sensitive subclass of central excitatory amino acid receptors were examined using [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10- imine (MK-801) binding in rat brain synaptic membrane preparations as a biochemical measure. The binding in membrane preparations not extensively washed was markedly inhibited not only by competitive NMDA antagonists such as (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic, D-2-amino-5-phosphonovaleric and D-2-amino-7-phosphonoheptanoic acids, but also by competitive antagonists at the strychnine-insensitive glycine (Gly) site including 7-chlorokynurenic acid and 6,7-dichloroquinoxaline-2,3-dione. Among several proposed ligands for alpha-adrenergic receptors tested, ifenprodil most potently inhibited the binding in these membrane preparations due to a decrease in the density of the binding sites without significantly affecting the affinity. Ifenprodil also inhibited the binding of [3H]N-[1-(2-thienyl)cyclohexyl]piperidine as well as of [3H]MK-801 to open NMDA channels in a concentration-dependent manner at concentrations above 10 nM in membrane preparations extensively washed but not treated by a detergent, with a Hill coefficient of less than unity. Further treatment of extensively washed membrane preparations with a low concentration of Triton X-100 resulted in an almost complete abolition of [3H]MK-801 binding, and the binding was restored to the level found in membrane preparations not extensively washed following the addition of both L-glutamic acid (Glu) and Gly. Ifenprodil was effective in inhibiting [3H]MK-801 binding via reducing both initial association and dissociation rates in Triton-treated membrane preparations, irrespective of the presence of Glu and Gly added. The binding in Triton-treated membrane preparations was additionally potentiated by the polyamine spermidine in a concentration-dependent manner at concentrations above 10 microM in the presence of both Glu and Gly at maximally effective concentrations. Ifenprodil invariably diminished the abilities of these three stimulants to potentiate [3H]MK-801 binding at concentrations over 1 microM in a manner that the maximal responses each were reduced. These results suggest that ifenprodil does not interfere with the NMDA receptor complex as a specific isosteric antagonist at the polyamine domain in contrast to the prevailing view.     

Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells

In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high-affinity glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 100 microM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of[3H]MK-801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC-exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC-exposed cells, however, the acute glutamate-induced NMDA receptor-mediated calcium fluxes were strongly diminished and no toxicity was observed. The down-regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK-801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.     

Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [(3)H]MK-801 binding site were delineated along with the enhancement of [(3)H]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.