Phorbol Ester Differentiates the Levels of [<Superscript>3</Superscript>H]MK-801 Binding in Rats Lines Selected for Differential Sensitivity to the Hypnotic Effects of Ethanol

These studies addressed the possible involvement between sensitivity to the hypnotic action of ethanol and function of the NMDA receptor. The studies were carried out using high-alcohol sensitive (HAS) and low-alcohol sensitive (LAS) rats, two rats having differential sensitivity to the acute hypnotic action of ethanol. The animal models were developed by a selective breeding experiment. Using a quantitative autoradiograph technique, it was demonstrated that [³H]MK-801 binding to the NMDA receptor was highest in hippocampus in both HAS and LAS rats, but significant [³H]MK-801 binding was also detected in cortex, caudate-putamen, and thalamus of HAS and LAS rats. The density of [³H]MK-801 binding was lower only in cerebellar granule layers of untreated HAS rats as compared to the same brain area in untreated LAS rats. Activation of protein kinase C (PKC) by 100 nM PDBu, increased [³H]MK-801 binding in cortex, caudate-putamen, thalamus, central gray, and cerebellum of HAS rats but activation of PKC did not influence [³H]MK-801 binding in LAS rats. These activation of PKC differentiates between [³H]MK-801 binding of HAS and LAS rats in frontal cortex (layer II-IV and cingulate), caudate-putamen, and ventral lateral thalamic nuclei. The basal level of PKC- mRNA was higher in HAS rats than that of LAS rats. These results suggest that the activation of PKC potentiates NMDA receptor function of the rat line which is more sensitive to alcohol (HAS) but does not affect [³H]MK-801 binding of alcohol resistant (LAS) rats.     

Pharmacological characterization of the N-methyl-d-aspartate (NMDA) receptor-channel in rodent and dog brain and rat spinal cord using [3H]MK-801 binding

The biochemical and pharmacological properties of [³H]MK-801 binding to the N-methyl-d-aspartate (NMDA) receptor-channel in homogenates of mouse, guinea pig and dog brain, dog cerebral cortex and rat spinal cord were determined using radioligand binding techniques. Specific [³H]MK-801 binding increased linearily with increasing tissue concentration and in general represented 80–93% of the total binding at 6–8 nM radioligand concentration. [³H]MK-801 interacted with brain and spinal homogenates with high affinity. The dissociation constants (K _d ) for all tissues studied were similar ranging between 7.9 and 11.9 nM, whereas the maximum number of binding sites (B_max) showed a wide, tissue-dependent range (0.1–6.75 pmol/mg protein). The rank order of tissue enrichment was found to be as follows: mouse brain>>dog cerebral cortex>>dog brain>> guinea pig brain>>rat spinal cord. Specific [³H]MK-801 binding in rodent and dog brain, dog cerebral cortex and rat spinal cord exhibited a similar pharmacological profile 9correlation coefficients=0.93–0.99). The rank order of potency of unlabelled compounds competing for [³H]MK-801 binding was: (+)MK-801>(–)MK-801>phencyclidine>(–)cyclazocine>>(+)cyclazocine ketamine>(+)N-allyl-N-normetazocine>(–)N-allyl-N-normetazocine>(–)pentazocine>(+)pentazocine. NMDA, Kainate, quisqualate and several other compounds failed to inhibit [³H]MK-801 binding at 100 M. In modulation studies conducted on extensively washed dog cortex membranes, Mg²⁺ ions stimulated [³H]MK-801 binding at 10 M-1 mM (EC₅₀=91.5 M) and then inhibited the binding from 1 mM to 10 mM (IC₅₀=3.1 mM). Glycine stimulated [³H]MK-801 binding at 30 nM-1 mM (EC₅₀=256 nM). In contrast, Zn²⁺ ions inhibited the binding of [³H]MK-801 binding site exhibited similar pharmacological and biochemical properties. These data appear to suggest that the pharmacological profile of the NMDA-receptor-channel is species and tissue independent.     

NMDA receptor function is enhanced in the hippocampus of aged rats

The density and functional activity of theN-methyl-D-aspartate (NMDA)-sensitive glutamate receptor was examined in various brain areas of 3-, 18- and 24-month-old rats. The total numbers of binding sites for the NMDA receptor antagonists [³H]CGP 39653 and [³H]MK 801 binding sites were decreased in the hippocampus, cerebral cortex and striatum of 18- and 24-month-old rats, relative to 3-month-old animals. In the hippocampus of 18-month-old rats, the reduced number of NMDA receptors was associated with an increased sensitivity of [³H]MK 801 binding to the stimulatory action of glycine and glutamate. Thus, 10 M glycine and 10 M glutamate increased [³H]MK 801 binding in the hippocampus of 18-month-old rats by 75 and 160%, respectively; in 3-month-old animals, the same concentration of these amino acids increased binding by 37 and 95%, respectively. The sensitivity of [³H]MK 801 binding to glycine and glutamate was not increased in the cerebral cortex and striatum of aged rats. Moreover, an increased efficacy of glycine and glutamate in stimulating the binding of [³H]MK 801 in the hippocampus was no longer apparent in the 24-month-old rats. The increased sensitivity of [³H]MK 801 binding to glycine and glutamate in the hippocampus of 18-month-old rats may reflect an increase in NMDA receptor activity to compensate for the decrease in receptor number.     

Long-Lasting Ibogaine Protection against NMDA-Induced Convulsions in Mice

Ibogaine, a putative antiaddictive drug, is remarkable in its apparent ability to downgrade withdrawal symptoms and drug craving for extended periods of time after a single dose. Ibogaine acts as a non-competitive NMDA receptor antagonist, while NMDA has been implicated in long lasting changes in neuronal function and in the physiological basis of drug addiction. The purpose of this study was to verify if persistent changes in NMDA receptors could be shown in vivo and in vitro after a single administration of ibogaine. The time course of ibogaine effects were examined on NMDA-induced seizures and [³H] MK-801 binding to cortical membranes in mice 30min, 24, 48, and 72h post treatment. Ibogaine (80 mg/kg, ip) was effective in inhibiting convulsions induced by NMDA at 24 and 72 hours post administration. Likewise, [³H] MK-801 binding was significantly decreased at 24 and 72 h post ibogaine. No significant differences from controls were found at 30min or 48h post ibogaine. This long lasting and complex pattern of modulation of NMDA receptors prompted by a single dose of ibogaine may be associated to its antiaddictive properties.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

Role of NMDA Receptors in Pentobarbital Tolerance/Dependence

Effects of continuous pentobarbital administration on binding characteristics of [³H]MK-801 in the rat brain were examined by autoradiography. Animals were rendered tolerant to pentobarbital using i.c.v. infusion of pentobarbital (300g/10l/hr for 7 days) by osmotic minipumps and dependent by abrupt withdrawal from pentobarbital. The levels of [³H]MK-801 binding were elevated in rats 24-hr after withdrawal from pentobarbital while there were no changes except in septum and anterior ventral nuclei in tolerant rats. For assessing the role of NMDA receptor in barbiturate action, an NMDA receptor antagonist (MK-801, 2.7 femto g/10l/hr) was co-infused with pentobarbital. The pentobarbital-infused group had a shorter duration of pentobarbital-induced loss of righting reflex (sleeping time) than that of the control group, and MK-801 alone did not affect the righting reflex. However, co-infusion of MK-801 blocked hyperthermia, and prolonged the onset of convulsions induced by t-butylbicyclophosphorothionate (TBPS) in pentobarbital withdrawal rats. In addition, elevated [³⁵S]TBPS binding was significantly attenuated by co-infusion with MK-801. These results suggest the involvement of NMDA receptor up-regulation in pentobarbital withdrawal and that the development of dependence can be attenuated by the treatment of subtoxic dose of MK-801.     

Distribution of Glutamate Receptors of the NMDA Subtype in Brains of Heterozygous and Homozygous Weaver Mutant Mice

In weaver mice, mutation of an G-protein inwardly rectifying K⁺ channel leads to a cerebellar developmental anomaly characterized by granule and Purkinje cell loss and, in addition, degeneration of dopaminergic neurons. To evaluate other deficits, glutamate receptors sensitive to N-methyl-d-aspartate (NMDA) were examined by autoradiography with [³H]MK-801 in 36 brain regions from heterozygous (wv/+) and homozygous (wv/wv) weaver mutants, and compared to wild type (+/+) mice. In wv/+ and wv/wv mutants labelling decreased in cortical regions, septum, hippocampus, subiculum, neostriatum, nucleus accumbens, superior colliculus and in the cerebellar granular layer. The reductions in [³H]MK-801 binding were particularly specific in the cerebellar granular layer of wv/wv mutants, but an ubiquitous altered NMDA receptor topology was revealed in other brain regions. Abnormal developmental signals, or aberrant cellular responses, may underlie widespread NMDA receptor reductions, while in cerebellar cortex they could be lacking due to the massive loss of cerebellar granule cells.     

Guanidinoethane sulphonic acid interferes with the binding of [3H]dizocilpine and neurotoxic action of AF64A

Summary The binding of [³H]dizocilpine [[³H]MK-801] to the N-methylD-aspartate receptor complex of well washed rat cortical membranes was reduced by guanidinoethane sulphonic acid (GES). Micromolar concentrations of GES, which were high relative to those of dizocilpine, inhibited in a concentration dependent manner the binding of [3H]dizocilpine. The inhibitory effect of GES on [3H]dizocilpine binding was slightly influenced by concentration of glutamate. The glutamate antagonist DL-2-amino-5phosphonovaleric acid blocked the effect GES at concentrations higher relative to GES. The inhibitory effect of GES was still present during spermidine-induced stimulation of [³H]dizocilpine binding. GES reduced the binding of the glycine antagonist [³H]5,7-dichlorokynurenic acid with an IC₅₀ of 530 M.. Intraperitoneal injections of GES (0.2mmol/kg) protected against both amnesia and decrease in the choline acetyltransferase activity following local injections of the neurotoxin AF64A into the nucleus basalis magnocellularis. GES given to lesioned rats during the training period in the spatial learning task gradually improved the performance to the level of sham operated rats. It is concluded that GES interferes with the transmitter and the dizocilpine binding sites of the NMDA receptor complex and has the capacity to protect against neurotoxic brain damage.     

Polyamines Modulate the Binding of [3H]MK-801 to the Solubilized N-Methyl-D-Aspartate Receptor

Spermine and spermidine enhance the binding of [³H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cyclohepten-5,10-imine ([³H]MK-801) to N-methyl-D-aspartate (NMDA) receptors in membranes prepared from rat brain. These polyamines also enhance binding of [³H]MK-801 to NMDA receptors that have been solubilized with deoxycholate. Other polyamines selectively antagonize this effect, a finding indicating that the polyamine recognition site retains pharmacological and structural specificity after solubilization. In the presence of spermidine, an increase in the affinity of the solubilized NMDA receptor for [³H]MK-801 is observed. However, the rates of both association and dissociation of [³H]MK-801 binding to solubilized NMDA receptors are accelerated when assays are carried out in the presence of spermidine. When kinetic data are transformed, pseudo-first-order association and first-order dissociation plots are nonlinear in the presence of spermidine, an observation indicating a complex binding mechanism. Effects of spermidine on solubilized NMDA receptors are similar to effects previously described in studies of membrane-bound receptors. The data indicate that polyamines interact with a specific recognition site that remains associated with other components of the NMDA receptor complex after detergent solubilization.     

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.     

Regional Profile of Developmental Changes in the Sensitivity of the N-Methyl-D-Aspartate Receptor to Polyamines

Abstract: The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [³H]MK-801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3-, 7-, 15-, 25-, and 60-day-old Sprague-Dawley rats. [³H]MK-801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 μM) caused maximal stimulation of [³H]MK-801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15–25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 μM) exhibited similar developmental and regional heterogeneity in its effects on [³H]MK-801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC₅₀ and E_max values for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative “polyamine site” inverse agonist 1,10-diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult. This lack of complementarity in the regional and developmental profiles of spermidine and diethylenetriamine, on the one hand, and 1,10-diaminodecane, on the other, suggests that their effects on [³H]MK-801 binding are mediated at different sites. The altered sensitivity of the NMDA receptor to polyamines during development could reflect the expression of molecular variants with different sensitivities to modulation by polyamines.     

Characterisation of [3H]MK-801 Binding and its Cooperative Modulation by PIG Brain Membranes

Abstract

Cooperative modulation of [³H]MK-801 binding to extensively washed pig cortical brain membranes in the presence of various concentrations of L-glutamate, glycine, spermine, CPP and DCKA was evaluated in association experiments. In saturation experiments [³H]MK-801 labelled a homogeneous population of binding sites with a K_d-value of 1.26 ± 0.18 nmol 1^?1 and a B_max-value of 2130 ± 200 fmol/mg protein. The pharmacological profile of this site was further evaluated in competition experiments with known NMDA receptor channel blockers. In nonequilibrium binding experiments EC₅₀-values of reference compounds acting at the L-glutamate, at the glycine, and at the polyamine site, were determined by increasing or decreasing [³H]MK-801 binding. Ifenprodil reduced [³H]MK-801 binding in a biphasic manner. All the data obtained are in agreement with results from [³H]MK-801 binding to rodent as well as human brain membranes. This study therefore strongly suggests, that pig cortical membranes are a suitable alternative to rodent brain membranes, and an acceptable substitute for human brain membranes in [³H]MK-801 binding experiments.     

Proliferation of thyrotropin releasing hormone receptors in specific brain regions during the development of hypertension in spontaneously hypertensive rats

The binding of [3H] [3-MeHis2] thyrotropin releasing hormone [( 3H]MeTRH) to brain membranes prepared from 8 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]MeTRH bound specifically to rat brain membranes at a single high affinity site. The density (Bmax value) of [3H]MeTRH binding sites was significantly greater (28%) in SHR rats compared to WKY rats. The apparent dissociation constants (Kd values) for the binding of [3H]MeTRH in SHR and WKY rats did not differ. Binding in the various brain regions revealed that the density of [3H]MeTRH was highest in the hypothalamus followed in decreasing order by pons + medulla, midbrain, cortex and striatum. The binding of [3H]MeTRH was approximately 25% greater in cortex, hypothalamus and striatum of SHR rats in comparison to WKY rats. The binding in pons + medulla, midbrain and pituitary of SHR and WKY rats did not differ. To assess the significance of increased binding sites for [3H]MeTRH in some brain regions of SHR rats, the binding studies were carried out during normotensive and hypertensive stages of postnatal age in the two strains. In 3 and 4 week old SHR rats there was neither an increase in blood pressure nor any increase in [3H]MeTRH binding in the hypothalamus and striatum as compared to age matched WKY rats. With the development of elevated blood pressure at 6 weeks, an increase in [3H]MeTRH binding in the hypothalamus and striatum of SHR rats in comparison to the tissues from WKY rats was observed. The results provide, for the first time, evidence for a parallel increase in the density of brain TRH receptors with elevation of blood pressure, and suggest that brain TRH receptors may play an important role in the pathophysiology of hypertension.     

NMDA and AMPA Receptors Evoke Transmitter Release from Noradrenergic Axon Terminals in the Rat Spinal Cord

N-methyl-D-aspartate (NMDA) stimulated release of [³H]noradrenaline (NA) from prelabelled rat spinal cord slices. The release was partially insensitive to tetrodotoxin (TTX) and was inhibited by the NMDA antagonist MK-801. Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) also evoked release of [³H]NA, which was enhanced by blocking AMPA receptor desensitization with cyclothiazide. AMPA-evoked release was inhibited by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)-quinoxaline (NBQX) but was not affected by TTX. NMDA and AMPA showed synergistic effects, indicating co-existence of NMDA and AMPA receptors on noradrenergic terminals. Kainate evoked [³H]NA release only at high concentrations and the release was not potentiated by blocking kainate receptor desensitization with concanavalin A. Thus, the results indicate that there are stimulatory presynaptic NMDA and AMPA receptors on noradrenergic axon terminals in the spinal cord and that they interact synergistically to evoke release of [³H]NA.     

Down-regulation of alpha 2 adrenoceptors in ventrolateral medulla of spontaneously hypertensive rats.

The binding of [3H]idazoxan [correction of idaxazon] to imidazole sites and [3H]rauwolscine to alpha 2 adrenoceptors of neuronal membranes prepared from cerebral cortex and ventrolateral medulla of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats was determined. [3H]idazoxan [correction of idaxazon] bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [3H]idazoxan [correction of idaxazon] in ventrolateral medulla and cerebral cortex was found to be similar in SHR and WKY rats. [3H]Rauwolscine bound to the membranes of cerebral cortex and ventrolateral medulla at a single high affinity site. The binding of [3H]rauwolscine in the cerebral cortex was found to be similar in SHR and WKY rats. However, in the ventrolateral medulla [3H]rauwolscine binding was found to be significantly lower in SHR as compared to WKY rats. The decreased binding was due a decrease (32%) in the Bmax value in SHR rats as compared to WKY rats. The Kd values were similar in SHR and WKY rats. It is concluded that imidazole binding sites are not affected while, alpha 2 adrenergic binding sites are decreased in the ventrolateral medulla of SHR rats and may be contributing to the regulation of blood pressure.     

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.     

Interference of S-Alkyl Derivatives of Glutathione with Brain Ionotropic Glutamate Receptors

The effects of glutathione, glutathione sulfonate and S-alkyl derivatives of glutathione on the binding of glutamate and selective ligands of ionotropic N-methyl-D-aspartate (NMDA) and non-NMDA receptors were studied with mouse synaptic membranes. The effects of glutathione and its analogues on ⁴⁵Ca²⁺ influx were also estimated in cultured rat cerebellar granule cells. Reduced and oxidized glutathione, glutathione sulfonate, S-methyl-, -ethyl-, -propyl-, -butyl- and -pentylglutathione inhibited the Na⁺-independent binding of L-[³H]glutamate. They strongly inhibited also the binding of (S)-2-amino-3-hydroxy-5-[³H]methyl-4-isoxazolepropionate [³H]AMPA (IC₅₀ values: 0.8–15.9 M). S-Alkylation of glutathione rendered the derivatives unable to inhibit [³H]kainate binding. The NMDA-sensitive binding of L-[³H]glutamate and the binding of 3-[(R)-2-carboxypiperazin-4-yl][1,2-³H]propyl-1-phosphonate ([³H]CPP, a competitive antagonist at NMDA sites) were inhibited by the peptides at micromolar concentrations. The strychnine-insensitive binding of the NMDA coagonist [³H]glycine was attenuated only by oxidized glutathione and glutathione sulfonate. All peptides slightly enhanced the use-dependent binding of [³H]dizocilpine (MK-801) to the NMDA-gated ionophores. This effect was additive with the effect of glycine but not with that of saturating concentrations of glutamate or glutamate plus glycine. The glutamate- and NMDA-evoked influx of ⁴⁵Ca²⁺ into cerebellar granule cells was inhibited by the S-alkyl derivatives of glutathione. We conclude that besides glutathione the endogenous S-methylglutathione and glutathione sulfonate and the synthetic S-alkyl derivatives of glutathione act as ligands of the AMPA and NMDA receptors. In the NMDA receptor-ionophore these glutathione analogues bind preferably to the glutamate recognition site via their -glutamyl moieties.     

A Possible Role of Glutathione as an Endogenous Agonist at the N-Methyl-d-Aspartate Recognition Domain in Rat Brain

Abstract: Glutathione, both reduced (GSH) and oxidized (GSSG), was effective in displacing binding of l -[³H]-glutamic acid (l -[³H]Glu) and dl -(E)-2-[³H]amino-4-propyl-5-phosphono-3-pentenoic acid ([³H]CGP-39653) in rat brain synaptic membranes, with less potent displacement of binding of dl -α-amino-3-hydroxy-5-[³H]-methylisoxazole-4-propionic and [³H]kainic acids. Liquid chromatographic analysis revealed that both GSH and GSSG were contaminated with l -Glu by <1%. Both GSH and GSSG potentiated (+)-5-[³H]methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine ([³H]MK-801) binding in a manner similar to that found with l -Glu. Pre-treatment with glutamate dehydrogenase (GDH) induced a marked rightward shift of the concentration-response curve for l -Glu in the presence of NAD without affecting that in its absence, whereas GDH was ineffective in affecting the potentiation by both GSH and GSSG even in the presence of NAD. In the presence of GSH at a maximally effective concentration, both glycine (Gly) and spermidine potentiated [³H]MK-801 binding to a somewhat smaller extent than that found in the presence of l -Glu at a maximally effective concentration. The potentiation of [³H]MK-801 binding by GSH was invariably attenuated by addition of CGP-39653, d -2-amino-5-phosphonovaleric acid (d -AP5), and 5,7-dichlorokynurenic acid (DCKA), whereas GSH was effective in diminishing potencies of CGP-39653, d -AP5, DCKA, and 6,7-dichloroquinoxaline-2,3-dione to inhibit [³H]MK-801 binding when determined in the presence of both l -Glu and Gly. These results suggest that glutathione may be an endogenous agonist selective for the N-methyl-d -aspartate (NMDA) recognition domain on the NMDA receptor ionophore complex.     

Regional Heterogeneity of Polyamine Effects on the N-Methyl-D-Aspartate Receptor in Rat Brain

Abstract: Polyamines have pronounced effects on N-methyl-D-aspartate (NMDA) receptors in vitro and may be important modulators of NMDA receptor activity in vivo. There is considerable regional heterogeneity in the effects of polyamines on [³H]MK-801 binding in rat brain sections. For example, spermidine enhances the binding of [³H]MK-801 to a much greater extent in the striatum than in the cortex. To further explore the basis for this regional heterogeneity, the effects of polyamines on [³H]MK-801 binding were measured in well-washed membranes prepared from frontal cortex and striatum. There was no difference in the concentration-response relationship for spermidine or the K_D for [³H]MK-801 in the presence of 75 μM spermidine, suggesting that the regional difference seen in tissue sections is due to an endogenous factor that is either removed or inactivated during the preparation of membranes. Comparison of spermidine concentration-response curves in washed and unwashed tissue sections revealed that washing selectively enhanced the E_max value in the ventromedial caudate putamen without changing the EC₅₀. This is consistent with the possibility that a noncompetitive polyamine antagonist is being removed from this region during washing. There was no regional variability in the effects of the putative inverse agonist 1, 10-diaminodecane, consistent with recent suggestions that this polyamine inhibits the NMDA receptor at a site distinct from the one at which polyamines act to enhance NMDA receptor function. Agents that modulate the redox state of the NMDA receptor did not eliminate the regional heterogeneity of polyamine effects. Furthermore, the stimulatory effect of glycine in these regions did not correlate with that of spermidine. These results suggest the existence of one or more endogenous factors that noncompetitively influence the effects of polyamines in a regionspecific manner.     

Allosteric Regulation of the N-Methyl-d-Aspartate Receptor-Linked Ion Channel Complex and Effects of Ethanol in Ethanol-Withdrawal Seizure-Prone and -Resistant Mice

Abstract: The effects of ethanol, glycine, and spermidine on the specific binding of [³H]MK-801 were characterized in Triton-treated membranes prepared from the hippocampus and cortex of ethanol-withdrawal seizure-prone (WSP) and -resistant (WSR) mice. Glycine, an allosteric agonist at the NMDA receptor-linked ion channel complex, caused an increase in specific [³H]MK-801 binding to hippocampal membrane preparations. There were no significant differences in EC₅₀ values between the selected lines for the effect of glycine (WSP, 391.7 ± 48.4 nM; WSR, 313.4 ± 77 nM) in the presence of 10 µM NMDA or in the maximal response to the agonist (WSP, 1.75 ± 0.26 pmol/mg of protein; WSR, 1.67 ± 0.22 pmol/mg of protein). The EC₅₀ values for the spermidine-induced increase in [³H]MK-801 binding in membranes from hippocampus in the absence (WSP, 11.7 ± 0.83 µM; WSR, 9.98 ± 1.29 µM) or in the presence of 10 µM glycine and 10 µM NMDA (WSP, 2.1 ± 0.35 µM; WSR, 2.37 ± 0.42 µM) also did not differ. Similar results were obtained in cortical membranes. Saturation isotherms indicated that there was no difference in the density of [³H]MK-801 binding sites, or in their affinity for the radioligand, between the mouse lines. In addition, administration of ethanol by inhalation (24 h) to WSP and WSR mice did not cause an increase in the density of [³H]MK-801 binding sites, and there was no difference in the density or affinity of binding sites between the mouse lines. Withdrawal from ethanol (6 h), which causes an increase in the severity of handling-induced convulsions in WSP mice, also did not alter the binding site density or affinity for radioligand. The results suggest that the characteristics of the NMDA receptor-linked ion channel complex in the tissue preparations described here do not differ in WSP and WSR mice. Thus, genetic differences in seizure susceptibility during ethanol withdrawal can be dissociated from the total density of hippocampal or cortex NMDA receptors under activating conditions.