MK-801 Binding Assays Research Articles

Repeated ethanol treatment in adolescent rats alters cortical NMDA receptor

Earlier we have reported that repeated ethanol treatment during adolescence causes long-lasting impairments in spatial learning and memory. The present study was undertaken to determine the cellular mechanisms underlying the persistent ethanol-induced cognitive dysfunction in adolescent male rats. Since in adult animals ethanol is known to affect the N-methyl- d-aspartate (NMDA) receptor–gated ion channel, the hypothesis tested here was that adolescent ethanol exposure modulates NMDA receptor (NR) regulation in the brain. Adolescent male rats were injected daily with ethanol (2 g/kg intraperitoneally) for 5 consecutive days. Control rats received isovolumetric saline for the same number of days. Groups of control and experimental rats were sacrificed 7 days after the last ethanol/saline administration, and NR activity was measured in specific brain regions (frontal cortex, hippocampus) using the [ 3H]MK-801 binding assay. In addition, some rats were sacrificed and their brains were used to investigate changes in NR pharmacology by measuring specific NR2 subunits immunohistochemically. Compared to saline-treated controls, ethanol-treated rats showed significant increases in [ 3H]MK-801 maximal binding in the frontal cortex. This was associated with increased cortical NR2B subunit protein. [ 3H]MK-801 binding in the hippocampus was minimally affected. These results indicate that ethanol exposure during the adolescent period produces brain region–specific alterations in NR activity. These changes are different from those reported in literature for ethanol administration during the perinatal period or adulthood. Together, these data suggest that adolescence represents a unique stage in brain development in its long-term sensitivity to ethanol.

Differential and irreversible CNS ontogenic reduction in maximal MK-801 binding site number in the NMDA receptor after acute hypoxic hypoxia

CNS exposure to hypoxia impairs excitatory and inhibitory neurotransmission. Our aim was to determine variations induced by normobaric acute hypoxic hypoxia (8% O 2 for 60 min) on the NMDA receptor complex, as well as their potential reversibility after normoxic recovery. To this end, [ 3H]MK-801 binding assays to a synaptic membrane fraction isolated from chick optic lobes were performed. Previous studies throughout development had disclosed a characteristic age-dependent pattern. Results at embryonic day (ED) 12 and 18 indicated two distinct MK-801 binding sites. Hypoxic treatment failed to alter either the high affinity site dissociation constant ( K d) or its maximal binding capacity ( B max), whereas the low affinity site B max was significantly decreased (50% and 30% at ED12 and 18, respectively), without alteration in its K d values. Hypoxic embryos restored for 48 h at ED12 to normoxic conditions displayed unchanged MK-801 binding reduction, unlike those treated likewise at ED18 whose values fully recovered control levels. To conclude, hypoxic treatment reduces low affinity MK-801 B max in the NMDA receptor which proves irreversible up to ED12. Such early neuronal vulnerability may be due to post-transcriptional changes, to endocytosis followed by receptor degradation, or alternatively to cell death.

Effects of olfactory bulbectomy on NMDA receptor density in the rat brain: [ 3H] MK-801 binding assay

Olfactory bulbectomy (OBX) transects the glutamatergic efferents from the olfactory bulbs, and the changes of glutamatergic N-methyl- d-aspartate (NMDA) receptor-mediated function are though to be involved in the behavioral deficits seen in OBX rats. In the present study, irritability scores in OBX male Wistar rats were correlated with discrete regional effects on NMDA receptor function measured using a [ 3H] MK-801 binding assay. Irritability scores, measured before and for 2 weeks after OBX, showed a gradual increase in irritability after OBX. A reduction of the NMDA receptor density was observed in the cerebral cortex and amygdala 16 days after OBX, but not in the striatum, olfactory tubercle, entorhinal cortex, and hippocampus. These results demonstrate that OBX causes changes in the NMDA receptor system in certain brain regions and suggest that these changes may be responsible for the behavioral deficits of OBX rats.

Morphine tolerance increases [3H]MK‐801 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord. (National Medical Defense Center, Taipei, Taiwan) Br J Anaesth 2000;85:587–591.

N‐Methyl‐D‐aspartate (NMDA) receptor antagonists and nitric oxide synthase (NOS) inhibitors inhibit morphine tolerance. In the present study, a lumbar subarachnoid polyethylene (PE10) catheter was implanted for drug administration to study alterations in NMDA receptor activity and NOS protein expression in a morphine‐tolerant rat spinal model. Antinociceptive tolerance induced by intrathecal morphine infusion (10 μg h−1) for 5 days. Co‐administered MK801 with morphine was used to inhibit the development of morphine tolerance. Lumbar spinal cord segments were removed and prepared for [3H]MK‐801 binding assays and NOS western blotting. The binding affinity of [3H]MK‐801 was higher in spinal cords of morphine‐related rats than in control rats. There was no difference in Bmax. Western blot analysis showed that constitutive expression of neuronal NOS protein in the morphine‐tolerant group was twice that in the control group. This up‐regulation was partially prevented by MK‐801. The results suggest that morphine tolerance affects NMDA receptor binding activity and increases nNOS expression in the rat spinal cord.Comment by Octavio Calvillo, M.D., Ph.D. Morphine tolerance may be due to receptor down‐regulation or receptor uncoupling; activation of the NMDA‐dependent pain‐facilitatory system may also play a role. It has been proposed that NMDA receptor activation may play a role in morphine tolerance. NMDA receptor antagonists and nitric oxide synthase [NOS] inhibitors may prevent morphine tolerance. Tolerance was induced in rats by intrathecal injection of morphine [10 ug/h] for 5 days, co‐administration of MK801 [NMDA antagonist] with morphine was used to prevent morphine tolerance. Lumbar spinal cord segments were removed and prepared for [H3]MK801 binding assays and NOS western blotting. The binding affinity of labeled MK801 was higher in spinal cords of morphine tolerant rats than in control rats. Western blot analysis showed that constitutive expression of neuronal NOS protein in the morphine tolerant rats was twice that in the control group, thus, up‐regulation was prevented by MK801. The results suggest that morphine tolerance affect NMDA receptor binding activity and increase neuronal protein expression in rat the spinal cord.

Morphine tolerance increases [3H]MK-801 binding affinity and constitutive neuronal nitric oxide synthase expression in rat spinal cord

N-Methyl-D-aspartate (NMDA) receptor antagonists and nitric oxide synthase (NOS) inhibitors inhibit morphine tolerance. In the present study, a lumbar subarachnoid polyethylene (PE10) catheter was implanted for drug administration to study alterations in NMDA receptor activity and NOS protein expression in a morphine-tolerant rat spinal model. Antinociceptive tolerance was induced by intrathecal (i.t.) morphine infusion (10 micrograms h-1) for 5 days. Co-administered (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK-801) (10 micrograms h-1 i.t.) with morphine was used to inhibit the development of morphine tolerance. Lumbar spinal cord segments were removed and prepared for [3H]MK-801 binding assays and NOS western blotting. The binding affinity of [3H]MK-801 was higher in spinal cords of morphine-tolerant rats (mean (SEM) KD = 0.41 (0.09) nM) than in control rats (1.50 (0.13) nM). There was no difference in Bmax. Western blot analysis showed that constitutive expression of neuronal NOS (nNOS) protein in the morphine-tolerant group was twice that in the control group. This up-regulation was partially prevented by MK-801. The results suggest that morphine tolerance affects NMDA receptor binding activity and increases nNOS expression in the rat spinal cord.

Na +, K + and Ca 2+ antagonize the glutamate- and glycine-induced decrease of [ 3H]MK-801 binding observed in the presence of Mg 2+ 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-5 H-dibenzo[ a,d]cyclohepten, 5,10-imine ([ 3H]MK-801) to NMDA receptors in the presence of 1 mM Mg 2+ 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 Ca 2+ 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 Mg 2+ and 10 mM Ca 2+ at pH 7.4, the cross-over values for Na +, K +, and Ca 2+ 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 Ca 2+-sensitive, glutamate-induced decreases in [ 3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10μM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1μM) antagonized the glutamate-induced decrease in [ 3H]MK-801 binding observed in presence of Mg 2+ 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 Ca 2+ there exist a low-affinity binding site for H +, Ca 2+, 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 Mg 2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca 2+ influx through activated NMDA receptor channels.

1-Aminocylopropane-1-carboxylic acid derivatives as ligands at the glycine-binding site of the N-methyl-D-aspartate receptor

Several 1-aminocyclopropane-1-carboxylic acid derivatives were prepared and tested for activity at the glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor complex. Structural modifications involved the amino group, the carboxylic function or position 2 of the ring. When tested in a [ 3H]-MK-801 binding assay in the presence of glutamic acid, some of the compounds were able to activate the receptor. Two of them ( 3e and 6) are selective ligands for the glycine site of the NMDA receptor.

The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets.

Bilirubin neurotoxicity can be mediated by numerous mechanisms due to its increased permeability in neuronal membranes. The present study tests the hypothesis that a prolonged bilirubin infusion modifies the N-methyl-D-aspartate (NMDA) receptor/ ion channel complex in the cerebral cortex of newborn piglets. Studies were performed in seven control and six bilirubin-exposed piglets, 2-4 d of age. Piglets in the bilirubin group received a 35 mg/kg bolus of bilirubin followed by a 4-h infusion (25 mg/kg/h) of a buffer solution containing 0.1 N NaOH, 5% human albumin, and 0.055 Na2HPO4 with 3 mg/mL bilirubin. The final mean bilirubin concentration in the bilirubin group was 495.9 +/- 85.5 mumol/L (29.0 +/- 5.0 mg/dL). The control group received a bilirubin-free buffer solution. Sulfisoxazole was administered to animals in both groups. P2 membrane fractions were prepared from the cerebral cortex. [3H]MK-801 binding assays were performed to study NMDA receptor modification. The Bmax in the control and bilirubin groups were 1.20 +/- 0.10 (mean +/- SD) and 1.32 +/- 0.14 pmol/mg protein, respectively. The value for Kd in the control brains was 6.97 +/- 0.80 nM compared with 4.80 +/- 0.28 nM in the bilirubin-exposed brains (p < 0.001). [3H]Glutamate binding studies did not show a significant difference in the Bmax and Kd for the NMDA-specific glutamate site in the two groups. The results show that in vivo exposure to bilirubin increases the affinity of the receptor (decreased Kd) for [3H]MK-801, indicating that bilirubin modifies the function of the NMDA receptor/ion channel complex in the brain of the newborn piglet. We speculate that the affinity of bilirubin for neuronal membranes leads to bilirubin-mediated neurotoxicity, resulting in either short- or long-term disruption of neuronal function.

Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) in newborn piglets

Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min prior to hypoxia, which was induced by lowering the FiO2 to 5-7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO2, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P2 membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [3H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [3H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [3H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors (Bmax) and receptor binding affinity (Kd) during hypoxia. CPP treatment also decreased receptor affinity (increased Kd) for [3H]MK-801 binding during normoxia and hypoxia. Assays of [3H]glutamate binding revealed that hypoxia decreased both the Bmax and the Kd of the NMDA receptor for [3H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [3H]glutamate Bmax or Kd during normoxia. We conclude that hypoxia decreases the Bmax and Kd of the NMDA receptor glutamate recognition site for [3H]glutamate and the ion channel site for [3H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [3H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.

Effect of cerebral hypoxia on NMDA receptor binding characteristics after treatment with 3-(2-carboxypiperazin-4-yl) propyl-1-phosphonic acid (CPP) in newborn piglets

Previous studies have shown that hypoxia modifies the NMDA receptor/ion channel complex in cortical brain cell membranes of newborn piglets. The present study tests the hypothesis that blockade of the glutamate recognition site of the NMDA receptor with the competitive antagonist 3-(2-carboxypiperazin-4-yl)propyl-l-phosphonic acid (CPP) prevents modification of the receptor during hypoxia. Twenty seven anesthetized, ventilated newborn piglets were randomized into four groups: 7 normoxic (Nx), 6 CPP-treated normoxic (CPP-Nx), 8 hypoxic (Hx) and 6 CPP-treated hypoxic (CPP-Hx). Treatment groups received CPP 2 mg/kg i.v. The CPP-Hx group received CPP 30 min: prior to hypoxia, which was induced by lowering the FiO 2, to 5–7% for 1 h. Physiologic data showed no change in heart rate, blood pressure, arterial blood gas values, glucose or lactate following CPP administration. During hypoxia there was a significant decrease in PaO 2, pH and an increase in lactate compared to baseline values. The CPP-Hx group had significantly higher lactate levels than the Hx group during hypoxia. P 2 membrane fractions were prepared and thoroughly washed. Characteristics of the NMDA receptor ion channel were determined by [ 3H]MK-801 binding assays and characteristics of the glutamate recognition site by specific NMDA-displaceable [ 3H]glutamate binding assays. Brain tissue ATP and PCr levels confirmed tissue hypoxia, and were not preserved by CPP administration. [ 3H]MK-801 binding assays revealed that CPP treatment attenuated the hypoxia-induced decrease in the number of receptors ( B max) and receptor binding affinity ( K d) during hypoxia. CPP treatment also decreased receptor affinity (increased K d) for [ 3H]MK-801 binding during normoxia and hypoxia. Assays of [ 3H]glutamate binding revealed that hypoxia decreased both the B max and the Kd of the NMDA receptor for [ 3H]glutamate and both were preserved by CPP treatment prior to hypoxia. CPP had no effect on [ 3H]glutamate B max or K d during normoxia. We conclude that hypoxia decreases the B max and K d of the NMDA receptor glutamate recognition site for [ 3H]glutamate and the ion channel site for [ 3H]MK-801 in newborn piglets. These changes are prevented by CPP administration prior to hypoxia. The different effects of CPP binding during normoxia and hypoxia suggest a use-dependent mechanism for CPP binding during hypoxia, possibly through an hypoxia-induced alteration of the high-affinity binding site for CPP. During both normoxia and hypoxia CPP binding appeared to induce a conformational change in the receptor causing a decrease in binding affinity for [ 3H]MK-801. CPP administration did not preserve brain tissue ATP or PCr levels during hypoxia and may alter cellular metabolism in addition to its action at the NMDA receptor. However, even with depletion of the energy precursors ATP and PCr, and with higher lactate levels in the CPP-Hx group, CPP was able to maintain NMDA receptor binding characteristics during hypoxia and may decrease excitotoxic cellular damage from hypoxia.

Analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione: structure-activity relationship at N-methyl-D-aspartate receptor glycine sites.

A series of aromatic and azepine ring-modified analogs of 3-hydroxy-1H-1-benzazepine-2,5-dione (HBAD) were synthesized and evaluated as antagonists at NMDA receptor glycine sites. Aromatic ring-modified HBADs were generally prepared via a Schmidt reaction with substituted 2-methoxynaphthalene-1,4-diones followed by demethylation. Electrophilic aromatic substitution of benzazepine 3-methyl ethers gave 7-substituted analogs. The preparation of multiply substituted 2-methoxynaphthalene-1,4-diones was effected via Diels-Alder methodology utilizing substituted butadienes with 2-methoxybenzoquinones followed by aromatization. Structural modifications, such as elimination of the aromatic ring, removal of the 3-hydroxyl group, and transfer of the hydroxyl group from C-3 to C-4, were also studied. An initial evaluation of NMDA antagonism was performed using a [3H]MK801 binding assay. HBADs demonstrating NMDA antagonist activity as indicated by inhibition of [3H]MK801 binding were further evaluated employing a [3H]-5,7-dichlorokynurenic acid (DCKA) glycine site binding assay. Selected HBADs were characterized for functional antagonism of NMDA and AMPA receptors using electrophysiological assays in Xenopus oocytes and cultured rat cortical neurons. Antagonist potency of HBADs showed good correlation between the different assay systems. HBADs substituted at the 8-position possessed the highest potency with the 8-methyl (5), 8-chloro (6), and 8-bromo (7) analogs being the most active. For HBAD 6, the IC50 in [3H]-DCKA binding assays was 0.013 microM and the Kb values for antagonism of NMDA receptors in oocytes (NR1a/2C) and cortical neurons were 0.026 and 0.048 microM, respectively. HBADs also antagonized AMPA-preferring non-NMDA receptors expressed in oocytes but at a lower potency than corresponding inhibition of NMDA receptors. HBADs demonstrating a high potency for NMDA glycine sites showed the highest steady-state selectivity index relative to AMPA receptors. Substitution at the 6-, 7-, and 9-positions generally reduced or eliminated glycine site affinity. Moving the hydroxyl group from C-3 to C-4 reduced receptor affinity, and potency was eliminated by the removal of the aromatic ring or the hydroxyl group. These data indicate that the HBAD series has specific structural requirements for high receptor affinity. With the exception of substitution at C-8, modified HBADs generally have a lower affinity at NMDA receptor glycine sites than the parent compound 3. Mouse maximum electroshock-induced seizure studies show that the three HBADs selected for testing have in vivo potency with the 6,8-dimethyl analog (52) being the most potent (ED50 = 3.9 mg/kg, iv).

Alteration of [3H]MK-801 binding associated with the N-methyl-D-Aspartate receptor complex by acute ethanol in rat cortex and hippocampus in vitro.

We investigated the effect of ethanol on specific binding of [3H]MK-801 to the intrachannel phencyclidine (PCP) receptor site, as an index of change in the functional response of the N-methyl-D-Aspartate (NMDA)-associated ion channel. Saturation binding experiments were performed on synaptic membrane homogenates from adult rat cortex and hippocampus. [3H]MK-801 binding assays were conducted under conditions of basal, 10 microM glutamate, or 10 microM glutamate + 30 microM D-serine, with and without 50 or 100 mM ethanol. Association experiments of [3H]MK-801 binding (5 nM) were conducted under conditions of 0 or 10 microM glutamate, with varying concentrations of glycine (0.01, 0.10, and 10 microM) with and without 100 mM ethanol. Ethanol (50 and 100 mM) significantly decreased the percentage of high-affinity (open-channel state) MK-801 receptors with a concomitant increase in percentage of low-affinity receptors, but did not change high- and low-affinity constants of the two binding states. An ethanol-induced increase in the closed-channel receptor density in basal and activated conditions was suggested by the saturation experiments. Association experiments further explained this finding, in that ethanol (100 mM) significantly decreased fast component (open-channel) [3H]MK-801 binding in conditions of glycine (0.01-10 microM) only and activated conditions of glutamate + glycine (0.01-0.10 microM). However, the observed fast and slow kinetic rate constants of [3h]MK-801 binding, as well as total specific binding (fast + slow components), were not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological profile of the metabolites and potential metabolites of the anticonvulsant remacemide

Remacemide hydrochloride ((±)-2-amino- N-(1-methyl-1,2-diphenylethyl)-acetamide hydrochloride or FPL 12924AA) is a novel compound undergoing clinical trials for patients with generalized tonic/clonic and complex partial epilepsy. Remacemide exhibits efficacy against maximal electroconvulsive shock (MES) in rodents and seizures elicited by N- methyl- d , l-aspartate (NMDLA) in mice. Using rat synaptic membrane fractions, remacemide was shown to possess relatively weak noncompetitive binding to the ionic channel site of the NMDA ( N- methyl- d-aspartic acid ) receptor complex. With the hypothesis that activity against NMDLA-elicited seizures might be reflected by transformation to a more active metabolic species, the aim of the present study was to evaluate potential pharmacological effects of the 9 identified metabolites of remacemide which were all found in human and dog urine. Moreover, specific entities were recognized in plasma (including the rat's), as well as dog and rat cerebrospinal fluid. Five putative metabolites were also examined. A major route of metabolic transformation of remacemide in rats yields the formation of a pharmacologically active more potent desglycine derivative, namely FPL 12495 (±). Potency over the parent compound is revealed in the MES test in mice and rats, the NMDA-induced convulsions/ mortality test in mice, and especially involving in vitro displacement of MK801 binding to the channel subsite of the NMDA receptor. The S isomer (FPL 12859) of this desglycinate is even more potent, while the R isomer is less potent than the corresponding racemate. Unlike the non-competitive NMDA antagonist, MK801, these desglycinates did not prevent kindled seizures. Three other identified metabolites show efficacy in the mouse and rat in vivo tests, namely the N-hydroxy-desglycinate (FPL 15053) and the p-hydroxy-desglycinates (FPL 14331 and FPL 14465). FPL 15053 exhibited modest activity in all tests. The only in vivo activity exhibited by the 2 p-hydroxy-desglycinates was evidenced in the MES test following i.p. and i.v. dosing. However, FPL 14331 was active in the MK801 binding assay. An oxoacetate metabolite, FPL 15455, failed to demonstrate any biological activity. Of potential metabolites tested 2 β-Miydroxy-desglycinates (FPL 14991 and FPL 14981) displayed modest activity in the MES test, however, only FPL 14981 prevented NMDLA-induced convulsions/mortality in mice and was 2-fold more active regarding MK801 binding. The hydroxy-methyl derivative of remacemide (FPL 13592) and its desglycinate (FPL 15112) prevented MES-induced convulsions only after i.v. administration; only the desglycine derivative displaced MK801 binding. FPL 14467 ( p-dihydroxy-desglycine) was inactive in vivo and weak in the MK801 binding assay. The discrepancy between in vivo/in vitro activities for many of the compounds is presumably due to pharmacokinetic factors.

S-9-5 - Selective inhibition of NMDA receptor function by nootropic drugs: evaluation of the sites of action using mRNA-injected Xenopus oocyte responses and [3H]MK-801 binding assay.

S-9-5 - Selective inhibition of NMDA receptor function by nootropic drugs: evaluation of the sites of action using mRNA-injected Xenopus oocyte responses and [3H]MK-801 binding assay.

Synthesis and bioactivity of a new class of rigid glutamate analogs. Modulators of the N-methyl-D-aspartate receptor

A variety of derivatives of azetidine-2,4-dicarboxylic acid were synthesized and examined for their ability to stimulate 45Ca2+ uptake in cultures of cerebellar granule cells. Of the compounds tested, the cis-azetidine-2,4-dicarboxylic acid (10f) was found to be the most potent agent in potentiating glutamate, aspartate, or N-methyl-D-aspartate (NMDA) stimulated 45Ca2+ uptake at the NMDA receptor. The mechanism of action of 10f was further investigated in [3H]MK-801 binding assays and [3H]GABA release from cultured embryonic rat forebrain neurons. All of the results from the functional studies of azetidine 10f are consistent with a selectivity of action at the NMDA receptor. Moreover, azetidine 10f appears to exhibit a dual type of action, behaving as a glutamate-like agonist at higher concentrations and as a positive modulator at concentrations below 50 microM.