Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.

Glutamate, d-(-)-2-Amino-5-Phosphonopentanoic Acid, and N-Methyl-d-Aspartate Do Not Directly Modulate Glycine Receptors.

Replication studies play an essential role in corroborating research findings and ensuring that subsequent experimental works are interpreted correctly. A previously published paper indicated that the neurotransmitter glutamate, along with the compounds N-methyl-d-aspartate (NMDA) and d-(-)-2-amino-5-phosphonopentanoic acid (AP5), acts as positive allosteric modulators of inhibitory glycine receptors. The paper further suggested that this form of modulation would play a role in setting the spinal inhibitory tone and influencing sensory signaling, as spillover of glutamate onto nearby glycinergic synapses would permit rapid crosstalk between excitatory and inhibitory synapses. Here, we attempted to replicate this finding in primary cultured spinal cord neurons, spinal cord slice, and Xenopus laevis oocytes expressing recombinant human glycine receptors. Despite extensive efforts, we were unable to reproduce the finding that glutamate, AP5, and NMDA positively modulate glycine receptor currents. We paid careful attention to critical aspects of the original study design and took into account receptor saturation and protocol deviations such as animal species. Finally, we explored possible explanations for the experimental discrepancy. We found that solution contamination with a high-affinity modulator such as zinc is most likely to account for the error, and we suggest methods for preventing this kind of misinterpretation in future studies aimed at characterizing high-affinity modulators of the glycine receptor. SIGNIFICANCE STATEMENT: A previous study indicates that glutamate spillover onto inhibitory synapses can directly interact with glycine receptors to enhance inhibitory signalling. This finding has important implications for baseline spinal transmission and may play a role when chronic pain develops. However, we failed to replicate the results and did not observe glutamate, d-(-)-2-amino-5-phosphonopentanoic acid, or N-methyl-d-aspartate modulation of native or recombinant glycine receptors. We ruled out various sources for the discrepancy and found that the most likely cause is solution contamination.

Stabilization of Ca2+-permeable AMPA receptors at perisynaptic sites by GluR1-S845 phosphorylation.

AMPA receptor (AMPAR) channel properties and function are regulated by its subunit composition and phosphorylation. Certain types of neural activity can recruit Ca(2+)-permeable (CP) AMPARs, such as GluR1 homomers, to synapses likely via lateral diffusion from extrasynaptic sites. Here we show that GluR1-S845 phosphorylation can alter the subunit composition of perisynaptic AMPARs by providing stability to GluR1 homomers. Using mice specifically lacking phosphorylation of the GluR1-S845 site (GluR1-S845A mutants), we demonstrate that this site is necessary for maintaining CP-AMPARs. Specifically, in the GluR1-S845A mutants, CP-AMPARs were absent from perisynaptic locations mainly due to lysosomal degradation. This regulation was mimicked by acute desphosphorylation of the GluR1-S845 site in wild-type mice by NMDA application. Furthermore, long-term depression (LTD) was associated with a reduction in perisynaptic CP-AMPAR levels. Our findings suggest that GluR1-S845 is necessary for maintaining CP-AMPARs on the surface, especially at perisynaptic sites, and suggest that the regulation of these receptors is involved in synaptic plasticity.

Reaction of threonine synthase with the substrate analogue 2-amino-5-phosphonopentanoate: implications into the proton transfer at the active site.

Threonine synthase catalyses the conversion of O-phospho-l-homoserine and a water molecule to l-threonine and has the most complex catalytic mechanism among the pyridoxal 5'-phosphate-dependent enzymes. In order to study the less-characterized earlier stage of the catalytic reaction, we studied the reaction of threonine synthase with 2-amino-5-phosphonopentanoate, which stops the catalytic reaction at the enamine intermediate. The global kinetic analysis of the triphasic spectral changes showed that, in addition to the theoretically expected pathway, the carbanion is rapidly reprotonated at Cα to form an aldimine distinct from the external aldimine directly formed from the Michaelis complex. The Kd for the binding of inhibitor to the enzyme decreased with increasing pH, showing that the 2-amino-group-unprotonated form of the ligand binds to the enzyme. On the other hand, the rate constants for the proton migration steps within the active site are independent of the solvent pH, indicating that protons are shared by the active dissociative groups and are not exchanged with the solvent during the course of catalysis. This gives an insight into the role of the phosphate group of the substrate, which may increase the basicity of the ε-amino group of the catalytic lysine residue in the active site.

Increased expression of the NR2A NMDA receptor subunit in the prefrontal cortex of rats reared in isolation.

A hypofunction of the N-methyl-D-aspartate (NMDA) receptor has been implicated in the pathophysiology of schizophrenia. Compelling evidence of altered NMDA receptor subunit expression in the schizophrenic brain has not, however, so far emerged. Rats reared in isolation exhibit several characteristics, including disturbed sensory gating, which resemble those seen in schizophrenia. To explore the possibility that NMDA receptor dysfunction may contribute to the behavioral and neurochemical consequences of rearing rats in isolation, we compared NMDA receptor subunit expression in brains of rats which were housed in isolation and which displayed a deficit in prepulse inhibition of the acoustic startle response with that of socially housed controls. An initial microarray analysis revealed a 1.26-fold increase in NR2A transcript in the prefrontal cortex, but not in the nucleus accumbens, of rats reared in isolation compared with those housed socially. In contrast, NR1, NR2B, NR2C, NR2D, NR3A, and NR3B subunit expression was unchanged in either brain area. In a second cohort of animals, in situ hybridization revealed increased NR2A mRNA expression in the medial prefrontal cortex, an observation that was substantiated by increased [(3)H]CGP39653 binding suggesting that NR2A receptor subunit protein expression was also elevated in the medial prefrontal cortex of the same animals. No changes in expression of NR1 or NR2B subunits were observed at both mRNA and protein level. Altered NR2A subunit expression in the medial prefrontal cortex of rats reared in isolation suggests that NMDA receptor dysfunction may contribute to the underlying pathophysiology of this preclinical model of aspects of schizophrenia.

Molecular Basis of Bacillus subtilis ATCC 6633 Self-Resistance to the Phosphono-oligopeptide Antibiotic Rhizocticin.

Rhizocticins are phosphono-oligopeptide antibiotics that contain a toxic C-terminal ( Z) -l -2-amino-5-phosphono-3-pentenoic acid (APPA) moiety. APPA is an irreversible inhibitor of threonine synthase (ThrC), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-phospho-l-homoserine to l-threonine. ThrCs are essential for the viability of bacteria, plants, and fungi and are a target for antibiotic development, as de novo threonine biosynthetic pathway is not found in humans. Given the ability of APPA to interfere in threonine metabolism, it is unclear how the producing strain B. subtilis ATCC 6633 circumvents APPA toxicity. Notably, in addition to the housekeeping APPA-sensitive ThrC ( BsThrC), B. subtilis encodes a second threonine synthase (RhiB) encoded within the rhizocticin biosynthetic gene cluster. Kinetic and spectroscopic analyses show that PLP-dependent RhiB is an authentic threonine synthase, converting O-phospho-l-homoserine to threonine with a catalytic efficiency comparable to BsThrC. To understand the structural basis of inhibition, we determined the crystal structure of APPA bound to the housekeeping BsThrC, revealing a covalent complex between the inhibitor and PLP. Structure-based sequence analyses reveal structural determinants within the RhiB active site that contribute to rendering this ThrC homologue resistant to APPA. Together, this work establishes the self-resistance mechanism utilized by B. subtilis ATCC 6633 against APPA exemplifying one of many ways by which bacteria can overcome phosphonate toxicity.

The effect of extracts of Searsia species on epileptiform activity in slices of the mouse cerebral cortex.

ETHNOPHARMACOLOGICAL RELEVANCE: Searsia dentata and Searsia pyroides are used in traditional South African medicine to treat convulsions and epilepsy. Previous studies have demonstrated that extracts of these plants comprise compounds that bind to the flumazenil-sensitive site on the GABA(A) receptor. However, their use as anticonvulsant medicinal plants cannot be adequately explained by these findings. AIMS: The aim of this study was to examine the possible involvement of the glutamatergic system of extracts from the plants. MATERIALS AND METHODS: The mouse cortical wedge preparation was used for functional characterization of the extracts. The affinity towards the NMDA and the AMPA receptor was investigated using classical [(3)H]-GP39653 and [(3)H]-AMPA binding assays, respectively. RESULTS: The extracts of Searsia dentata and Searsia pyroides inhibited the spontaneous epileptiform discharges in mouse cerebral cortical slices with ED(50) of 0.62 and 1.67mg dry extract/mL, respectively. Both extracts displaced [(3)H]-GP39653 binding and significantly inhibited the NMDA-induced response during co-administration in cortical slices. CONCLUSION: In this study, the NMDA receptor antagonistic effect of the crude ethanolic extracts of these two South African medicinal plants was demonstrated.

Crystal structures of cystathionine gamma-synthase inhibitor complexes rationalize the increased affinity of a novel inhibitor.

Cystathionine gamma-synthase catalyzes the committed step of methionine biosynthesis. This pathway is unique to microorganisms and plants, rendering the enzyme an attractive target for the development of antimicrobials and herbicides. We solved the crystal structures of complexes of cystathionine gamma-synthase (CGS) from Nicotiana tabacum with inhibitors of different compound classes. The complex with the substrate analog dl-E-2-amino-5-phosphono-3-pentenoic acid verifies the carboxylate-binding function of Arg423 and identifies the phosphate-binding pocket of the active site. The structure shows the function of Lys165 in specificity determination and suggests a role for the flexible side-chain of Tyr163 in catalysis. The importance of hydrophobic interactions for binding to the active-site center is highlighted by the complex with 3-(phosphonomethyl)pyridine-2-carboxylic acid. The low affinity of this compound is due to the non-optimal arrangement of the functional groups binding to the phosphate and carboxylate-recognition site, respectively. The newly identified inhibitor 5-carboxymethylthio-3-(3'-chlorophenyl)-1,2,4-oxadiazol, in contrast, shows the highest affinity to CGS reported so far. This affinity is due to binding to an additional active-site pocket not used by the physiological substrates. The inhibitor binds to the carboxylate-recognition site, and its tightly bent conformation enables it to occupy the novel binding pocket between Arg423 and Ser388. The described structures suggest improvements for known inhibitors and give guidelines for the development of new lead compounds.

Estradiol selectively regulates agonist binding sites on the N-methyl-D-aspartate receptor complex in the CA1 region of the hippocampus.

Estradiol alters cognitive function and lowers the threshold for seizures in women and laboratory animals. Both of these activities are modulated by the excitatory neurotransmitter glutamate in the hippocampus. To assess the hypothesis that estradiol increases the sensitivity of the hippocampus to glutamate activation by increasing glutamate binding sites, the densities of N-methyl-D-aspartate (NMDA) agonist sites (determined by NMDA displaced glutamate), competitive antagonist sites (CGP 39653), noncompetitive antagonist sites (MK801) as well as the non-NMDA glutamate receptors for kainate and AMPA (using kainate and CNQX, respectively) were measured using autoradiographic procedures. Two days of estradiol treatment increased the density of NMDA agonist, but not of competitive nor noncompetitive NMDA antagonist binding sites exclusively in the CA1 region of the hippocampus. The density of noncompetitive NMDA antagonist sites, however, was decreased in the dentate gyrus by estradiol treatment. Ovarian steroids had no effect on the density of kainate or AMPA receptors in any region of the hippocampus examined. These data indicate that the agonist and antagonist binding sites on the NMDA receptor/ion channel complex are regulated independently by an as yet unidentified mechanism, and that this regulation exhibits regional specificity in the hippocampus. The increase in NMDA agonist sites with ovarian hormone treatment should result in an increase in the sensitivity of the hippocampus to glutamate activation which may mediate some of the effects of estradiol on learning and epileptic seizure activity.

Mechanistic distinctions between excitotoxic and acidotic hippocampal damage in an in vitro model of ischemia.

Excitotoxicity is believed to underlie the selective loss of vulnerable neurons after transient ischemia, while lactic acidosis seems to be the principal feature and probable cause of tissue infarcts. Primary hippocampal cultures containing both neurons and astrocytes derived from fetal rats were used to examine the relative contributions of and interactions between excitotoxic and acidotic cell injury. Hypoxia-induced damage was energy dependent and involved the N-methyl-D-aspartate (NMDA) receptor. Glucose above 1 mM could completely protect against hypoxia-induced injury in a pH range of 7.4-6.5, while the NMDA receptor antagonist D,L-2-amino-5-phosphonovaleric acid (500 microM) during the posthypoxic period provided only partial protection in the absence of glucose. Astrocyte cultures were undamaged by ischemic-like treatment in this pH range, suggesting that hypoxia-induced cell death in mixed cultures was restricted to neurons. Lowering the extracellular pH to 7.0 and 6.5 caused no neuronal damage in normoxic controls, but in each case provided significant protection against hypoxic neuronal injury. In contrast, a second type of neurotoxicity was observed after a 6-h exposure to pH 6.0, while exposure to pH 5.5 was required to kill astrocytes. This acidotic damage appeared to be energy independent and did not involve the NMDA receptor. These results suggest that excitotoxic neuron death has an energetic component and that acidosis may produce both protective and damaging effects in the hippocampus during ischemic insults.

Cocaine-induced kindling is associated with elevated NMDA receptor binding in discrete mouse brain regions.

The present study was undertaken to investigate the involvement of N-methyl-D-aspartate (NMDA) type of glutamate receptors in the induction and maintenance of kindling generated by daily cocaine (35 mg/kg) injections to Swiss Webster mice. In addition, the regulation of NMDA receptor binding following the development of sensitization to horizontal locomotor activity produced by daily injections of a low dose of cocaine (15 mg/kg for 5 days) was investigated. Three days following the administration of the high dose of cocaine (35 mg/kg) a marked augmentation in cocaine-induced horizontal and vertical activities was observed (induction phase). Subsequently, after 10 days of cocaine administration, mice developed stage 5 seizures (Racine scale). Binding of [3H]CGP 39653 to the NMDA receptors revealed a marked increase in receptor densities in the striatum, amygdala and hippocampus associated with the induction phase. The elevation of NMDA receptor binding in the striatum and amygdala was sustained for 10 days following the induction phase. The pattern of altered NMDA receptor binding following the expression of cocaine kindled seizures was different. One day after the expression of kindled seizures NMDA receptor binding was elevated in striatum, amygdala, hippocampus and frontal cortex. However, only the elevation of NMDA receptor binding in the amygdala and hippocampus was sustained for 10 days following the expression of cocaine kindled seizures. In the brains of mice sensitized to the low dose of cocaine (15 mg/kg) no change in NMDA receptor binding was observed compared with control values. The present findings suggest the following: (a) The induction of cocaine kindling is associated with increased NMDA receptor binding activity in the striatum, amygdala and hippocampus; (b) the maintenance of cocaine kindling depends on increased NMDA receptor binding in the amygdala and hippocampus; (c) sensitization to cocaine-induced horizontal locomotor activity may be independent of elevation in NMDA receptor binding.

Potent quinoxaline-spaced phosphono alpha-amino acids of the AP-6 type as competitive NMDA antagonists: synthesis and biological evaluation.

A series of alpha-amino-3-(phosphonoalkyl)-2-quinoxalinepropanoic acids was synthesized and evaluated for NMDA receptor affinity using a [3H] CPP binding assay. Functional antagonism of the NMDA receptor complex was evaluated in vitro using a stimulated [3H]TCP binding assay and in vivo by employing an NMDA-induced seizure model. Some analogues also were evaluated in the [3H]-glycine binding assay. Several compounds of the AP-6 type show potent and selective NMDA antagonistic activity both in vitro and in vivo. In particular alpha-amino-7-chloro-3-(phosphonomethyl)-2-quinoxalinepropanoic acid (1) displayed an ED50 of 1.1 mg/kg ip in the NMDA lethality model. Noteworthy is alpha-amino-6,7-dichloro-3-(phosphonomethyl)-2-quinoxalinepropanoic++ + acid (3) with a unique dual activity, displaying in the NMDA receptor binding assay an IC50 of 3.4 nM and in the glycine binding assay an IC50 of 0.61 microM.

Synthesis and binding properties of 2-amino-5-phosphono-3-pentenoic acid photoaffinity ligands as probes for the glutamate recognition site of the NMDA receptor.

Four omega-benzoylated (E)-2,10-diamino-4-(phosphonomethyl)dec-3-enoic acids have been synthesized and tested in vitro for binding affinity to the glutamate recognition site of the NMDA (N-methyl-D-aspartate) receptor. The omega-4-azidosalicylamide derivative 24 was iodinated to give the photoaffinity ligand 25 (CGP 55802 A) which showed an IC50 value of 34 nM in the in vitro [3H]CGP 39653 binding assay. This compound and its radioactive 125I-form are the first photoaffinity ligands for the NMDA receptor with high affinity to the glutamate recognition site.

Potent, orally active, competitive N-methyl-D-aspartate (NMDA) receptor antagonists are substrates for a neutral amino acid uptake system in Chinese hamster ovary cells.

A series of enantiomerically pure (phosphonomethyl)-substituted phenylalanine derivatives related to SDZ EAB 515 (1) were prepared as competitive N-methyl-D-aspartate (NMDA) receptor antagonists. Unlike most known competitive NMDA antagonists, analogs in this series with the S-configuration are potent NMDA antagonists whereas analogs with the unnatural R-configuration are weak NMDA antagonists, as determined by receptor binding experiments and their anticonvulsant action in mice. Examination in a previously reported competitive NMDA pharmacophore model revealed that receptor affinity can be explained partially by a cavity that accommodates the biphenyl ring of 1, while the biphenyl ring of the R-enantiomer 2 extends into a disallowed steric region. We proposed that analogs with the natural S-configuration and a large hydrophobic moiety would have an advantage in vivo over analogs with an R-configuration by being able to use a neutral amino acid uptake system to enhance both peripheral adsorption and transport into the brain. Examination in a system L neutral amino acid transport carrier assay shows that 1 competes with L-Phe for transport in an apparent competitive and stereospecific manner (estimated Ki = 50 microM). The 1- and 2-naphthyl derivatives 3a,3b were found to be among the most potent, competitive NMDA antagonists yet discovered, being ca. 15-fold more potent than 1 in vitro and in vivo, with a long duration of action. The title compound 3a had potent oral activity in MES (ED50 = 5.0 mg/kg). 3a also retains its ability to compete, albeit more weakly than 1 (estimated Ki = 200 microM), for L-Phe uptake to CHO cells. In this series, analogs with the R-configuration are not substrates for the system L neutral amino acid transport carrier. These results provide evidence that central nervous system active agents can be designed as substrates of a neutral amino acid transporter as a means to enhance penetration of the blood-brain barrier.

A comparative analysis of the neuroprotective properties of competitive and uncompetitive N-methyl-D-aspartate receptor antagonists in vivo: implications for the process of excitotoxic degeneration and its therapy.

Injection of the N-methyl-D-aspartate receptor agonist, quinolinic acid, into the rat striatum in vivo results in the degeneration of cholinergic and GABAergic neurons, as determined seven days later using the marker enzymes, choline acetyltransferase and glutamate decarboxylase, respectively. Such damage was dose-dependently prevented by CGP 37849 or MK-801 (competitive and uncompetitive N-methyl-D-aspartate receptor antagonists, respectively) administered systemically or intrastriatally at the same time as quinolinic acid. The neuroprotective activity of CGP 37849 was associated with the D-enantiomer, CGP 40116 (ED50 7.5 mg/kg i.p.), which was approximately 1.5-fold and 3.5-fold more potent than the related compounds, D-CPPene and CGS 19755, respectively. CGP 37849 was a weaker neuroprotectant than MK-801 (ED50 0.8 mg/kg i.p) when administered systemically, but was dramatically more potent following coinjection with quinolinic acid (ED50's 0.2 and 117 nmol, respectively). When injected intrastriatally 0.5-2 h post-quinolinic acid, CGP 37849 was protective over the entire period studied, whereas MK-801 was less effective at all post-quinolinic acid injection times. The finding that CGP 37849 is neuroprotective when administered intrastriatally 1-2 h post-quinolinic acid supports the hypothesis that a period exists following excitotoxic insult in which neurons are not committed to die, and can be rescued by blockade of ongoing N-methyl-D-aspartate receptor activation. Competition studies indicated that, when coinjected with 100-400 nmol quinolinic acid into the striatum, CGP 37849 exhibited kinetics predicted of a competitive N-methyl-D-aspartate receptor antagonist (declining neuroprotective potency with increasing doses of agonist), whereas MK-801 displayed a complex picture, with weak protective activity at low doses of quinolinic acid. Following systemic administration, neither antagonist was markedly affected by the dose of excitotoxin. When given i.p. at up to 6 h post-quinolinic acid, CGP 37849 and MK-801 showed essentially identical profiles of post-insult protection; degeneration of cholinergic neurons was reduced significantly throughout the entire post-insult period, whereas GABAergic neurons were protected only when drugs were administered 2 h or earlier post-quinolinic acid. The data indicate that competitive and uncompetitive N-methyl-D-aspartate receptor antagonists are effective neuroprotectants in vivo, and that parameters such as drug lipophilicity or mechanism of action at the receptor do not impinge upon their properties as systemically active cerebroprotectants.

Regionally different N-methyl-D-aspartate receptors distinguished by ligand binding and quantitative autoradiography of [3H]-CGP 39653 in rat brain.

1. Binding of D,L-(E)-2-amino-4-[3H]-propyl-5-phosphono-3-pentenoic acid ([3H]-CGP 39653), a high affinity, selective antagonist at the glutamate site of the N-methyl-D-aspartate (NMDA) receptor, was investigated in rat brain by means of receptor binding and quantitative autoradiography techniques. 2. [3H]-CGP 39653 interacted with striatal and cerebellar membranes in a saturable manner and to a single binding site, with KD values of 15.5 nM and 10.0 nM and receptor binding densities (Bmax values) of 3.1 and 0.5 pmol mg-1 protein, respectively. These KD values were not significantly different from that previously reported in the cerebral cortex (10.7 nM). 3. Displacement analyses of [3H]-CGP 39653 in striatum and cerebellum, performed with L-glutamic acid, 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) and glycine showed a pharmacological profile similar to that reported in the cerebral cortex. L-Glutamic acid and CPP produced complete displacement of specific binding with Ki values not significantly different from the cerebral cortex. Glycine inhibited [3H]CGP 39653 binding with shallow, biphasic curves, characterized by a high and a low affinity component. Furthermore, glycine discriminated between these regions (P < 0.005, one-way ANOVA), since the apparent Ki of the high affinity component of the glycine inhibition curve (KiH) was significantly lower (Fisher's protected LSD) in the striatum than the cortex (33 nM and 104 nM, respectively). 4. Regional binding of [3H]-CGP 39653 to horizontal sections of rat brain revealed a heterogeneous distribution of binding sites, similar to that reported for other radiolabelled antagonists at the NMDA site (D-2-[3H]-amino-5-phosphonopentanoic acid ([3H]-D-AP5) and [3H]-CPP). High values of binding were detected in the hippocampal formation, cerebral cortex and thalamus, with low levels in striatum and cerebellum. 5. [3H]-CGP 39653 binding was inhibited by increasing concentrations of L-glutamic acid, CPP and glycine. L-Glutamic acid and CPP completely displaced specific binding in all regions tested, with similar IC50 values throughout. Similarly, glycine was able to inhibit the binding in all areas considered: 10 microM and 1 mM glycine reduced the binding to 80% and 65% of control (average between areas) respectively. The percentage of specific [3H]-CGP 39653 binding inhibited by 1 mM glycine varied among regions (P < 0.05, two-ways ANOVA). Multiple comparison, performed by Fisher's protected LSD method, showed that the inhibition was lower in striatum (72% of control), with respect to cortex (66% of control) and hippocampal formation (58% of control). 6. The inhibitory action of 10 microM glycine was reversed by 100 microM 7-chloro-kynurenic acid (7-CKA), a competitive antagonist of the glycine site of the NMDA receptor channel complex, in all areas tested. Moreover, reversal by 7-CKA was not the same in all regions (P < 0.05, two-ways ANOVA). In fact, in the presence of 10 microM glycine and 100 microM 7-KCA, specific [3H]-CGP 39653 binding in the striatum was 131% of control, which was significantly greater (Fisher's protected LSD) than binding in the hippocampus and the thalamus (104% and 112% of control, respectively). 7. These results demonstrate that [3H]-CGP 39653 binding can be inhibited by glycine in rat brain regions containing NMDA receptors; moreover, they suggest the existence of regionally distinct NMDA receptor subtypes with a different allosteric mechanism of [3H]-CGP 39653 binding modulation through the associated glycine site.

Antinociceptive effects after intrathecal administration of phosphodiester-, 2'-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse.

In the present study, we have compared the antinociceptive effect of three different types of antisense oligodeoxynucleotides targeting the N-methyl-D-aspartate (NMDA) R1-subunit in mice. The probes were administrated intrathecally three times during a period of 5 days (1, 5 or 25 microg/injection), followed by evaluation using the formalin test. The antinociceptive effect was correlated to in vitro receptor binding in spinal cord sections. The tissue distribution was studied after a single injection of fluorescein-conjugated probes. The phosphodiester probe showed superficial tissue penetration after 30 min and disappeared within 2 h. The probe did, however, significantly reduce both receptor binding in laminae I and II (by 36-44% compared to saline) as well as pain behavior (32% compared to saline), without apparent side effects. The mismatched probe was ineffective at 25 microg, while some reductions in receptor binding and pain behavior were seen after 5 microg. The C-5-propyne-modified phosphorothioate probe showed pronounced tissue penetration and cellular uptake as soon as 30 min after injection which was still detectable after 24 h. Immediately after injection of the highest dose, long-lasting hind-limb paralysis was observed. Receptor binding was reduced but not in a dose-related manner. Pain behavior was significantly reduced by 40% following 25 microg of antisense probe but not after lower doses or 25 microg of mismatched probe. The 2'-O-allyl-modified probe did not significantly reduce receptor binding or pain behavior. Thus, only the phosphodiester probe showed a significant correlation between reduction in pain behavior and receptor binding. These findings demonstrate that antisense technology is associated with specificity problems, but still could provide a valuable tool to study the role of different target proteins in the drug discovery process.

Effects of L-trans-pyrrolidine-2,4-dicarboxylate and L-threo-3-hydroxyaspartate on the binding of [3H]L-aspartate, [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and [3H]kainate studied by autoradiography in rat forebrain.

L-trans-Pyrrolidine-2,4-dicarboxylate (L-t-PDC) and L-threo-3- hydroxyaspartate (L-t-3OHA), compounds known to interact strongly with the Na(+)-dependent high affinity uptake of excitatory amino acids in central nervous tissue, were tested as potential inhibitors of binding to glutamate receptors and transport sites in frozen sections of rat brain. [3H] alpha-amino-3-hydroxy- 5-methyl-4-isoxazolepropionate (AMPA), [3H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and [3H] kainate were used as ligands for the binding sites on the "non-NMDA" classes of glutamate receptors and [3H]DL-(E)-2-amino-4-propyl-5-phosphono-3-pentenoate (CGP 39653) was used to label NMDA receptor binding sites. The Na(+)-dependent glutamate-uptake site was marked by [3H]L-aspartate. The autoradiograms, obtained by exposing 3H-sensitive film to sections of rat forebrain preincubated with 3H-labelled ligands, were scanned by laser beam and quantified. Distribution patterns of the receptor and transporter sites visualized by the 3H-labelled ligands were compatible with previously published results. [3H]CNQX binding, however, was found to be significantly decreased by Na+.L-t-3OHA was about an order of magnitude stronger than L-t-PDC as an inhibitor of [3H]L-aspartate binding. Neither of the compounds had any important effect at the "non-NMDA" receptor binding sites but L-t-3OHA was a weak inhibitor of [3H]CGP 39653 binding (< 40% at 100 microM). The results suggest that, at low nanomolar concentrations, both compounds are likely to be selective for Na(+)-dependent high affinity glutamate transporter sites. Moreover, L-t-3OHA seems to have a sufficiently high affinity for the site to be almost certainly useful, if available in a 3H-labelled form, as a ligand in autoradiographic studies.

Inhibition of [3H]CGP 39653 binding to NMDA receptors by a P2 antagonist, suramin.

Suramin, which has been used for more than 50 years as an anthelmintic in humans, has recently been shown to inhibit P2 purinoceptors in the CNS and to block glutamate-evoked excitatory potentials in hippocampus. We now report that suramin inhibits (IC50 approximately 100 microM) the binding of [3H]CGP 39653, a ligand specific for NMDA-type glutamate receptors in brain. Consequently, suramin at concentrations used sometimes in physiological experiments may act as both P2x and NMDA antagonist. Since both NMDA receptors and P2x-subtype of purinoceptors form Ca(2+)-selective channels, suramin may block Ca2+ entry into neurones depolarized by either ATP, L-glutamate or any other neurotransmitter or drug acting via P2x or NMDA receptors.

A novel fluorescent GSH-adduct binds to the NMDA receptor.

In an attempt to develop various fluorescent probes to label glutathione (GSH) receptors, we have serendipitously synthesized a probe that binds to and antagonizes the NMDA receptor. Probe 1, a GSH adduct, displaces the competitive NMDA antagonist [3H]-CGP 39653 with a higher affinity than NMDA or cysteine in rat synaptic membranes. In recording experiments from a rat cortical 'wedge' preparation, Probe 1 reversibly blocks both NMDA- and cysteine-induced depolarization. In mixed astrocyte-neuron tissue culture preparations, Probe 1 labels parts of both cell bodies as well as processes. The present data suggest that Probe 1 binds to the NMDA receptor and antagonizes channel function.