Aminoglycoside neurotoxicity involves NMDA receptor activation

Previous studies have led to the hypothesis that the ototoxicity produced by aminoglycoside antibiotics involves the excitotoxic activation of cochlear NMDA receptors. If this hypothesis is correct, then these antibiotics should also injure neurons within the brain. Because aminoglycosides do not readily penetrate the blood brain barrier, we examined the effects of the aminoglycoside neomycin following intrastriatal injection. Neomycin (10-250 nmol) produced dose-dependent striatal damage manifested as an increased gliosis as measured by: (1) [3H]PK-11195 binding, (2) staining for the astrocytic marker glial fibrillary acidic protein (GFAP) and (3) staining for OX-6, an MHC class II antigen expressed by microglia and macrophages. Co-injection of subthreshhold doses of NMDA potentiates the striatal damage produced by neomycin (10 nmol). Moreover, neomycin-induced striatal damage is attenuated by a combination of the NMDA antagonists ifenprodil and 5, 7-dichlorokynurenic acid. Intrastriatal administration of compounds structurally related to neomycin, but devoid of modulatory actions at NMDA receptors (paromamine and 2-deoxystreptamine), fail to produce neuronal damage. These data support the hypothesis that aminoglycoside-induced ototoxicity is, in part, an excitotoxic process involving the activation of NMDA receptors. Moreover, aminoglycosides may damage the central nervous system in individuals with compromised blood brain barriers.     

5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: novel, systemically active and broad spectrum antagonists for NMDA/glycine, AMPA, and kainate receptors

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) binding to N-methyl-D-aspartate (NMDA)/glycine receptors, [3H]-(S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding to AMPA receptors, and [3H]kainate ([3H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a-e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a-e display 2-12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and approximately 2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a-e, having IC50 values of 0.69, 1.3, and 2.4 microM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A+) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 microM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a-e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg i.v., respectively. The high in vivo potency of QXs 12a-e is particularly surprising given their low log P values (approximately -2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.     

Effect of spinal cord transection on N-methyl-D-aspartate receptors in the cord

Spinal cord injury can lead to an exaggeration of transmission through spinal pathways, resulting in muscle spasticity, chronic pain, and abnormal control of blood pressure and bladder function. These conditions are mediated, in part, by N-methyl-D-aspartate (NMDA) receptors on spinal neurons, but the effects of cord injury on the expression or function of these receptors is unknown. Therefore, antibodies to the NMDA-R1 receptor subunit and binding of [3H]MK-801 were used to assess NMDA receptors in the spinal cord. Receptor density in rats with intact spinal cords was compared to that in rats 1 and 2 weeks after spinal cord transection (SCT) at the mid-thoracic level. At 1 and 2 weeks after SCT, [3H]MK-801 binding was reduced in most laminae in cord segments caudal to the injury, whereas no decrease in amount of R1 subunit immunoreactivity was observed. No significant changes in [3H]MK-801 binding and NMDA-R1 immunoreactivity could be seen rostral to the transection. Since [3H]MK-801 binding requires an open ion channel, the discrepancy between [3H]MK-801 binding and immunocytochemistry may indicate a loss of functional receptors without a consistent change in their total number. Therefore, the exaggerated reflexes that are well established in rats 2 weeks after cord injury must be mediated by a mechanism that withstands attenuation of NMDA receptor function.     

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).     

Survey of food safety awareness among HIV positive individuals

Though opioids are known to have neuroprotective properties, little information is available on the functional state of opioidergic receptors following focal cerebral ischaemia. The present study investigated the evolution of the Bmax and Kd for [3H]DAMGO, [3H]DADLE, and [3H]U69,593, respectively, for the mu, delta, and kappa opioidergic receptors after permanent focal cerebral ischaemia in mice. While the various Kd were unchanged, mu and delta Bmax values were precociously decreased in frontoparietal cortices, earlier than kappa receptors, reflecting infarct extension with time. The Bmax values for mu and delta receptors were also altered in non-infarcted tissues, such as tissues at risk (e.g., temporal auditory cortex) and exofocal (e.g., contralateral and non-infarcted) cortices. These results suggest that, in non-infarcted areas, the observed changes reflect functional modifications to focal ischaemia.     

Global cerebral ischemia and reperfusion alters NMDA receptor binding in canine brain

We employed a canine model to test whether binding to the N-methyl-D-aspartate (NMDA) class of glutamate receptor channels is altered by global cerebral ischemia and/or reperfusion. Ischemia was induced by 10-min cardiac arrest, followed by restoration of spontaneous circulation for periods of 0, 0.5, 2, 4, and 24 h. In vitro autoradiography was performed on frozen brain sections with three radioligands: [3H]glutamate (under conditions to label the NMDA site), [3H]glycine, and [3H]MK-801. Modest decreases in [3H]glutamate and [3H]MK-801 binding were seen in several regions of hippocampus, and parietal and temporal cortex at early times after reperfusion, with values returning toward control by 24 h. In the striatum, a different pattern was seen: [3H]glutamate and [3H]MK-801 binding increased 50-200% at 0.5-4 h after the start of reperfusion, returning toward control levels by 24 h. These increases correlate with findings of increased sensitivity to NMDA-stimulated release of dopamine from striatal tissue in the same model (Werling et al., 1993), and suggest that changes in tissue receptors may contribute to the selective vulnerability to ischemic damage during the first hours following reperfusion.     

Modulation by magnesium of N-methyl-D-aspartate receptors in developing human brain

AIM: To investigate age related alterations in glutamate N-methyl-D-aspartate (NMDA) receptor binding produced by the modulatory compounds glutamate, glycine, and magnesium (Mg2+) sulphate. METHODS: The effects produced by glutamate plus glycine, and Mg2+ on the binding of [3H]MK-801, a ligand for the N-methyl-D-aspartate ion channel phencyclidine site, were measured in membrane preparations made from prefrontal cortex from human neonate (n = 5), infant (n = 6), and adult (n = 6) necropsy brains. RESULTS: Neonatal brains had the least [3H]MK-801 binding, suggesting either a low density of NMDA receptors or a more restricted access of [3H]MK-801 to cation channel sites. Infant brains had the most [3H]MK-801 binding which was stimulated to a greater extent by L-glutamate (100 microM) and glycine (10 microM) than in neonatal and adult brains. MG2+ invariably inhibited [3H]MK-801 binding. However, the Mg2+ IC50 value was higher in neonatal brain (3.6 mM) than infant (1.4 mM) and adult (0.87 mM) brains. CONCLUSION: Infant brain may have excess NMDA receptors which are hyper responsive to glutamate and glycine. The lower potency of Mg2+ to inhibit [3H]MK-801 binding in neonatal cortex may be because newborn babies have NMDA receptors without the normal complement of Mg2+ sites. The findings suggest that therapeutic NMDA receptor block in neonates requires higher concentrations of magnesium sulphate in brain tissue.     

The use of neamine as a molecular template: inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3''-phosphotransferase type IIa

Aminoglycoside 3'-phosphotransferase type IIa [APH(3')-IIa] is a member of the family of bacterial aminoglycoside-modifying enzymes. Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics. Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3')-IIa. These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.     

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 microM). 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-(3)H]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 gamma-glutamyl moieties.     

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.     

Dilated fallopian tubes: MR imaging characteristics

A complex of four proteins isolated from neuronal membranes has ligand binding sites for N-methyl-d-aspartate (NMDA) receptor agonists and antagonists and forms NMDA-activated ion channels upon reconstitution into lipid membranes. In this study, the cDNA of a subunit of this complex containing binding sites for the competitive antagonists of NMDA receptors was cloned. The cDNA clone coded for a protein of 719 amino acids (78.9 kDa). The expressed protein had binding activity for the agonists l-[3H]glutamate and [3H]glycine, the antagonist (+/-)-[3H]-(E)-2-amino-4-propyl-5-phosphonopentanoic acid ([3H]CGP 39653), but not the ion channel inhibitors. The cloned cDNA had no homology to other cloned cDNAs. Northern blot analyses indicated high expression of an 3.8 kb poly(A+) RNA in brain, but not in other tissues. These findings indicate that proteins that have recognition sites for NMDA receptor activators and inhibitors and that differ from the well-characterized NMDA receptor proteins NR1-3 are expressed in mammalian brain.     

Fenfluramine potentiates canine pulmonary vasoreactivity to endothelin-1

Glutamate may act via an N-methyl-D-Aspartate (NMDA)-sensitive receptor site to destroy cholinergic neurons within the nucleus basalis magnocellularis in age-associated neurodegenerative diseases. Multiple interesting properties of the NMDA receptor are relevant to its excitotoxic actions, e.g., glutamate is ineffective unless a glycine (gly) modulatory site is also occupied. Thus, the antagonism of glutamate receptor-related toxicity by blockade of either the NMDA-sensitive recognition site or the gly binding site may therefore have therapeutic applications. The current study investigated the ability of four novel noncompetitive antagonists at these two sites: one NMDA open channel antagonist (MRZ 2/579: 1-amino-1,3,3,5,5-pentamethyl-cyclohexane hydrochloride), and three glyB receptor antagonists (MRZ 2/570: 8-bromo-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline salt; MRZ 2/57: 8-fluoro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline; MRZ 2/576: 8-chloro-4-hydroxy-1-oxo-1,2-dihydropyridaziono [4,5-beta] quinoline-5-oxide choline) administered acutely, to provide neuroprotection from a NMDA receptor agonist within the nucleus basalis magnocellularis of young rats. Injection of NMDA into the nucleus basalis magnocellularis significantly decreased cortical choline acetyltransferase activity. Acute administration (i.p.) of MRZ 2/579, 2/570, 2/571 and 2/576 provided significant neuroprotection from NMDA.     

Neurons and fractals: how reliable and useful are calculations of fractal dimensions?

alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are critically involved in several forms of synaptic plasticity proposed to be neural substrates for learning and memory, e.g., long-term potentiation and long-term depression (LTD). The present study was designed to determine changes in cerebellar AMPA receptors following classical conditioning of the eyeblink-nictitating membrane response (NMR) in the rabbit. Quantitative autoradiography was used to assess changes in ligand binding properties of cerebellar AMPA receptors following NMR conditioning elicited by pairing electrical stimulation of the pontine nuclei with an airpuff to the eye. [3H]AMPA and [3H]-6-cyano-7-nitroquinoxaline-2,3-dion (CNQX) binding were determined following preincubation of frozen-thawed brain tissue sections at 0 or 35 degreesC. With 0 degreesC preincubation, no significant differences in [3H]AMPA binding to cerebellar AMPA receptors were seen between any of the experimental groups tested. In contrast, preincubation at 35 degreesC revealed significant decreases in [3H]AMPA binding to the trained side of the cerebellar cortex resulting from paired presentations of the conditioned and the unconditioned stimuli, while unpaired presentations of the stimuli resulted in no significant effect. With 35 degreesC preincubation, there were no significant differences in [3H]CNQX binding between any of the experimental groups and no significant differences in [3H]AMPA binding in the untrained side of the cerebellum. These results indicate that NMR conditioning is associated with a selective modification of AMPA-receptor properties in brain structures involved in the storage of the associative memory. Furthermore, they support the hypothesis that cerebellar LTD, resulting from decreased synaptic efficacy at parallel fiber-Purkinje cell synapses mediated by a change in AMPA-receptor properties, is a form of synaptic plasticity that supports this type of learning.     

Attentional selection of motion states

Glutapyrone, a disodium salt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)- glutaric acid, is a representative of a novel 'class' of amino acid-containing 1,4-dihydropyridine (DHP) compounds developed at the Latvian Institute of Organic Synthesis, Riga, Latvia. Conceptually, the glutapyrone molecule can be regarded as a dipeptide-mimicking structure formed by the "free" amino acid (glutamate) moiety and "crypto" (built into the DHP cycle) amino acid ("GABA") elements. Both of these amino acids are joined by the peptide bond. This compound unlike classical DHPs lacks calcium antagonistic or agonistic properties. Our previous studies revealed a profound and long-term anticonvulsant, stress-protective and neurodeficit-preventive activities of glutapyrone. In view of structural properties the role of glutamatergic mechanisms in the mediation of central effects of glutapyrone was considered. In the present study glutapyrone at the concentration range of 1 microM(-1) mM failed to effect both NMDA ([3H]TCP) and non-NMDA ([3H]KA and [3H]AMPA) receptor ligand binding in the rat cortical membranes in vitro. The compound markedly enhanced motor hyperactivity induced by the NMDA antagonist PCP and the dopamine releasing compound D-amphetamine in the rats. Glutapyrone displayed activity in a variety of animal models relevant for affective/depressive disorders in humans i.e. reserpine-induced ptosis and hypothermia, forced swimming test and open field test. These data indicate that the unusually "broad" pharmacological spectrum of glutapyrone might involve concomitant actions on multiple neurotransmitter systems, particularly, GABA-ergic and the catecholamines. It is discussed whether these functional properties are secondary to action on intracellular events, predominantly, G protein-related since glutapyrone appears to lack direct interactions with a number of receptors including ionotropic glutamate and GABA(A)/Bzd receptors.     

Pharmacological characterization of N-methyl-D-aspartate receptors in spinal cord of rats with a chronic peripheral mononeuropathy

N-Methyl-D-aspartate (NMDA) receptor antagonists, acting in the spinal cord, are analgesic. However, the clinical utility of these antagonists is diminished by their adverse effects on cognition and behavior. To facilitate the development of spinal cord-selective NMDA receptor antagonists, we characterized ligand interactions at NMDA receptors in spinal cord of normal rats and rats with a chronic peripheral neuropathy. NMDA receptors in spinal cord were distinguished from those in cerebral cortex on the basis of differences in the potencies of competitive and noncompetitive antagonists and on the basis of differences in their response to spermidine. D(-)-2-Amino-5-phosphonopentanoic acid (AP-5) and (+)-(1-hydroxy-3-aminopyrrolidine-2-one) (HA-966) were more potent in inhibiting NMDA-dependent [3H]TCP binding in spinal cord while, conversely, MK-801 was more potent in inhibiting [3H]TCP binding to NMDA receptors in cerebral cortex. Spermidine increased [3H]TCP binding to NMDA receptors in cerebral cortex (39+/-8%) but not spinal cord (2+/-1%). Based on these properties, NMDA receptors in spinal cord more closely resembled those in cerebellum than those in cerebral cortex. Generation of a chronic neuropathy had no effect on the density of NMDA receptors in lumbar spinal cord. There were also no major changes in the potencies of competitive antagonists or channel blocking ligands, although there was a trend for kynurenic acid and D-CPP to be more potent in the spinal cords of neuropathic animals. These findings indicate that, in both normal and neuropathic pain states, NMDA receptors in spinal cord can be distinguished pharmacologically from those in cerebral cortex. These findings underscore the feasibility of developing spinal cord-selective NMDA receptor antagonists as novel analgesics.     

Reproductive and thyroid hormone levels in rats following 90-day dietary exposure to PCB 28 (2,4,4''-trichlorobiphenyl) or PCB 77 (3,3''4,4''-tetrachlorobiphenyl)

It has been reported that suramin, an anthelminthic, trypanocidal agent and an inhibitor of P2 receptors, may antagonise N-methyl-D-aspartate (NMDA) subtype of the excitatory amino acid receptors. Both NMDA receptors and P2X subclass of P2 receptors are ligand-gated Ca2+-selective channels and, since the increased influx of Ca2+ into neurons has been linked to neurotoxicity, simultaneous inhibition of P2X and NMDA receptors in vivo by suramin could represent an effective neuroprotective treatment. We have found that suramin inhibited the binding of [3H]CGP 39653 to NMDA receptor binding sites in vitro and reduced the frequency of NMDA channel openings in patch-clamp studies. Suramin (1 mM) had no effect on [3H]kainate binding in vitro. In vivo, intracerebroventricular (I.C.V.) injections of suramin (70 nmol/brain) antagonised convulsive effects of the NMDA agonist (RS)-(tetrazol-5-yl)-glycine (TZG, LY 285265). Suramin, however, did not prevent neurotoxic lesions in the hippocampus caused by I.C.V. administration of TZG. Increasing the dose of suramin resulted in death from severe respiratory depression.     

L-2-chloropropionic acid inhibits glutamate and aspartate release from rat cerebellar slices but does not activate cerebellar NMDA receptors: implications for L-2-chloropropionic acid-induced neurotoxicity

L-2-Chloropropionic acid (L-CPA), when orally administered at single high dose to rats produces a selective lesion in the cerebellum involving destruction of a high proportion of granule cells by a mechanism which involves N-methyl-D-aspartate (NMDA) receptors. Receptor binding studies demonstrated that L-CPA a had low affinity at the glutamate and glycine binding sites at NMDA receptors (530-660 microM), respectively, whereas L-CPA did not displace [3H]AMPA, [3H]NBQX or [3H]kainate from AMPA or kainate receptors. Whole cell-patch clamp experiments using cultured granule cells failed to demonstrate changes in membrane potential of cultured granule cells when either L-CPA (0.25 or 1 microM) was added alone to the bathing solution, or in combination with glycine (10 microM). Furthermore L-CPA did not alter the magnitude of the inward current produced by application of NMDA (100 microM)) to cultured granule cells, in the presence of glycine, as measured by patch clamp techniques. Experiments were also performed to discover whether L-CPA may alter the release of the excitatory amino acids from the cerebellum, which may then indirectly alter activity at glutamate receptors, leading to neuronal cell death. L-CPA (2 mM) did not affect either basal or stimulated (electrical or high potassium) endogenous aspartate release from superfused cerebellar slices nor did it alter the basal or stimulated release of [3H]aspartate from preloaded slices when introduced into the superfusion medium over 30 min. However, when cerebellar slices were preincubated with 2 mM L-CPA for 2 h at concentrations that are known to be neurotoxic to the brain in vivo, but not in vitro, the stimulated endogenous glutamate and aspartate net release was significantly attenuated, as compared to controls. Basal release was not significantly affected by the introduction of L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum. In conclusion, although L-CPA does not appear to directly alter NMDA receptor activity the L-CPA-induced cerebellar neurotoxicity may be related to the inhibition of excitatory amino acid release from the cerebellum.     

Alterations in N-methyl-D-aspartate receptor binding in dystonic hamster brains

The genetically dystonic hamster is an animal model of idiopathic dystonia that displays sustained abnormal movements and postures either spontaneously or in response to mild environmental stimuli. Previous pharmacological studies have shown that competitive and non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists exert potent antidystonic activity in this model, indicating that abnormal NMDA receptor function may be involved in the pathophysiology of this movement disorder. Autoradiographic analysis of NMDA receptor density in 67 brain regions, using the ligand [3H] N-(1-[2-thienyl]cyclohexyl)3,4-piperidine, which binds to the phencyclidine (PCP) site in the ion channel of the NMDA receptor channel complex, revealed that NMDA receptor binding is not substantially altered in dystonic hamster brains compared to age-matched controls. Nevertheless, there was a tendency towards enhanced binding during a dystonic attack in several regions, including a 25% increase in the ventrolateral thalamic nucleus (P < 0.05), which may be associated with altered basal ganglia output. While the data do not indicate widespread abnormalities in the PCP site of the NMDA complex, they do not exclude the possibility of more pronounced changes at other regulatory binding sites of the NMDA complex or other types of glutamate receptors in dystonia.     

Activation of PKC reverses apparent NMDA receptor reduction in ALS

The binding of [3H]MK-801 to NMDA receptors was reduced by 40-45% in the dorsal and ventral horns of spinal cords from patients who died with amyotrophic lateral sclerosis (ALS) compared with controls. These results reflect either neurone death with concomitant receptor loss or regulation-related receptor decreases independent of motoneurone degeneration. To distinguish between these possibilities we explored aspects of NMDA receptor regulation using phorbol ester to activate protein kinase C (PKC). Spinal cord sections were exposed to phorbol ester before incubation with [3H]MK-801 to determine levels of NMDA binding. Phorbol ester treatment increased [3H]MK-801 binding in both ALS and control tissue to almost identical levels of specific binding for both groups. The increased [3H]MK-801 binding could be completely blocked by concurrent exposure of spinal cord sections to H-7, a general protein kinase inhibitor. These results suggest that NMDA receptors in ALS spinal cord are decreased as a result of abnormal enzyme activity independent of motoneurone degeneration.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.