Autoantibodies associated with Type I diabetes mellitus persist after diagnosis in children

We have examined the effects of riluzole, a neuroprotective drug which stabilizes voltage-dependent sodium channels in their inactivated state and inhibits the release of glutamate in-vivo and in-vitro, on the release of newly taken up [3H]dopamine induced by ouabain, a potent and selective inhibitor of Na+/K+-ATPase in mouse striatal slices in-vitro. Riluzole potently (IC50 (concentration resulting in 50% inhibition) = 0.9+/-0.3 microM) and dose-dependently antagonized ouabain-stimulated [3H]dopamine release, the effect being observed at low concentrations. Tetrodotoxin (1 microM) and nomifensine (10 microM) also abolished ouabain-induced [3H]dopamine release. Blockade of glutamate receptors with dizocilpine (1 microM) and 6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione (YM-90K; 10 microM), alone or in combination, was without effect. Incubation of striatal slices with 50 microM La3+, which blocks voltage-dependent calcium channels, did not inhibit [3H]dopamine release induced by ouabain. The potent effects of riluzole observed in this model are probably related to its ability to block voltage-dependent sodium channels. The consequences of this activity are critically discussed in relation to the protective action of riluzole previously reported in various models of Parkinson's disease and other neurodegenerative disorders.     

Binding characteristics of a potent AMPA receptor antagonist [3H]Ro 48-8587 in rat brain

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.     

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.     

Comparative antagonism of kainate-activated kainate and AMPA receptors in hippocampal neurons

Native kainate receptors expressed by cultured hippocampal cells were studied in the whole-cell configuration of the patch-clamp technique by using a fast perfusion system. About 80% of the neurons expressed kainate receptors independently of the time in culture (0-4 days), which coincided with the number of cells immunoreactive for a monoclonal antibody against the GluR5/6/7 subunits. Three types of cells were considered: neurons in which the rapid application of kainate induced a rapidly desensitizing current, cells in which kainate induced a more slowly rising, non-desensitizing, response and those in which a mixture of both responses was apparent. Steady responses induced by 300 microM kainate were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) in a dose-dependent manner (IC50 = 0.92 microM). CNQX was less potent in blocking transient kainate-induced responses (IC50 = 6.1 microM). Responses to kainate, whether steady or transient, were also inhibited by NS102, showing poor selectivity for the transient response (IC50 = 4.1 and 2.2 microM respectively). The new alpha-amino-3-hydroxy-5-methyl-4-isoxazole (AMPA) receptor antagonist NS394 was very potent in inhibiting steady kainate-induced currents (IC50 = 0.45 microM), but was even more effective in preventing peak responses (IC50 = 0.13 microM). In contrast, cyclothiazide did not affect transient kainate-induced responses but did potentiate current induced by activation of AMPA receptors by AMPA or kainate. These results demonstrate the lack of complete selectivity amongst some available competitive antagonists for AMPA and kainate receptors, and indicate that kainate receptors expressed by hippocampal cells lack the cyclothiazide modulatory site present at AMPA receptors. In addition, the present data support the idea that low-affinity kainate binding sites in the brain correspond to receptor channels selectively activated by kainate.     

Delayed treatment with YM90K, an AMPA receptor antagonist, protects against ischaemic damage after middle cerebral artery occlusion in rats

The neuroprotective effect of an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist YM90K [6-(1H-imidazol-1-yl)-7-nitro-2,3(1H,4H)-quinoxalinedione monohydrochloride] has been examined in a rat middle cerebral artery occlusion model. Intravenous infusion of YM90K (2.5-20mgkg(-l)h(-l) for 4h) starting immediately after occlusion of the middle cerebral artery significantly reduced the cortical infarct volume 24h after occlusion compared with the control group. The protection at the highest dose was 39% (P < 0.05). Similar protective effects were observed when YM90K (20mgkg(-1)h(-1) for 4h) was delayed up to 2h after middle cerebral artery occlusion (45% reduction, P < 0.05). CNS1102 [N-(1-naphthyl)-N'-(3-ethylphenyl)-N'-methylguanidine hydrochloride], a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, also reduced the cortical infarct volume when 1.13mgkg(-1) was administered by intravenous bolus injection immediately after middle cerebral artery occlusion, followed by intravenous infusion at 0.785mgkg(-l)h(-1) for 4h (35% reduction, P<0.05). This neuroprotective effect was not observed when administration was delayed lh after middle cerebral artery occlusion. These results suggest that AMPA receptors might play a more important role than NMDA receptors in the late development of neuronal cell damage after focal cerebral ischaemia and that AMPA receptor blockade would be one beneficial strategy in treating acute stroke.     

Kainate excitotoxicity is mediated by AMPA- but not kainate-preferring receptors in embryonic rat hippocampal cultures

We investigated kainate-induced excitotoxicity in embryonic rat hippocampal cells cultured in a chemically defined medium. Treatment with kainate for 24 h resulted in neuronal death, as assessed by the release of lactate dehydrogenase into the culture media. This neurotoxic effect was kainate dose- and culture age-dependent. EC50 of kainate was 127 +/- 11 microM. 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo (f)quinoxaline (NBQX) completely blocked the toxicity, while MK801, an N-methyl-D-aspartate (NMDA) receptor antagonist, also blocked it but not completely. Furthermore, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) attenuated the kainate injury, while the selective and noncompetitive AMPA-preferring receptor antagonist 1-(4-aminophenyl)-4-methyl-7, 8-methylenedioxy-5H-2,3-benzo-diazepine (GYKI 52466) blocked it completely. Concanavalin A (ConA), which potentiates the response to kainate at kainate-preferring receptors, had little effect on kainate toxicity. Further, AMPA alone induced little toxicity, but produced remarkable toxicity when cyclothazide was used to block the desensitization of AMPA-preferring receptors. These results indicate that kainate excitotoxicity in hippocampal cultures is mediated by AMPA- but not kainate-preferring receptors, and that it involves NMDA-receptor-mediated toxicity. The non-desensitizing response at AMPA-preferring receptors may play an important role in kainate-induced excitotoxicity.     

Structure--activity studies for alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid receptors: acidic hydroxyphenylalanines

Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid (AMPA) receptors may have therapeutic potential as psychotropic agents. A series of mononitro- and dinitro-2- and 3-hydroxyphenylalanines was prepared, and their activity compared with willardiine, 5-nitrowillardiine, AMPA, and 2,4,5-trihydroxyphenylalanine (6-hydroxydopa) as inhibitors of specific [3H]AMPA and [3H]kainate binding in rat brain homogenates. The most active compounds were highly acidic (pKa 3-4), namely, 2-hydroxy-3,5-dinitro-DL-phenylalanine (13; [3H]AMPA IC50 approximately equal to 25 microM) and 3-hydroxy-2,4-dinitro-DL-phenylalanine (19; [3H]AMPA IC50 approximately equal to 5 microM). Two other dinitro-3-hydroxyphenylalanines, and 3,5-dinitro-DL-tyrosine, were considerably less active. Various mononitrohydroxyphenylalanines, which are less acidic, were also less active or inactive, and 2- and 3-hydroxyphenylalanine (o- and m-tyrosine) were inactive. Compounds 13 and 19, DL-willardiine (pKa 9.3, [3H]AMPA IC50 = 2 microM), and 5-nitro-DL-willardiine (pKa 6.4, [3H]AMPA IC50 = 0.2 microM) displayed AMPA > kainate selectivity in binding studies. Compound 19 was an AMPA-like agonist, but 13 was an antagonist in an AMPA-evoked norepinephrine release assay in rat hippocampal nerve endings. Also, compound 13 injected into the rat ventral pallidum antagonized the locomotor activity elicited by systemic amphetamine.     

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.     

The synergistic effects of vitamin E and selenium in iron-overloaded mouse hearts

In the present study, we report that low concentrations of the glutamate ionotropic agonist kainate decreased the turnover of [3H]-phosphoinositides ([3H]-InsPs) induced by muscarinic receptors in the chick embryonic retina. When 100 microM carbachol was used, the estimated IC50 value for kainate was 0.2 microM and the maximal inhibition of approximately 50% was obtained with 1 microM or higher concentrations of the glutamatergic agonist. Our data also show that veratridine, a neurotoxin that increases the permeability of voltage-sensitive sodium channels, had no effect on [3H]-InsPs levels of the embryonic retina. However, 50 microM veratridine, but not 50 mM KCl, inhibited approximately 65% of the retinal response to carbachol. While carbachol increased [3H]-InsPs levels from 241.2 +/- 38.0 to 2044.5 +/- 299.9 cpm/mg protein, retinal response decreased to 861.6 +/- 113.9 cpm/mg protein when tissues were incubated with carbachol plus veratridine. These results suggest that the accumulation of phosphoinositides induced by activation of muscarinic receptors can be inhibited by the influx of Na+ ions triggered by activation of kainate receptors or opening of voltage-sensitive sodium channels in the chick embryonic retina.     

Heteroaryl analogues of AMPA. 2. Synthesis, absolute stereochemistry, photochemistry, and structure-activity relationships

We have previously shown that (S)-2-amino-3-(3-hydroxy-5-phenyl-4-isoxazolyl)propionic acid [(S)-APPA, 2] is a weak agonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors, specifically activated by (S)-AMPA (1), whereas (S)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-4-isoxazolyl]propionic acid [(S)-2-Py-AMPA, 5] and (RS)-2-amino-3-[3-hydroxy-5-(2-thiazolyl)-4-isoxazolyl]propionic acid (4) are potent AMPA agonists. On the other hand, (R)-APPA (3) and (R)-2-Py-AMPA (6) have been shown to be weak AMPA antagonists. We now report the synthesis of 2-Py-AMPA (7a) and the isomeric compounds 3-Py-AMPA (7b) and 4-Py-AMPA (7c) as well as the 7a analogues, (RS)-2-amino-3-[3-hydroxy-5-(6-methyl-2-pyridyl)-4-isoxazolyl]p ropion ic acid (7d) and (RS)-2-amino-3-[3-hydroxy-5-(2-quinolinyl)-4-isoxazolyl]propionic acid (7e). Furthermore, (RS)-2-amino-3-[3-hydroxy-5-(2-furyl)-4-isoxazolyl]propionic acid (2-Fu-AMPA, 7f) and its 5-bromo-2-furyl derivative (7g) were synthesized, and (S)-2-Fu-AMPA (8) and (R)-2-Fu-AMPA (9) were prepared by semipreparative chiral HPLC resolution of 7f. HPLC analyses and circular dichroism spectroscopy indicated the absolute stereochemistry of 8 and 9 to be S and R, respectively. This was confirmed by an X-ray crystallographic analysis of 9.HCl. In receptor binding (IC50 values) and rat cortical wedge electrophysiological (EC50 values) studies, 7c (IC50 = 5.5 +/- 0.6 microM; EC50 = 96 +/- 5 microM) was shown to be markedly weaker than 7a (IC50 = 0.57 +/- 0.16 microM; EC50 = 7.4 +/- 0.2 microM) as an AMPA agonist, whereas 7b,d,e were inactive. The very potent AMPA agonist effect of 7f (IC50 = 0.15 +/- 0.03 microM; EC50 = 1.7 +/- 0. 2 microM) was shown to reside exclusively in 8 (IC50 = 0.11 +/- 0.01 microM; EC50 = 0.71 +/- 0.11 microM), whereas 9 did not interact significantly with AMPA receptors, either as an agonist or as an antagonist. 8 was shown to be photochemically active and is a potential photoaffinity label for the recognition site of the AMPA receptors. Compound 7g turned out to be a very weak AMPA receptor agonist (IC50 = 12 +/- 0.7 microM; EC50 = 160 +/- 15 microM). None of these new compounds showed detectable effects at N-methyl-d-aspartic acid (NMDA) or kainic acid receptors in vitro. The present studies have emphasized that the presence of a heteroatom in the 2-position of the heteroaryl 5-substituent greatly facilitates AMPA receptor agonist activity.     

Neuroprotective effects of NMDA receptor glycine recognition site antagonism: dependence on glycine concentration

High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1-10 microM) before 500 microM glutamate, 30 microM NMDA, or 300 microM kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 mM) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 microM-1 mM) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 microM ACEA 1021 was similar for glutamate and NMDA (18 +/- 3 and 29 +/- 9 microM, respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

Pharmacological differentiation of kainate receptors on neonatal rat spinal motoneurones and dorsal roots

The objectives of this study, conducted on neonatal rat spinal cord and dorsal roots in vitro, were to characterise the actions of a range of willardiine analogues on GluR5-containing kainate receptors present in dorsal roots, to determine whether GluR5-containing receptors are also present on motoneurones, and to differentiate responses mediated by kainate receptors from those mediated by AMPA receptors on motoneurones. (S)-5-Trifluoromethyl-willardiine, (S)-5-iodowillardiine, (S)-5-iodo-6-azawillardiine and ATPA were found to be potent agonists of kainate receptors on dorsal roots (EC50 values 0.108 +/- 0.002, 0.127 +/- 0.010, 0.685 +/- 0.141 and 1.3 +/- 0.3 microM, respectively) being more potent but of lower efficacy than kainate (EC50 value 14.8 +/- 1.8 microM). (S)-5-Iodo-6-azawillardiine blocked kainate-induced depolarisations of the dorsal root, probably via its desensitising action. Kainate-induced responses of dorsal roots were weakly antagonised by (RS)-3,5-dicarboxyphenylglycine (DCPG) (apparent KD 1.5 +/- 0.4 mM). Kainate receptors containing GluR5 subunits do not appear to be present on motoneurones since (RS)-3,5-DCPG (1 mM) potentiated rather than antagonised kainate-induced depolarisations of motoneurones. Although (S)-5-iodowillardiine (a potent and selective agonist at GluR5-containing kainate receptors) depolarised motoneurones (EC50 value 5.8 +/- 0.6 microM), such depolarisations were antagonised by both (RS)-3,4- and (RS)-3,5-DCPG, which are selective AMPA receptor antagonists at motoneurones, showing a KD value of 73 microM (Schild slope, 0.96 +/- 0.09) and an apparent KD value of 123 +/- 38 microM, respectively. This accords with the previously reported activity of willardiine analogues at AMPA receptors. Since neither (RS)-3,4- nor (RS)-3,5-DCPG antagonised kainate-induced motoneuronal depolarisations but cyclothiazide enhanced and GYK153655 blocked these responses it is possible that a component of the kainate response may be mediated by a population of DCPG-insensitive AMPA receptors on motoneurones. However, it is also possible that a population of kainate receptors other than those containing GluR5 subunits, are responsible for these effects. The new compounds introduced in this study are likely to be useful tools for studying the physiological role of kainate receptors in CNS function.     

Synthesis of the four isomers of 4-aminopyrrolidine-2,4-dicarboxylate: identification of a potent, highly selective, and systemically-active agonist for metabotropic glutamate receptors negatively coupled to adenylate cyclase

The four isomers of 4-aminopyrrolidine-2,4-dicarboxylate (APDC) were prepared and evaluated for their effects at glutamate receptors in vitro. (2R,4R)-APDC (2a), an aza analog of the nonselective mGluR agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (1S,3R)-ACPD, 1), was found to possess relatively high affinity for metabotropic glutamate receptors (mGluRs) (ACPD-sensitive [3H]glutamate binding IC50 = 6.49 +/- 1.21 microM) with no effects on radioligand binding to NMDA, AMPA, or kainate receptors up to 100 microM. None of the other APDC isomers showed significant mGluR binding affinity, indicating that this interaction is highly stereospecific. Both 1 and 2a were effective in decreasing forskolin-stimulated cAMP formation in the adult rat cerebral cortex (EC50 = 8.17 +/- 2.21 microM for 1; EC50 = 14.51 +/- 5.54 microM for 2a); however, while 1 was also effective in stimulating basal tritiated inositol monophosphate production in the neonatal rat cerebral cortex (EC50 = 27.7 +/- 5.2 microM), 2a (up to 100 microM) was ineffective in stimulating phosphoinositide hydrolysis in this tissue preparation, further supporting our previous observations that 2a is a highly selective agonist for mGluRs negatively coupled to adenylate cyclase. Microelectrophoretic application of either 1 or 2a to intact rat spinal neurons produced an augmentation of AMPA-induced excitation (95 +/- 10% increase for 1, 52 +/- 6% increase for 2a). Intracerebral injection of 1 (400 nmol) produced characteristic limbic seizures in mice which are not mimicked by 2a (200-1600 nmol, ic). However, the limbic seizures induced by 1 were blocked by systemically administered 2a in a dose-dependent manner (EC50 = 271 mg/kg, ip). It is concluded that (2R,4R)-APDC (2a) is a highly selective, systemically-active agonist of mGluRs negatively coupled to adenylate cyclase and that selective activation of these receptors in vivo can result in anticonvulsant effects.     

Differential expression of extracellular matrix and cell adhesion molecules in the olfactory nerve and glomerular layers of adult rats

A series of five 6,7-disubstituted 1,4-dihydro-2,3-quinoxalinediones was prepared, two of which are known microbial flavin metabolites and three of which are potential flavin metabolites. Four of the five compounds inhibited specific binding of [3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropanoic acid ([3H]AMPA), [3H]kainic acid, and [3H]6-cyano-1,4-dihydro-7-nitro-2,3-quinoxalinedione ([3H]CNQX) in rat brain homogenate fractions, with IC50 values in the low micromolar range (the fifth compound competed only with [3H]CNQX). Two of the compounds were moderately potent AMPA antagonists in an in vitro functional test.     

Characterization of (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine binding in rat brain

[(2S,2'R,3'R)-2-(2',3'-[3H]Dicarboxycyclopropyl)glycine ([3H]DCG IV) binding was characterized in vitro in rat brain cortex homogenates and rat brain sections. In cortex homogenates, the binding was saturable and the saturation isotherm indicated the presence of a single binding site with a K(D) value of 180 +/- 33 nM and a Bmax of 780 +/- 70 fmol/mg of protein. The nonspecific binding, measured using 100 microM LY354740, was <30%. NMDA, AMPA, kainate, L(-)-threo-3-hydroxyaspartic acid, and (S)-3,5-dihydroxyphenylglycine were all inactive in [3H]DCG IV binding up to 1 mM. However, several compounds inhibited [3H]DCG IV binding in a concentration-dependent manner with the following rank order of potency: LY341495 = LY354740 > DCG IV = (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S,2'S)-2-methyl-2-(2-carboxycyclopropyl)glycine > L-glutamate = ibotenate > quisqualate > (RS)-alpha-methyl-4-phosphonophenylglycine = L(+)-2-amino-3-phosphonopropionic acid > (S)-alpha-methyl-4-carboxyphenylglycine > (2S)-alpha-ethylglutamic acid > L(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-L-aspartyl-L-glutamic acid inhibited the binding in a biphasic manner with an IC50 of 0.2 microM for the high-affinity component. The binding was also affected by GTPgammaS, reducing agents, and CdCl2. In parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1, 3, and 4 > 2, 5, and 6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, subiculum, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for pre- and postsynaptic metabotropic glutamate mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to GTPgammaS show that [3H]DCG IV binding is probably to group II metabotropic glutamate receptors in rat brain.     

Pharmacology and toxicology of ATOA, an AMPA receptor antagonist and a partial agonist at GluR5 receptors

(RS)-2-Amino-3-[3-(carboxymethoxy)-5-tert-butyl-4-isoxazolyl]propi onic acid (ATOA) has previously been described as an antagonist at (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptors with an IC50 value of 150 microM towards AMPA-induced depolarisation in the rat cortical wedge preparation. ATOA has now been shown also to be a partial agonist at recombinant GluR5 receptors, expressed in Xenopus oocytes, with an EC50 value of 170 microM and a relative efficacy of 0.17 +/- 0.04 compared with responses produced by kainic acid (1.0). Using cultured cerebral cortical neurones as a test system and leakage of lactate dehydrogenase (LDH) as an indicator of cell damage, ATOA was shown to be cytotoxic (ED50 > 300 microM), though much less toxic than the structurally related dual AMPA and GluR5 agonist, (RS)-2-amino-3-(3-hydroxy-5-tert-butyl-4-isoxazolyl)propionic acid (ATPA) (ED50 = 14 +/- 2 microM). The toxic effect of ATPA was sensitive to 6,7-dinitroquinoxaline-2,3-dione (DNQX) but was not significantly reduced by the selective AMPA receptor antagonist, (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA). The toxicity of ATOA (1 mM) could not be significantly attenuated by co-administration of AMOA (300 microM) or DNQX (25 microM). A structure-activity analysis indicates that the tert-butyl group of ATPA and ATOA facilitates the interaction of these compounds with GluR5 receptors.     

In vivo and in vitro evaluation of AMPA receptor antagonists in rat hippocampal neurones and cultured mouse cortical neurones

The effects of four glutamate receptor antagonists on alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)- and N-methyl-D-aspartate (NMDA)-responses were evaluated using both in vitro and in vivo electrophysiological techniques: whole cell patch-clamp recordings from cultured mouse cortical neurones and microiontophoresis in the rat hippocampus. The compounds tested were NBQX (2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline), GYKI 52466 (1-(4-amino-phenyl)-4-methyl-7,8-methyl-endioxyl-5H-2,3-benzodiaze pine), PNQX (pyrido[3, 4-f]quinoxaline-2,3-dione, 1,4,7,8,9,10-hexahydro-9-methyl-6-nitro-, methanesulfonate), NS377 (7-ethyl-5-phenyl-1,6,7,8-tetrahydro-1,7-diaza-as-indacene-2 ,3-dione), and MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenz(a,d)cycloheptene-5,10-imine hydrogen maleate). In vitro, the IC50 values (in microM) for inhibition of AMPA-evoked inward currents were approximately 0.4 for NBQX, approximately 7.5 for GYKI 52466, approximately 1 for PNQX and approximately 15 for NS377. PNQX and NS377 also inhibited NMDA-induced currents with IC50 values at approximately 5 and approximately 18 microM, respectively, while NBQX at 60 microM and GYKI 52466 at 100 microM had only weak effects. The ED50 values in micromol/kg i.v. for inhibition of AMPA-evoked hippocampal neuronal spike activity in vivo were approximately 32 for NBQX, approximately 19 for GYKI 52466, approximately 17 for PNQX and approximately 11 for NS377 with efficacy values (maximal inhibition) between 71% and 81%. The ED50 values (in [Lmol/kg i.v.) and efficacy values for inhibition of NMDA-evoked hippocampal neuronal spike activity were approximately 28 with an efficacy of 61% for NBQX, approximately 16 with 35% for PNQX and approximately 6 with 61% for NS377. GYKI 52466 did not significantly affect NMDA responses, whereas MK-801 showed NMDA specificity in vivo.     

Effects of beta-amyloid-(25-35) peptides on radioligand binding to excitatory amino acid receptors and voltage-dependent calcium channels: evidence for a selective affinity for the glutamate and glycine recognition sites of the NMDA receptor

The neurotoxic fragment corresponding to residues 25-35 of the beta-amyloid (A beta) peptide [A beta-(25-35)] has been shown to exert effects on (+)-[3H]5-methyl-10, 11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-imine maleate ([3H]MK-801) binding to the cation channel of the N-methyl-D-aspartate (NMDA) receptor. In the present study, we investigated whether the amidated and carboxylic acid C-terminated forms of A beta-(25-35) [A beta-(25-35-NH2) and A beta-(25-35-COOH), respectively] exert effects on other excitatory amino acid receptor and cation channel types in rat cortical membranes. Both A beta-(25-35-NH2) and A beta-(25-35-COOH) gave statistically significant dose-dependent inhibitions of [3H]glutamate and [3H]glycine binding to the agonist recognition sites of the NMDA receptor. Ten microM A beta-(25-35-NH2) and A beta-(25-35-COOH) gave 25% and 20% inhibitions of [3H]glutamate binding and 75% and 70% inhibitions of [3H]glycine binding, respectively. A beta-(25-35-NH2), but not A beta-(25-35-COOH), gave a small (ca. 17% at 10 microM) statistically significant increase of [3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) binding. [3H]kainate binding was not significantly affected by either peptide. Similarly, neither peptide affected either the maximal level or EC50 value for calcium stimulation of [3H]nitrendipine binding. It is concluded that A beta-(25-35) shows slight affinity for the agonist recognition sites of the NMDA receptor, but not for other excitatory amino acid receptor types or for L-type voltage-dependent calcium channels.     

Postsynaptic factors in the expression of long-term potentiation (LTP): increased glutamate receptor binding following LTP induction in vivo

Several lines of evidence indicate that LTP in the hippocampus is associated with a change in the properties of postsynaptic glutamate receptors. In the present study, we used quantitative autoradiography to examine the binding properties of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate subclasses of glutamate receptors in frozen brain sections obtained from rats in which perforant-path LTP was induced in vivo. Induction of LTP resulted in a selective increase in [3H]AMPA binding in those hippocampal subfields receiving perforant-path axons. Increases in [3H]AMPA binding in dentate gyrus (stratum moleculare) were highly correlated with the magnitude of LTP recorded in this structure. Scatchard analyses of [3H]AMPA and 6-cyano-7-nitro-[3H]quinoxaline-2,3-dione (an AMPA receptor antagonist) binding in the dentate gyrus indicated that LTP induction resulted in an increase in the number of AMPA receptor binding sites. No changes in the binding of 3H-labeled N-[1-(thienyl)cyclohexyl]piperidine (an N-methyl-D-aspartate receptor antagonist) were observed in any hippocampal subfield. These results suggest that a modification in postsynaptic AMPA receptors plays a role in the expression of synaptic enhancement following LTP induction in the hippocampus.     

NMDA and non-NMDA receptor antagonists protect against excitotoxic injury in the rat spinal cord

The neuroprotective properties of the N-methyl-D-aspartate (NMDA) antagonist dizocilpine (MK-801) and the non-NMDA antagonists 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline (NBQX) and alpha-methyl-4-carboxyphenylglycine (MCPG) were evaluated against neuronal injury produced by the intraspinal injection of NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). Forty-nine animals were divided into eight groups in order to evaluate the effects of different drug combinations: (a) NMDA; (b) NMDA + MCPG; (c) NMDA + NBQX; (d) NMDA + MK-801; (e) AMPA; (f) AMPA + MCPG; (g) AMPA + MK-801; and (h) AMPA + NBQX. Drugs were microinjected into spinal segments T12-L3 through a micropipette attached to a Hamilton microliter syringe. Spinal cords were evaluated after a survival period of 48 h at which time NMDA and AMPA were found to produce morphological changes over the concentration ranges of 125-500 mM and 75-500 microM, respectively. Neuronal loss following injections of NMDA + MK-801 or AMPA + NBQX was significantly less than that following injections of NMDA or AMPA alone. By contrast, neuronal loss following co-injections of NMDA or AMPA with inappropriate antagonists, i.e., NMDA + NBQX/MCPG or AMPA + MCPG/MK-801, was not significantly different from that produced by NMDA or AMPA. The results suggest that elevations in spinal levels of glutamate followed by prolonged activation of NMDA and AMPA receptor subtypes initiate an excitotoxic cascade resulting in neuronal injury. Blockade of NMDA and AMPA effects by MK-801 and NBQX respectively confirms the well documented neuroprotective effects of these drugs and lends support to the potential importance of NMDA and especially AMPA receptor antagonists as therapeutic agents in the treatment of acute spinal cord injury.