Differential roles of mGlu8 receptors in the regulation of glutamate and gamma-aminobutyric acid release at periaqueductal grey level

We investigated the role of group III metabotropic glutamate (mGlu) receptors on glutamate and GABA releases at the periaqueductal grey (PAG) level by using in vivo microdialysis in rats. Intra-PAG perfusion of either L-(+)-2-amino-4-phosphonobutyric acid (L-AP4, 100-300 microM), (RS)-4-phosphonophenylglycine ((RS)-PPG, 100-300 microM) selective agonists of group III mGlu receptors, or (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG, 50-100 microM), a selective agonist of mGlu8 receptor, increased glutamate and decreased GABA extracellular concentrations. (RS)-alpha-methylserine-O-phosphate (MSOP, 0.5 mM), a selective group III receptor antagonist, perfused in combination with (S)-3,4-DCPG, L-AP4 or (RS)-PPG, antagonised the effects induced by these agonists on both extracellular glutamate and GABA values. alpha-Methyl-3-methyl-4-phosphonophenylglycine (UBP1112, 300 microM), a group III mGlu receptor antagonist, perfused in combination with (RS)-PPG or (S)-3,4-DCPG, antagonised the effects induced by these agonists. Intra-PAG perfusion with forskolin (100 microM), an activator of adenylate cyclase, increased dialysate glutamate and GABA levels. Moreover, intra-PAG perfusion with N-[2-(p-bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89) (100 microM), a protein kinase (PKA) inhibitor, abolished the effect of (S)-3,4-DCPG on both glutamate and GABA releases. H-89, per se, did not modify glutamate release but reduced extracellular GABA value at the higher dosage used (200 microM). These data suggest that group III mGlu receptors in the PAG modulate the releases of glutamate and GABA conversely. In particular, both the facilitation of glutamate and the inhibition of GABA releases require the participation of coupling to adenylate cyclase and the subsequent activation of the PKA pathway.     

Systemic administration of the potent mGlu8 receptor agonist (S)-3,4-DCPG induces c-Fos in stress-related brain regions in wild-type,but not mGlu8 receptor knockout mice

The effect of a novel and potent metabotropic glutamate 8 (mGlu8) receptor agonist, (S)-3,4-dicarboxyphenylglycine (DCPG), was studied in vivo in mouse brain. c-Fos expression was used as a marker of neuronal activity in specific brain regions 2 h after systemic (S)-3,4-DCPG (3-100 mg/kg, i.p.). The selectivity of (S)-3,4-DCPG on mGlu8 receptors was determined in mGlu8 receptor knockout mice. In wild-type mice, (S)-3,4-DCPG (100 mg/kg) significantly increased c-Fos expression in several stress-related brain regions: paraventricular nucleus of the hypothalamus, central nucleus of the amygdala, lateral parabrachial nucleus and locus coeruleus. In the central nucleus of the amgydala, more than 92% of c-Fos positive neurons were identified as GABAergic inhibitory neurons after (S)-3,4-DCPG. Moreover, (S)-3,4-DCPG significantly induced c-Fos in the superficial gray layer of the superior colliculus, a central visual region. c-Fos expression was unchanged by (S)-3,4-DCPG in mGlu8 receptor knockout mice. Our results indicate that systemic (S)-3,4-DCPG alters neuronal excitability in specific brain regions via mGlu8 receptor. The prominent activation of stress areas suggests a role for mGlu8 receptors in the central integration of stress responses. Furthermore, our results indicate that systemic (S)-3,4-DCPG can be used as a tool to explore behavioral and cellular consequences of mGlu8 receptor activation.     

Reduction of excitatory transmission in the retino-collicular pathway via selective activation of mGlu8 receptors by DCPG

We have previously shown that activation of Group III metabotropic glutamate (mGlu) receptors modulates synaptic transmission in the superior colliculus. We thus investigated the effect of the selective mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine (DCPG) on excitatory synaptic transmission in the superficial superior colliculus (SC) using an in vitro slice preparation of the rat SC. Field EPSPs evoked by optic tract stimulation under conditions of GABA receptor blockade were reduced by DCPG by up to 67.8+/-5.46% (EC(50) 1.25+/-0.56 microM), and this effect could be antagonised by LY341495 at a concentration (300 nM) known to be effective at mGlu8 receptors but not at mGlu4 or mGlu7 receptors. The broad-spectrum (mGlu4/mGlu7/mGlu8) agonist L-2-amino-4- phosphonobutyric acid (L-AP4) produced similar reductions of synaptic transmission (maximal reduction 68.6+/-2.33%; EC(50) 5.7+/-2.61 microM). These data are consistent with previous results which show that mGlu8 receptor activation can reduce synaptic transmission in the spinal cord, and indicate that similar mechanisms operate in other brain areas. Furthermore, this indicates that the mGlu8 receptor may have a role in the modulation of visual transmission in the superior colliculus.     

Anticonvulsant activity of 3,4-dicarboxyphenylglycines in DBA/2 mice

The 3,4-dicarboxyphenylglycines (3,4-DCPG) inhibit sound-induced seizures in DBA/2 mice with the racemate being notably more potent than either isomer (ED(50) (nmol, i.c.v.)): (RS)-3,4-DCPG (0.004; 86 mg/kg, i.p.)>the mGlu(8) agonist (S)-3,4-DCPG (0.11)>the AMPA antagonist (R)-3,4-DCPG (0.38). A potentiation of anticonvulsant activity between AMPA and mGlu(8) receptors was confirmed by combining (R)-3,4-DCPG with the mGlu(8) agonist (RS)-4-phosphonophenylglycine. This potentiating mechanism provides a novel strategy for the treatment of epileptic seizures.     

Effects of (S)-3,4-DCPG, an mGlu8 receptor agonist, on inflammatory and neuropathic pain in mice

In this study, the effect of (S)-3,4-dicarboxyphenylglycine (DCPG), a selective mGlu8 receptor agonist, has been investigated in inflammatory and neuropathic pain models in order to elucidate the role of mGlu8 receptor in modulating pain perception. Inflammatory pain was induced by the peripheral injection of formalin or carrageenan in awake mice. Systemic administration of (S)-3,4-DCPG, performed 15 min before formalin, decreased both early and delayed nociceptive responses of the formalin test. When this treatment was carried out 15 min after the peripheral injection of formalin it still reduced the late hyperalgesic phase. Similarly, systemic (S)-3,4-DCPG reduced carrageenan-induced thermal hyperalgesia and mechanical allodynia when administered 15 min before carrageenan, but no effect on pain behaviour was observed when (S)-3,4-DCPG was given after the development of carrageenan-induced inflammatory pain. When microinjected into the lateral PAG (RS)-alpha-methylserine-O-phoshate (MSOP), a group III receptor antagonist, antagonised the analgesic effect induced by systemic administration of (S)-3,4-DCPG in both of the inflammatory pain models. Intra-lateral PAG (S)-3,4-DCPG reduced pain behaviour when administered 10 min before formalin or carrageenan; both the effects were blocked by intra-lateral PAG MSOP. (S)-3,4-DCPG was ineffective in alleviating thermal hyperalgesia and mechanical allodynia 7 days after the chronic constriction injury of the sciatic nerve, whereas it proved effective 3 days after surgery. Taken together these results suggest that stimulation of mGlu8 receptors relieve formalin and carrageenan-induced hyperalgesia in inflammatory pain, whereas it would seem less effective in established inflammatory or neuropathic pain.     

Potential antipsychotic and extrapyramidal effects of (R,S)-3,4-dicarboxyphenylglycine [(R,S)-3,4-DCPG], a mixed AMPA antagonist/mGluR8 agonist

An involvement of glutamatergic transmission in schizophrenia has been postulated for several years. According to that view, hypofunction of NMDA receptors and a compensatory increase in glutamate release which overstimulates non-NMDA receptors contributes to psychotic symptoms. Therefore, potential antipsychotic drugs are searched for among compounds which block AMPA receptors and inhibit glutamate release. (R,S)-3,4-dicarboxyphenylglycine [(R,S)-3,4-DCPG] is a mixed antagonist of AMPA receptors and agonist of an autoreceptor, i.e. metabotropic glutamate receptor 8. The aim of the study was to look for putative antipsychotic properties of (R,S)-3,4-DCPG in the model of locomotor stimulation induced by amphetamine or phencyclidine in mice. Moreover, a risk of extrapyramidal side-effects induced by this compound was examined, as capability to induce catalepsy in the bar test and to increase the proenkephalin mRNA expression, measured autoradiographically in striatal slices by in situ hybridization. (R,S)-3,4-DCPG (80 mg/kg i.p.) decreased the amphetamine (2.5 mg/kg s.c.)-but not phencyclidine (3 mg/kg s.c.)-induced hyperactivity. That dose of (R,S)-3,4-DCPG did not decrease the spontaneous locomotor activity of mice. However, a dose of 100 mg/kg ip of that compound evoked catalepsy and enhanced the catalepsy and striatal proenkephalin mRNA expression induced by haloperidol (1-2 mg/kg i.p.). The study seems to suggest that (R,S)-3,4-DCPG may possess antipsychotic properties at doses close to those evoking extrapyramidal side-effects which speaks for its rather typical than atypical neuroleptic profile.     

JNJ16259685, a highly potent, selective and systemically active mGlu1 receptor antagonist

We examined the pharmacological profile of (3,4-dihydro-2H-pyrano[2,3]b quinolin-7-yl) (cis-4-methoxycyclohexyl) methanone (JNJ16259685). At recombinant rat and human metabotropic glutamate (mGlu) 1a receptors, JNJ16259685 non-competitively inhibited glutamate-induced Ca2+ mobilization with IC50 values of 3.24+/-1.00 and 1.21+/-0.53 nM, respectively, while showing a much lower potency at the rat and human mGlu5a receptor. JNJ16259685 inhibited [3H]1-(3,4-dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-2-phenyl-1-ethanone ([3H]R214127) binding to membranes prepared from cells expressing rat mGlu1a receptors with a Ki of 0.34+/-0.20 nM. JNJ16259685 showed no agonist, antagonist or positive allosteric activity toward rat mGlu2, -3, -4 or -6 receptors at concentrations up to 10 microM and did not bind to AMPA or NMDA receptors, or to a battery of other neurotransmitter receptors, ion channels and transporters. In primary cerebellar cultures, JNJ16259685 inhibited glutamate-mediated inositol phosphate production with an IC50 of 1.73+/-0.40 nM. Subcutaneously administered JNJ16259685 exhibited high potencies in occupying central mGlu1 receptors in the rat cerebellum and thalamus ( ED50=0.040 and 0.014 mg/kg, respectively). These data show that JNJ16259685 is a selective mGlu1 receptor antagonist with excellent potencies in inhibiting mGlu1 receptor function and binding and in occupying the mGlu1 receptor after systemic administration.     

Lack of the antianxiety-like effect of (S)-3,4-DCPG, an mGlu8 receptor agonist, after central administration in rats

Substances acting as agonists of group III mGlu receptors were shown to induce an antianxiety-like effect after intrahippocampal administration to rats. The purpose of the present study was to establish whether the selective mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG) induced an anxiolytic-like effect after injection into the basolateral amygdala nuclei or the CA1 region of the hippocampus in the conflict drinking Vogel test in rats. The obtained results indicate that (S)-3,4-DCPG (10, 50 and 100 nmol/rat) produces no anticonflict effect in rats. We conclude that selective stimulation of mGlu8 receptors (a subtype of group III mGluRs) does not evoke anxiolytic-like activity, and that the mGlu8 receptors are of no significance for anxiolytic-like effects of group III mGluR agonists.     

Metabotropic glutamate autoreceptors of the mGlu(5) subtype positively modulate neuronal glutamate release in the rat forebrain in vitro

In the present study we have examined the role of presynaptic group I metabotropic glutamate (mGlu) receptors in the control of neuronal glutamate release using rat forebrain slices pre-loaded with [(3)H]D-aspartate. We have also addressed the question of which group I mGlu receptor subtype, mGlu(1) or mGlu(5), mediates the facilitatory response observed by the use of a range of established and some more novel agonists and antagonists showing selectivity for these receptors. The electrically-stimulated release of pre-loaded [(3)H]D-aspartate from rat forebrain slices was markedly potentiated by the potent group I mGlu receptor agonist, L-quisqualic acid (L-QUIS), in a concentration-dependent manner (EC(50) 17.31 microM). This response was inhibited by the mGlu receptor antagonists (S)-MCPG (100 microM) and (RS)-MTPG (100 microM) but not by the AMPA-type ionotropic glutamate receptor antagonist, NBQX (100 microM). The selective group I mGlu receptor agonist (S)-3, 5-dihydroxyphenylglycine ((S)-DHPG) also enhanced electrically-stimulated efflux of label, although responses diminished with high (10-100 microM) concentrations of the agonist. Maximum responses were fully restored when (S)-DHPG (10 microM) was applied in the presence of the proposed mGlu(5) receptor desensitization inhibitor, cyclothiazide (10 microM). The positive modulatory response to (S)-DHPG (1 microM) was powerfully inhibited by (S)-MCPG (IC(50) 0.08 microM) but was resistant to the mGlu(1) receptor antagonists, (RS)-AIDA (1-500 microM), CPCCOEt (0.1-100 microM) and (+)-2-methyl-4-carboxyphenylglycine (LY367385) (0.1-10 microM). The recently developed, selective mGlu(5) receptor agonist (RS)-2-chloro-5-hydroxyphenylglycine ((RS)-CHPG) enhanced electrically-stimulated [(3)H]D-aspartate efflux from rat forebrain slices with a similar concentration-response profile to that of (S)-DHPG. Responses to this receptor subtype-selective agonist were also blocked by (S)-MCPG (IC(50) 1.13 microM) but were unaffected by (RS)-AIDA (500 microM), CPCCOEt (100 microM) or LY367385 (10 microM). These results indicate that the positive modulation of neuronal glutamate release seen in the rat forebrain in the presence of group I mGlu receptor agonists is mediated by presynaptically located mGlu(5) glutamate autoreceptors. The pharmacological profile of these receptors appears to be distinct from that of postsynaptic mGlu receptors. Novel antagonists acting at these presynaptic receptors may provide new drugs for the experimental therapy of a range of acute or chronic neurodegenerative disorders.     

Group I mGlu receptors potentiate synaptosomal [3H]glutamate release independently of exogenously applied arachidonic acid

In the current study, we have characterized group I metabotropic glutamate (mGlu) receptor enhancement of 4-aminopyridine (4AP)-evoked [3H]glutamate release from rat cerebrocortical synaptosomes. The broad spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD, 10 microM) increased 4AP-evoked [3H]glutamate release (143.32+/-2.73% control) only in the presence of exogenously applied arachidonic acid; an effect reversed by the inclusion of bovine serum albumin (BSA, fatty acid free). In contrast, the selective group I mGlu receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) potentiated (EC50 = 1.60+/-0.25 microM; Emax = 147.61+/-10.96% control) 4AP-evoked [3H]glutamate release, in the absence of arachidonic acid. This potentiation could be abolished by either the selective mGlu1 receptor antagonist (R,S)-1-aminoindan-1,5-dicarboxylic acid (AIDA, 1 mM) or the selective PKC inhibitor (Ro 31-8220, 10 microM) and was BSA-insensitive. The selective mGlu5 receptor agonist (R,S)-2-chloro-5-hydroxyphenylglycine (CHPG, 300 microM) was without effect. DHPG (100 microM) also potentiated both 30 mM and 50 mM K+ -evoked [3H]glutamate release (121.60+/-12.77% and 121.50 +/-4.45% control, respectively). DHPG (100 microM) failed to influence both 4AP-stimulated 45Ca2+ influx and 50 mM K+ -induced changes in synaptosomal membrane potential. Possible group I mGlu receptor suppression of tonic adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptor activity is unlikely since 4AP-evoked [3H]glutamate release was insensitive to the selective inhibitory receptor antagonists 8-cyclopentyl-1,3-dimethylxanthine, (R,S)-alpha-cyclopropyl-4-phosphonophenylglycine or CGP55845A, respectively. These data suggest an 'mGlu1 receptor-like' receptor potentiates [3H]glutamate release from cerebrocortical synaptosomes in the absence of exogenously applied arachidonic acid. This PKC dependent effect is unlikely to be via modulation of synaptosomal membrane potential or voltage-activated Ca2+ channels and not via a suppression of tonically active inhibitory adenosine A1 receptor, group II/III mGlu receptors or GABA(B) receptors.     

The group II metabotropic glutamate receptor agonist, DCG-IV, alleviates akinesia following intranigral or intraventricular administration in the reserpine-treated rat

1. This study examined whether activation of group II metabotropic glutamate (mGlu) receptors in the substantia nigra pars reticulata (SNr) could reverse akinesia in a rodent model of Parkinson's disease (PD). 2. Male Sprague Dawley rats, stereotaxically cannulated above either the SNr or third ventricle, were rendered akinetic by injection of reserpine (5 mg kg-1 s.c.). Eighteen hours later, the rotational behaviour induced by unilateral injection of the group II mGlu receptor agonist, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV), was examined. 3. Following intranigral injection, DCG-IV (0.125-0.75 nmol in 0.1 microliter) produced a dose-dependent increase in net contraversive rotations (n = 6-8 animals per dose), reaching a maximum of 395 +/- 51 rotations 60 min-1 after 0.75 nmol. The effects of DCG-IV (0.5 nmol) were inhibited by 63.0 +/- 9.0% following 30 min pre-treatment with the group II mGlu receptor antagonist, (2S)-alpha-ethylglutamic acid (EGLU; 100 nmol in 0.2 microliter; n = 6). 4. Following intraventricular injection, DCG-IV (0.125-1.5 nmol in 2 microliters) produced a dose-dependent increase in bilateral locomotor activity (n = 6-7 animals per dose), reaching a maximum of 180 +/- 21 locomotor units 30 min-1 after 0.5 nmol. Pre-treatment with EGLU (200 nmol in 2 microliters) inhibited the effects of DCG-IV (0.5 nmol) by 68.2 +/- 12.3% (n = 5). 5. These data show that activation of group II mGlu receptors in the SNr provides relief of akinesia in the reserpinized rat model of PD. The reversal seen following intraventricular administration supports the likely therapeutic benefit of systemically-active group II mGlu receptor agonists in PD.     

The novel antagonist 3-CBW discriminates between kainate receptors expressed on neonatal rat motoneurones and those on dorsal root C-fibres

1. The natural product willardiine is a selective AMPA receptor agonist. We report that an N(3)-substituted analogue of willardiine, (S)-3-(4-carboxybenzyl)willardiine 3-CBW, antagonizes AMPA and kainate receptors expressed on motoneurones and dorsal root C-fibres, respectively. 2. Reduction of the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) has been used as a novel method to compare AMPA receptor antagonists. 3-CBW, NBQX and GYKI53655 depressed the fDR-VRP with IC(50) values of 10.3+/-2.4, 0.214+/-0.043 and 4.03+/-0.31 micro M, respectively. That 3-CBW depressed the fDR-VRP by acting at AMPA and not metabotropic glutamate receptors was demonstrated by the lack of effect of LY341495 (100 micro M). 3. The Schild plot for antagonism of responses to (S)-5-fluorowillardiine on motoneurones by 3-CBW had a slope of 1.11+/-0.13 giving a pA(2) value of 4.48. The Schild plot for antagonism of kainate responses on the dorsal root by 3-CBW had a slope of 1.05+/-0.05 giving a pA(2) value of 4.96. 4. On neonatal rat motoneurones 3-CBW (200 micro M) almost completely abolished responses to AMPA while responses to NMDA, kainate and DHPG were 101.6+/-11.6%, 39.4+/-5.8% and 110.5+/-9.0% of control, respectively. 3-CBW can therefore be used to isolate kainate receptor responses from those mediated by AMPA receptors. 5 3-CBW antagonized kainate-induced responses on dorsal root C-fibres with a pA(2) value of 4.96 whereas kainate receptor mediated responses (isolated by including GYKI53655 in the medium) on motoneurones were not completely blocked by 200 micro M 3-CBW, substantiating evidence that kainate receptors on neonatal rat motoneurones differ from those on dorsal root C-fibres.     

Synthesis, pharmacological characterization, and molecular modeling of heterobicyclic amino acids related to (+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid (LY354740): identification of two new potent, selective, and systemically active agonists for group II metabotropic glutamate receptors

As part of our ongoing research program aimed at the identification of highly potent, selective, and systemically active agonists for group II metabotropic glutamate (mGlu) receptors, we have prepared novel heterobicyclic amino acids (-)-2-oxa-4-aminobicyclo[3.1. 0]hexane-4,6-dicarboxylate (LY379268, (-)-9) and (-)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY389795, (-)-10). Compounds (-)-9 and (-)-10 are structurally related to our previously described nanomolar potency group II mGlu receptor agonist, (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740 monohydrate, 5), with the C4-methylene unit of 5 being replaced with either an oxygen atom (as in (-)-9) or a sulfur atom (as in (-)-10). Compounds (-)-9 and (-)-10 potently and stereospecifically displaced specific binding of the mGlu2/3 receptor antagonist ([3H]LY341495) in rat cerebral cortical homogenates, displaying IC50 values of 15 +/- 4 and 8.4 +/- 0.8 nM, respectively, while having no effect up to 100 000 nM on radioligand binding to the glutamate recognition site on NMDA, AMPA, or kainate receptors. Compounds (-)-9 and (-)-10 also potently displaced [3H]LY341495 binding from membranes expressing recombinant human group II mGlu receptor subtypes: (-)-9, Ki = 14.1 +/- 1.4 nM at mGlu2 and 5.8 +/- 0.64 nM at mGlu3; (-)-10, Ki = 40.6 +/- 3.7 nM at mGlu2 and 4.7 +/- 1.2 nM at mGlu3. Evaluation of the functional effects of (-)-9 and (-)-10 on second-messenger responses in nonneuronal cells expressing human mGlu receptor subtypes demonstrated each to be a highly potent agonist for group II mGlu receptors: (-)-9, EC50 = 2.69 +/- 0.26 nM at mGlu2 and 4.58 +/- 0.04 nM at mGlu3; (-)-10, EC50 = 3.91 +/- 0.81 nM at mGlu2 and 7.63 +/- 2. 08 nM at mGlu3. In contrast, neither compound (up to 10 000 nM) displayed either agonist or antagonist activity in cells expressing recombinant human mGlu1a, mGlu5a, mGlu4a, or mGlu7a receptors. The agonist effects of (-)-9 and (-)-10 at group II mGlu receptors were not totally specific, however, as mGlu6 agonist activity was observed at high nanomolar concentrations for (-)-9 (EC50 = 401 +/- 46 nM) and at micromolar concentrations (EC50 = 2 430 +/- 600 nM) for (-)-10; furthermore, each activated mGlu8 receptors at micromolar concentrations (EC50 = 1 690 +/- 130 and 7 340 +/- 2 720 nM, respectively). Intraperitoneal administration of either (-)-9 or (-)-10 in the mouse resulted in a dose-related blockade of limbic seizure activity produced by the nonselective group I/group II mGluR agonist (1S,3R)-ACPD ((-)-9 ED50 = 19 mg/kg, (-)-10 ED50 = 14 mg/kg), indicating that these molecules effectively cross the blood-brain barrier following systemic administration and suppress group I mGluR-mediated limbic excitation. Thus, heterobicyclic amino acids (-)-9 and (-)-10 are novel pharmacological tools useful for exploring the functions of mGlu receptors in vitro and in vivo.     

Group 1 mGlu receptors elevate [Ca2+]i in rat cultured cortical type 2 astrocytes: [Ca2+]i synergy with adenosine A1 receptors.

Brain macroglia are known to express a diverse array of neurotransmitter receptors whose signal transduction pathways may be subject to heteroreceptor 'cross-talk'. In the current study we have examined group 1 mGlu receptor-evoked [Ca2+]i signalling, and possible heteroreceptor cross-talk, in cultured type 2 astrocytes. The selective group 1 metabotropic glutamate (mGlu) receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG) elevated [Ca2+]i (EC50 = 1.7 +/- 0.6 microM); an effect reversed by the selective mGlu receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (IC50 = 52.7 +/- 8.7 microM). DHPG-evoked [Ca2+]i responses were abolished by (1) thapsigargin (100 nM), implicating the involvement of internal Ca2+ stores in group 1 mGlu [Ca2+]i responses and (2) the removal of extracellular Ca2+. When applied alone, the selective adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA, 100 nM) failed to influence [Ca2+]i. However, in the presence of 1 microM DHPG, CPA potently (EC50 = 12.3 +/- 1.9 nM) increased [Ca2+]i responses. In the presence of 100 nM CPA, the efficacy of DHPG was doubled without any significant change in the DHPG EC50 value. This effect was reversed by either the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (IC50 = 50.3 +/- 19.9 nM) or overnight incubation with Pertussis toxin (100 ng/ml). We conclude that (1) type 2 astrocytes contain group 1 mGlu receptors coupled to [Ca2+]i signalling and (2) co-activation of adenosine A1 receptors enhances group 1 mGlu-evoked [Ca2+]i responses in these cells via a Gi/o G protein-mediated mechanism.     

Studying specific effects of nootropic drugs on glutamate receptors in the rat brain

The influence of nootropic drugs of different groups (piracetam, phenotropil, nooglutil, noopept, semax, meclofenoxate, pantocalcine, and dimebon) on the binding of the corresponding ligands to AMPA, NMDA, and mGlu receptors of rat brain has been studied by the method of radio-ligand binding in vitro. It is established that nooglutil exhibits pharmacologically significant competition with a selective agonist of AMPA receptors ([G-3H]Ro 48-8587) for the receptor binding sites (with IC50 = 6.4 +/- 0.2 microM), while the competition of noopept for these receptor binding sites was lower by an order of magnitude (IC50 = 80 +/- 5.6 microM). The heptapeptide drug semax was moderately competitive with [G-3H]LY 354740 for mGlu receptor sites (IC50 = 33 +/- 2.4 microM). Dimebon moderately influenced the specific binding of the ligand of NMDA receptor channel ([G-3H]MK-801) at IC50 = 59 +/- 3.6 microM. Nootropic drugs of the pyrrolidone group (piracetam, phenotropil) as well as meclofenoxate, pantocalcine (pantogam) in a broad rage of concentrations (10(-4)-10(-10) M) did not affect the binding of the corresponding ligands to glutamate receptors (IC50 100 pM). Thus, the direct neurochemical investigation was used for the first time to qualitatively characterize the specific binding sites for nooglutil and (to a lower extent) noopept on AMPA receptors, for semax on metabotropic glutamate receptors, and for dimebon on the channel region of NMDA receptors. The results are indicative of a selective action of some nootropes on the glutamate family.     

Actions of LY341495 on metabotropic glutamate receptor-mediated responses in the neonatal rat spinal cord

1. The group II metabotropic glutamate (mGlu) receptor antagonist (2S,1'S,2'S)-2-(2-carboxycyclopropyl)-2-(9H-xanthen-9-yl)glycine (LY341495) also has activity at group I and III mGlu receptors at higher concentrations and can be used to discriminate between mGlu receptor subtypes. We report the antagonist action of LY341495 on glutamate receptors expressed in the neonatal rat spinal cord preparation and the use of this antagonist to investigate the group III mGlu receptor subtypes responsible for mediating the depression of synaptic transmission in the spinal cord mediated by the group III mGlu receptor agonists (S)-2-amino-4-phosphonobutanoic acid ((S)-AP4) and (1S,3R,4S)-1-aminocyclopentane-1,2,4-tricarboxylic acid (ACPT-I). 2. LY341495 antagonised mGlu receptor agonist-induced responses in the spinal cord with a rank order of potency of group II > group III > group I, which is the same as that observed in human cloned mGlu receptor cell lines. Antagonism of group II and III mGlu receptor-mediated effects were time dependent when low-nanomolar concentrations of LY341495 were used. Although the rank order of potency of LY341495 was the same on native rat and cloned human mGlu receptors, there was a compression in the selectivity between group II and III mGlu receptors, expressed in the spinal cord. 3. In agreement with a previous study on cloned ionotropic glutamate receptors 100 microM LY341495 had little or no effect on N-methyl-D-aspartate, (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid or kainate receptor-mediated responses on motoneurones. 4. LY341495 exhibited low-nanomolar potency antagonist activity against (S)-AP4 and ACPT-I suggesting that these agonists are activating predominantly mGlu8 and that mGlu4 receptors do not play a role in modulating synaptic transmission in the pathways stimulated in the experiments described here.     

Suppression of alcohol self-administration and cue-induced reinstatement of alcohol seeking by the mGlu2/3 receptor agonist LY379268 and the mGlu8 receptor agonist (S)-3,4-DCPG

Glutamatergic neurotransmission has been suggested to modulate cue-induced drug-seeking behavior. Here we examined the effects of metabotropic glutamate receptor agonists on alcohol self-administration and cue-induced reinstatement. Rats were trained to self-administer 10% w/v ethanol under an FR1 schedule of reinforcement during 30-min sessions. In the reinstatement experiments, ethanol and a non-rewarding quinine solution (available on alternating days) were paired with olfactory stimuli (S+/S-) as well as light (CS+) or tone (CS-) stimuli. Following extinction training, reinstatement of responding was induced by the ethanol-associated stimuli (S+/CS+). The mGlu2/3 receptor agonist LY379268 (0, 1, 3 and 5 mg/kg i.p.) and the mGlu8 receptor agonist (S)-3,4-DCPG (0, 5, 10 and 15 mg/kg i.p.) attenuated alcohol self-administration and reinstatement at doses that decreased also spontaneous locomotor activity. The results suggest that metabotropic glutamate receptors may have a role in the modulation of alcohol seeking and self-administration. However, further studies with ligands with fewer motor-suppressant side effects are needed.     

A novel, competitive mGlu(5) receptor antagonist (LY344545) blocks DHPG-induced potentiation of NMDA responses but not the induction of LTP in rat hippocampal slices

1. We have investigated the pharmacological properties of LY344545, a structurally related epimer of the broad spectrum competitive metabotropic glutamate receptor antagonist, LY341495. We have found that LY344545 also antagonizes competitively nearly all mGlu receptor subtypes, but with a wide spectrum of activity. The order of potency for the human receptor isoforms was mGlu(5a) (IC(50) of 5. 5+/-0.6 microM)>mGlu(2)=mGlu(3)>mGlu(1alpha)=mG lu(7)>mGlu(6)=mGlu(8). No significant mGlu(4) receptor antagonist activity was detected at the highest concentration used (100 microM). 100 microM LY344545 displaced 50+/-5% of [(3)H]-CGP39653 binding, but less than 30% of [(3)H]-kainate or [(3)H]-AMPA in radioligand binding assays. 2. LY344545 antagonized L-glutamate stimulated Ca(2+) release in CHO cells transfected with mGlu receptors in a concentration dependent manner with a 10 fold higher affinity for the rat mGlu(5a) receptor (K:(i)=2.1+/-0.6 microM) compared to the rat mGlu(1alpha) receptor (K:(i)=20.5+/-2.1 microM). 50 microM (1S, 3R)-ACPD-induced Ca(2+) rises in hippocampal CA1 neurones were also antagonized (IC(50)=6. 8+/-0.7 microM). 3. LY344545 antagonized 10 microM (S)-3,5-DHPG-induced potentiation of NMDA depolarizations in CA1 neurones (EC(50)=10. 6+/-1.0 microM). At higher concentrations (> or =100 microM), LY344545 was an NMDA receptor antagonist. 4. LY344545 also blocked the induction, but not the expression, of LTP at CA3 to CA1 synapses with an IC(50)>300 microM. This effect is consistent with its weak activity at NMDA receptors. 5. These results demonstrate that the binding of ligands to mGlu receptor subtypes is critically dependent on the spatial orientation of the same molecular substituents within a given chemical pharmacophore. The identification of LY344545 as the first competitive antagonist to show selectivity towards mGlu(5) receptors supports the potential to design more selective and potent competitive antagonists of this receptor. 6. These results further indicate that mGlu receptor-mediated potentiation of NMDA responses is not essential for the induction of LTP.     

Structural requirements for novel willardiine derivatives acting as AMPA and kainate receptor antagonists

1. The natural product willardiine is an AMPA receptor agonist. We have examined the structural changes required to convert willardiine into an antagonist at AMPA and kainate receptors. Structure-activity analysis has been carried out to discover the structural features required to increase the potency and/or selectivity of the antagonists at AMPA or kainate receptors. 2. Reduction of the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) has been used to investigate AMPA receptor antagonist activity. To examine antagonist activity at kainate receptors, the ability of compounds to depress kainate-induced depolarisations of dorsal root fibres was assessed. 3. Blocking ionisation of the uracil ring by adding a methyl group to the N(3) position was not sufficient to convert willardiine into an antagonist. However, willardiine derivatives with a side-chain bearing a carboxylic acid group at the N(3)-position of the uracil ring could antagonise AMPA and kainate receptors. 4. S stereochemistry was optimal for antagonism. When compounds with differing interacidic group chain lengths were compared, a group chain length of two methylene groups was preferable for AMPA receptor antagonism in the series of compounds bearing a carboxyalkyl side chain (UBP275, UBP277 and UBP279 reduced the fDR-VRP with IC(50) values of 287+/-41, 23.8+/-3.9 and 136+/-17 micro M, respectively). For kainate receptor antagonism, two or three methylene groups were almost equally acceptable (UBP277 and UBP279 reduced dorsal root kainate responses with apparent K(D) values of 73.1+/-4.5 and 60.5+/-4.1 micro M, respectively). 5. Adding an iodo group to the 5-position of UBP277 and UBP282 enhanced activity at kainate receptors (UBP291 and UBP301 antagonised kainate responses on the dorsal root with apparent K(D) values of 9.83+/-1.62 and 5.94+/-0.63 micro M, respectively). 6. The most useful antagonist identified in this study was UBP301, which was a potent and approximately 30-fold selective kainate receptor antagonist. UBP282 may also be of use in isolating a non-GluR5-mediated kainate response.     

Novel 1-hydroxyazole bioisosteres of glutamic acid. Synthesis, protolytic properties, and pharmacology.

A number of 1-hydroxyazole derivatives were synthesized as bioisosteres of (S)-glutamic acid (Glu) and as analogues of the AMPA receptor agonist (R,S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA, 3b). All compounds were subjected to in vitro pharmacological studies, including a series of Glu receptor binding assays, uptake studies on native as well as cloned Glu uptake systems, and the electrophysiological rat cortical slice model. Compounds 7a,b, analogues of AMPA bearing a 1-hydroxy-5-pyrazolyl moiety as the distal carboxylic functionality, showed only moderate affinity for [3H]AMPA receptor binding sites (IC(50) = 2.7 +/- 0.4 microM and IC(50) = 2.6 +/- 0.6 microM, respectively), correlating with electrophysiological data from the rat cortical wedge model (EC(50) = 280 +/- 48 microM and EC(50) = 586 +/- 41 microM, respectively). 1-Hydroxy-1,2,3-triazol-5-yl analogues of AMPA, compounds 8a,b, showed high affinity for [3H]AMPA receptor binding sites (IC(50) = 0.15 +/- 0.03 microM and IC(50) = 0.13 +/- 0.02 microM, respectively). Electrophysiological data showed that compound 8a was devoid of activity in the rat cortical wedge model (EC(50) > 1000 microM), whereas the corresponding 4-methyl analogue 8b was a potent AMPA receptor agonist (EC(50) = 15 +/- 2 microM). In accordance with this disparity, compound 8a was found to inhibit synaptosomal [3H]D-aspartic acid uptake (IC(50) = 93 +/- 25 microM), as well as excitatory amino acid transporters (EAATs) EAAT1 (IC(50) = 100 +/- 30 microM) and EAAT2 (IC(50) = 300 +/- 80 microM). By contrast, compound 8b showed no appreciable affinity for Glu uptake sites, neither synaptosomal nor cloned. Compounds 9a-c and 10a,b, possessing 1-hydroxyimidazole as the terminal acidic function, were devoid of activity in all of the systems tested. Protolytic properties of compounds 7a,b, 8b, and 9b were determined by titration, and a correlation between the pK(a) values and the activity at AMPA receptors was apparent. Optimized structures of all the synthesized ligands were fitted to the known crystal structure of an AMPA-GluR2 construct. Where substantial reduction or abolition of affinity at AMPA receptors was observed, this could be rationalized on the basis of the ability of the ligand to fit the construct. The results presented in this article point to the utility of 1-hydroxypyrazole and 1,2,3-hydroxytriazole as bioisosteres of carboxylic acids at Glu receptors and transporters. None of the compounds showed significant activity at metabotropic Glu receptors.