Characterization of metabotropic glutamate receptor-stimulated phosphoinositide hydrolysis in rat cultured cerebellar granule cells.

1. The pharmacology of excitatory amino acid (EAA)-stimulated phosphoinositide (PI) hydrolysis, monitored via [3H]-inositol monophosphate accumulation, was investigated in primary cultures of rat cerebellar granule cells. 2. EAA-stimulated PI hydrolysis peaked after 4-5 days in vitro and subsequently declined. 3. The agonist order of potency was found to be (EC50): L-quisqualic acid (Quis) (2 microM) >> L-glutamate (50 microM) > (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) (102 microM). L-Glutamate (Emax = 873% of basal activity) elicited the largest stimulation of PI hydrolysis, whereas Quis (Emax = 603%) and (1S,3R)-ACPD (Emax = 306%) produced somewhat lower stimulations. 4. Several phenylglycine derivatives were found to be active in inhibiting 2 microM Quis-stimulated PI hydrolysis, in order of potency (IC50): (S)-4-carboxy-3-hydroxyphenylglycine (41 microM) > or = (S)-4-carboxyphenylglycine (51 microM) >> (+)-alpha-methyl-4-carboxyphenylglycine (243 microM). 5. Cultured cerebellar granule cells of the rat appear to have Group I mGluR pharmacology similar to that reported for cloned mGluR1 and provide an ideal system for investigating novel mGluR1 ligands in a native environment.     

Stimulatory effects of the putative metabotropic glutamate receptor antagonist L-AP3 on phosphoinositide turnover in neonatal rat cerebral cortex.

1. The effects of the metabotropic glutamate receptor (mGluR) antagonist, L-2-amino-3-phosphonopropionate (L-AP3) on phosphoinositide turnover in neonatal rat cerebral cortex slices has been investigated. 2. At concentrations of < or = 300 microM, L-AP3 inhibited total [3H]-inositol phosphate ([3H]-InsPx) and Ins(1,4,5)P3 mass responses stimulated by the selective mGluR agonist, 1-amino-cyclopentane-1S, 3R-dicarboxylic acid (1S, 3R-ACPD). Comparison with the competitive mGluR antagonist (+/-)-alpha-methyl-4-carboxyphenylglycine ((+/-)-MCPG) clearly demonstrated that L-AP3 caused inhibition by a mechanism that was not competitive, as L-AP3 decreased the maximal response to 1S, 3R-ACPD (by approximately 40% at 300 microM L-AP3) without significantly affecting the concentration of 1S, 3R-ACPD required to cause half-maximal stimulation of the [3H]-InsPx response. 3. In contrast, at a higher concentration L-AP3 (1 mM) caused a large increase in [3H]-InsPx accumulation which was similar in magnitude in both the absence and presence of 1S, 3R-ACPD (300 microM). D-AP3 (1 mM) had no stimulatory effect alone and did not affect the response evoked by 1S, 3R-ACPD. L-AP3 (1 mM) also caused a large increase in Ins(1,4,5)P3 accumulation. The magnitude of the response (4-5 fold increase over basal) approached that evoked by a maximally effective concentration of 1S, 3R-ACPD, but differed substantially in the time-course of the response. The stimulatory effects of 1S, 3R-ACPD and L-AP3 on Ins(1,4,5)P3 accumulation were also similarly affected by decreases in extracellular calcium concentration. 4. Detailed analysis of the inositol phospholipid labelling pattern and the inositol (poly)phosphate isomeric species generated following addition of L-AP3 was also performed. In the continued presence of myo-[3H]-inositol, L-AP3 (1 mM) stimulated a significant increase in phosphatidylinositol labelling, but not that of the polyphosphoinositides, and the inositol (poly)phosphate profile suggested that substantial Ins(1,4,5)P3 metabolism occurs via both 5-phosphatase and 3-kinase routes. 5. A significant stimulatory effect of L-AP3 (1 mM) on [3H]-InsPx accumulation was also observed in neonatal rat hippocampus, and cerebral cortex and hippocampus slices prepared from adult rat brain. 6. These data demonstrate that whilst L-AP3 antagonizes mGluR-mediated phosphoinositide responses at concentrations of < or = 300 microM, higher concentrations substantially stimulate this response. The ability of (+/-)-MCPG (1 mM) to attenuate significantly L-AP3-stimulated [3H]-InsPx accumulation, suggests that both the inhibitory and stimulatory effects of L-AP3 may be mediated by mGluRs.     

Excitatory amino acid receptor-stimulated phosphoinositide turnover in primary cerebrocortical cultures.

1. Characterization of excitatory amino acid-induced accumulation of [3H]-phosphoinositides was carried out in primary cerebrocortical cultures isolated from foetal rats. 2. All of the excitatory amino acid receptor agonists examined caused concentration-dependent enhancement of phosphoinositide (PI) formation. The most potent excitatory amino acid receptor agonists were quisqualate, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD), ibotenate and glutamate with mean EC50 values of 0.9 +/- 0.4 microM, 15 +/- 5 microM, 15 +/- 3 microM and 41 +/- 8 microM respectively. 3. The selective ionotropic receptor antagonists kynurenic acid (1 mM), 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX, 10 microM) and (+/-)-4-(3-phosphonopropyl)-2 piperazinecarboxylic acid (CPP, 100 microM), failed to block responses to quisqualate, (1S,3R)-ACPD or glutamate. D,L-2-Amino-3-phosphonopropionate (D,L-AP3) did not block 1S,3R-ACPD or quisqualate-induced PI turnover, but had an additive effect with quisqualate or (1S,3R)-ACPD. 4. Exposure of cultures to agonists in the absence of added extracellular calcium reduced the maximal quisqualate response by approximately 45%, revealing a two-component concentration-response curve. Concentration-response curves to ibotenate and glutamate became flattened by omission of extracellular calcium, whereas (1S,3R)-ACPD-stimulated PI turnover was unaffected. 5. Pretreatment of cultures with pertussis toxin markedly inhibited PI responses evoked by (1S,3R)-ACPD. 6. These results suggest that excitatory amino acid-stimulated PI turnover in cerebrocortical cultures is independent of ionotropic receptor activation and is mediated via specific G-protein-linked metabotropic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-dependent changes in receptor-stimulated phosphoinositide turnover in the rat hippocampus.

To study the changes in the hippocampal cholinergic system of chronologically old and behaviorally impaired animals, old (21 months of age) and young (3 months of age) male, Fischer-344 rats were used. The aged animals were tested on a reference memory task (Morris water maze) and found to be functionally impaired as compared to the young controls. Carbachol-stimulated phosphoinositide metabolism was measured in hippocampal slices from young and old rats. Slices were prelabeled with 3H-inositol for 120 min and subjected to muscarinic stimulation in the presence of lithium. Following extraction of the slices with acidified solvent mixture, the inositolphosphates present in the aqueous fraction were isolated by ion exchange chromatography. Receptor-stimulated release of inositolphosphates (IPs) was found to be increased in the hippocampus of older animals. This age-related enhancement of IP release was in contrast to the decrease in choline acetyltransferase (CHAT) activity in the hippocampus. We postulate that alterations in the G-protein coupling with the muscarinic receptor leads to an increase in the phosphoinositide turnover in part as a compensatory mechanism for neuronal cell death and reduced transmitter levels.     

Regulation of phosphoinositide turnover in neonatal rat cerebral cortex by group I- and II- selective metabotropic glutamate receptor agonists

1. The interactive effects of different metabotropic glutamate (mGlu) receptor subtypes to regulate phosphoinositide turnover have been studied in neonatal rat cerebral cortex and hippocampus by use of agonists and antagonists selective between group I and II mGlu receptors.
2. The group II-selective agonist 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC; 100 μM) had no effect on basal total inositol phosphate ([³H]-InsP_x) accumulation (in the presence of Li⁺) in myo-[³H]-inositol pre-labelled slices, but enhanced the maximal [³H]-InsP_x response to the group I-selective agonist (S)-3,5-dihydroxyphenylglycine (DHPG) by about 100% in both hippocampus and cerebral cortex. In cerebral cortex the enhancing effect of 2R,4R-APDC occurred with respect to the maximal responsiveness and had no effect on EC₅₀ values for DHPG (-log EC₅₀ (M): control, 5.56±0.05; +2R,4R-APDC, 5.51±0.08). 2R,4R-APDC also caused a significant enhancement of the DHPG-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) mass response over an initial 0–300 s time-course.
3. The enhancing effects of 2R,4R-APDC on DHPG-stimulated [³H]-InsP_x accumulation were observed in both the presence and nominal absence of extracellular Ca²⁺, and irrespective of whether 2R,4R-APDC was added before, simultaneous with, or subsequent to DHPG. Furthermore, increasing the tissue cyclic AMP concentration up to 100 fold had no effect on DHPG-stimulated Ins(1,4,5)P₃ accumulation in the absence or presence of 2R,4R-APDC.
4. 2R,4R-APDC and (2S, 1′R, 2′R, 3′R)-2-(2,3-dicarboxylcyclopropyl)glycine (DCG-IV), the latter agent in the presence of MK-801 to prevent activation of NMDA-receptors, each inhibited forskolin-stimulated cyclic AMP accumulation by about 50%, with respective EC₅₀ values of 1.3 and 0.04 μM (-log EC ₅₀ (M): 2R,4R-APDC, 5.87±0.09; DCG-IV, 7.38±0.05). In the presence of DHPG (30 μM), 2R,4R-APDC and DCG-IV also concentration-dependently increased [³H]-InsP_x accumulation with respective EC₅₀ values of 4.7 and 0.28 μM (-log EC₅₀ (M): 2R,4R-APDC, 5.33±0.04; DCG-IV, 6.55±0.09) which were 3–7 fold rightward-shifted relative to the adenylyl cyclase inhibitory responses.
5. The group II-selective mGlu receptor antagonist {"type":"entrez-nucleotide","attrs":{"text":"LY307452","term_id":"1257780057","term_text":"LY307452"}}LY307452 (30 μM) caused parallel rightward shifts in the concentration-effect curves for inhibition of forskolin-stimulated adenylyl cyclase, and enhancement of DHPG-stimulated [³H]-InsP_x accumulation, by 2R,4R-APDC yielding similar equilibrium dissociation constants (K_ds, 3.7±1.1 and 4.1±0.4 μM respectively) for each response.
6. The ability of 2R,4R-APDC to enhance receptor-mediated [³H]-InsP_x accumulation appeared to be agonist-specific; thus although DHPG (100 μM) and the muscarinic cholinoceptor agonist carbachol (10 μM) stimulated similar [³H]-InsP_x accumulations, only the response to the former agonist was enhanced by co-activation of group II mGlu receptors.
7. These data demonstrate that second messenger-generating phosphoinositide responses stimulated by group I mGlu receptors are positively modulated by co-activation of group II mGlu receptors in cerebral cortex and hippocampus. The data presented here are discussed with respect to the possible mechanisms which might mediate the modulatory activity, and the physiological and pathophysiological significance of such crosstalk between mGlu receptors.

     

Actions of a metabotropic glutamate receptor agonist in immature and adult rat cerebellum.

The electrophysiological actions of the metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD) on Purkinje and granule cells were studied in immature and adult cerebellar slices. ACPD elicited a depolarising response when applied to Purkinje cells (EC50 approximately 20 microM). Granule cells hyperpolarised when exposed to low (3-10 microM) concentrations of ACPD; higher concentrations produced a depolarisation (EC50 approximately 40 microM) that was rapidly curtailed by a hyperpolarisation. The hyperpolarisation was abolished when Ca2+ was removed. In Purkinje cells, the amplitude of the depolarisation was greater in adult slices compared to those in immature slices. The responses were not blocked by ionotropic glutamate receptor antagonists or (L)-2-amino-3-phosphonopropionate (AP3).     

Modulatory effects of NMDA on phosphoinositide responses evoked by the metabotropic glutamate receptor agonist 1S,3R-ACPD in neonatal rat cerebral cortex.

1. The effect of NMDA-receptor stimulation on phosphoinositide signalling in response to the metabotropic glutamate receptor agonist 1-aminocyclopentane-1S,3R-dicarboxylic acid (1S,3R-ACPD) has been examined in neonatal rat cerebral cortex slices. 2. Total [3H]-inositol phosphate ([3H]-InsPx) accumulation, in the presence of 5 mM LiCl, in [3H]-inositol pre-labelled slices was concentration-dependently increased by 1S,3R-ACPD (EC50 16.6 microM) and, at a maximally effective concentration, 1S,3R-ACPD (300 microM) increased [3H]-InsPx accumulation by 12.8 fold over basal values. 3. [3H]-InsPx accumulation stimulated by 1S,1R-ACPD was enhanced by low concentrations of NMDA (3-30 microM), but not by higher concentrations (> 30 microM). [3H]-InsPx accumulations stimulated by 1S,3R-ACPD in the absence or presence of 10 microM NMDA were linear with time, at least over the 15 min period examined; however, in the presence of 100 microM NMDA the initial enhancement of 1S,3R-ACPD-stimulated phosphoinositide hydrolysis progressively decreased with time. 4. In the presence of a maximal enhancing concentration of NMDA (10 microM), the response to 1S,3R-ACPD (300 microM) was increased 1.9 fold and the EC50 for agonist-stimulated [3H]-InsPx accumulation decreased about 4 fold. The enhanced response to the metabotropic agonist was concentration-dependently inhibited by competitive and uncompetitive antagonists of NMDA-receptor activation. 5. 1S,3R-ACPD also stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) mass accumulation with an initial peak response (5-6 fold over basal) at 15 s decaying to a smaller (2 fold), but persistent elevated accumulation (1-10 min).(ABSTRACT TRUNCATED AT 250 WORDS)     

Homologous and heterologous regulation of receptor-stimulated phosphoinositide hydrolysis

Signal transduction at a diverse range of pharmacologically distinct receptors is effected by the enhanced turnover of inositol phospholipids, with the attendant formation of inositol 1,4,5-trisphosphate and diacylglycerol. Although considerable progress has been made in recent years towards the identification and characterization of the individual components of this pathway, much less is known of mechanisms that may underlie its regulation. In this review, evidence is presented for the potential regulation of inositol lipid turnover at the level of receptor, phosphoinositide-specific phospholipase C and substrate availability in response to either homologous or heterologous stimuli. Available data indicate that the extent of receptor-stimulated inositol lipid hydrolysis is regulated by multiple mechanisms that operate at different levels of the signal transduction pathway.     

Molecular basis for the differential agonist affinities of group III metabotropic glutamate receptors

Agonist stimulation of group III metabotropic glutamate receptors (mGluRs) induces an inhibition of neurotransmitter release from neurons. The group III mGluRs are pharmacologically defined by activation with the glutamate analog L-amino-4-phosphonobutyric acid (L-AP4). The affinities of these receptors for L-AP4 and glutamate vary over approximately a 1500-fold concentration range. The goal of this study was to elucidate the molecular basis for this dispersion of agonist affinities for the group III receptors mGluR4, mGluR6, and mGluR7. [3H]L-AP4 binding was present in human embryonic kidney cells transfected with the high-affinity mGluR4 receptor but not in cells transfected with mGluR6 or the low-affinity mGluR7 receptor. Analysis of mGluR4/mGluR6 receptor chimeras revealed that replacement of the first 35 amino acids of mGluR6 with the first 50 amino acids of mGluR4 was sufficient to impart [3H]L-AP4 binding to mGluR6. Homology models of mGluR4 and mGluR7 were used to predict amino acids that may affect ligand affinity. Mutations were made in mGluR7 to convert selected residues into the equivalent amino acids present in the high-affinity mGluR4 receptor. The mGluR7 N74K mutation caused a 12-fold increase in affinity in a functional assay, whereas the N74K mutation in combination with mutations in residues 258 to 262, which lie outside the binding pocket, caused a 112-fold increase in affinity compared with unmutated mGluR7. Our results demonstrate that the binding site residues at position lysine 74 in mGluR4, glutamine 58 in mGluR6, and asparagine 74 in mGluR7 are key determinants of agonist affinity and that additional residues situated outside of the binding pocket, including those present in the extreme amino terminus, also contribute to agonist affinity and the pharmacological profiles of the group III mGluRs.     

Opioid agonist and antagonist activities of morphindoles related to naltrindole.

A series of naltrindole-related ligands (4-10) with an N-methyl,N-phenethyl,N-cinnamyl, or an unsubstituted basic nitrogen were synthesized and tested for opioid agonist and antagonist activity in smooth muscle preparations and in mice. The nor compounds (4 and 6) and the phenethyl derivatives (5 and 8) displayed full agonist activity (IC50 = 85-179 nM) in the mouse vas deferens preparation (MVD) while the other members of the series exhibited partial agonist or weak antagonist activity. In the guinea pig ileum preparation (GPI), all compounds except 8 were partial agonists. The ligands that were evaluated in mice were found to produce antinociception that was not selectively mediated via delta opioid receptors. However, two of these ligands (4 and 5) appeared to be delta-selective opioid receptor antagonists at subthreshold doses for antinociception. The finding that all of the compounds bind selectively to delta opioid receptors in guinea pig brain membranes together with the in vitro pharmacology and in vivo antagonist studies suggests that the lack of delta agonist selectivity in vivo may be due to a number of factors, including a basic difference between the delta receptor system in the MVD and in the mouse brain. Further, it is suggested that the constellation of message and address components in the morphindole nucleus may tend to stabilize delta receptors in the brain in antagonist state.     

(S)-homoquisqualate: a potent agonist at the glutamate metabotropic receptor.

The synthetic quisqualate analogue, (S)-homoquisqualate was examined for activity at the glutamate metabotropic receptor, in relation to its ability to stimulate phosphoinositide hydrolysis in rat pup cerebro-cortical slices. The compound produced a robust increase in hydrolysis (EC50 = 50.2 +/- 1.6 microM), which, in common with responses to quisqualate and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate ((1S,3R)-ACPD), was antagonized uncompetitively by L-2-amino-3-phosphonopropionate (L-AP3). In contrast to quisqualate which exhibits low efficacy, (S)-homoquisqualate behaves as a full agonist at the metabotropic receptor.     

Coupling of metabotropic glutamate receptors to phosphoinositide mobilisation and inhibition of cyclic AMP generation in the guinea-pig cerebellum.

1. The effects of metabotropic glutamate receptor (mGluR) agonists on cyclic nucleotide and phosphoinositide turnover were investigated in adult guinea-pig cerebellar slices by use of radioactive precursors. 2.L-Glutamate, 1-aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD) and RS-3,5-dihydroxyphenylglycine (DHPG) evoked concentration-dependent increases in the accumulation of [3H]-inositol phosphates with pEC50 values of 2.98 +/- 0.02, 4.45 +/- 0.06 and 4.47 +/- 0.07, respectively. Maximal responses to these agents were 43 +/- 8, 52 +/- 12 and 84 +/- 11% of the response to 1 mM histamine, respectively. 3. The phosphoinositide response to 1S,3R-ACPD was antagonized in the presence of (+)-alpha-methyl-4-carboxyphenylglycine, with a calculated pKi value of 3.55 +/- 0.03. 4. Forskolin-stimulated accumulation of [3H]-cyclic AMP was not significantly altered in the presence of 10 microM DCG-IV or 300 microM 1S,3R-ACPD. Similarly, 300 microM 1S,3R-ACPD failed to alter isoprenaline-(1 microM) or 2-chloroadenosine (2-CA, 30 microM)-stimulated accumulation of [3H]-cyclic AMP. 5. Forskolin-stimulated accumulation of [3H]-cyclic AMP was concentration-dependently inhibited in the presence of L-glutamate and L-serine-O-phosphate (L-SOP) with pIC50 values of 2.91 +/- 0.17 and 2.86 +/- 0.04 with maximal inhibitions of 47 +/- 2 and 92 +/- 3%, respectively. L-2-Amino-4-phosphonobuty-rate (L-AP4) inhibited the forskolin response without saturating, evoking an inhibition of 71 +/- 7% at 3 mM. 6. 2-CA-evoked accumulation of [3H]-cyclic AMP was also inhibited by L-glutamate and L-SOP with pIC50 values of 2.71 +/- 0.03 and 2.72 +/- 0.08 and maximal inhibitions of 51 +/- 5 and 99 +/- 0%, respectively. L-AP4 inhibited the 2-CA response without saturating, evoking an inhibition of 68 +/- 1% at 3 mM. 7. Isoprenaline-evoked accumulation of [3H]-cyclic AMP was inhibited by L-glutamate and L-SOP with pIC50 values of 3.21 +/- 0.01 and 2.96 +/- 0.08 and maximal inhibitions of 88 +/- 2 and 93 +/- 3%, respectively. 8. These results suggest that the guinea-pig cerebellum expresses Group I and Group III mGluRs coupled to phosphoinositide turnover and inhibition of cyclic AMP generation, respectively.     

Cyclic AMP potentiates receptor-stimulated phosphoinositide hydrolysis in human neuroepithelioma cells.

A stimulatory role for cAMP in the regulation of receptor-activated phosphoinositide hydrolysis has been examined in human SK-N-MCIXC and SK-N-MCIIE neuroepithelioma cells. The addition of optimal concentrations of oxotremorine-M, norepinephrine, endothelin-1, and ATP enhanced the release of inositol phosphates by 2-9-fold after activation of muscarinic, alpha 1-adrenergic, endothelin, and P2 nucleotide receptors, respectively. All combinations of these agonists elicited a release of inositol phosphates that was at least additive. However, the combined presence of oxotremorine-M and norepinephrine resulted in a phosphoinositide hydrolysis that was 30% greater than additive. This potentiation of inositol lipid hydrolysis resulted from an increased activity of the muscarinic receptor after the addition or norepinephrine and persisted after alpha 1-adrenergic receptor blockade. The enhancement of muscarinic receptor-stimulated inositol phosphate release could be quantitatively mimicked by inclusion of the beta-adrenergic agonist isoproterenol (EC50 approximately 0.1 microM), but not by alpha 1- or alpha 2-adrenergic agonists. Potentiation of oxotremorine-M-stimulated inositol lipid hydrolysis observed in the presence of either norepinephrine or isoproterenol was reduced in the absence of added Ca2+. Addition of either norepinephrine or isoproterenol to SK-N-MCIXC cells also resulted in a 16-fold increase in cAMP concentration. Although the cell-permeant 8-chloro-4-phenylthio-cAMP had a small inhibitory effect on basal inositol phosphate release, its inclusion resulted in a 19-31% enhancement of muscarinic, endothelin, ATP, and alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis. We conclude 1) that, in SK-N-MCIXC cells, the addition of beta-adrenergic agonists selectively enhances muscarinic receptor-stimulated phosphoinositide hydrolysis through a cAMP-dependent process and 2) that the ability of exogenously added cAMP to enhance the activation of all four inositol lipid-linked receptors indicates that the effects of cAMP on inositol lipid hydrolysis are compartmentalized in these cells.     

Distinct mechanisms contribute to agonist and synaptically induced metabotropic glutamate receptor long-term depression

Metabotropic glutamate receptor mediated long-term depression (mGlu receptor LTD) is evoked in hippocampal area CA1 chemically by the agonist 3,5-Dihydroxyphenylglycine (DHPG, agonist LTD) and synaptically by paired-pulse low frequency stimulation (PP-LFS, synaptic LTD). We tested the hypothesis that different expression mechanisms regulate mGlu receptor LTD in 15-21 day old rats through neurophysiologic recordings in CA1. Several findings, in fact, suggest that agonist and synaptic mGlu receptor LTD are expressed through different mechanisms. First, neither LTD occluded the other. Second, a low calcium solution enhanced agonist LTD but did not alter synaptic LTD. Third, application of the mGlu receptor antagonist LY341495 (2S-2-amino-2-(1S,2S-2-carboxycyclopropyl-1-yl)-3-(xanth-9-yl)propanoic acid) reversed agonist LTD expression, but did not alter synaptic LTD. Finally, an N-type, voltage-dependent calcium channel antagonist, ω-conotoxin GVIA (CTX), reduced agonist LTD expression by 35%, but did not alter synaptic LTD. CTX also blocked the increase in the paired-pulse ratio associated with agonist LTD. This study cautions against assuming that agonist and synaptic LTD are equivalent as several components underlying their expression appear to differ. Moreover, the data suggest that agonist LTD, but not synaptic LTD, has a presynaptic, N-channel mediated component.     

Bradykinin analogues: differential agonist and antagonist activities suggesting multiple receptors.

Bradykinin analogues with specific antagonist activity in several bioassays were evaluated for effects on [3H]-bradykinin receptor binding sites and inositol phosphate production in neuroblastoma N1E-115 cells. The analogues varied in their affinities for bradykinin receptors in guinea-pig ileum and N1E-115 cell membranes, in their effects on uterine and ileal contractions and in their agonist or antagonist activity on phosphoinositide turnover in N1E-115 cells. These tissue specific effects suggest the presence of multiple bradykinin receptor subtypes.     

The disposition, metabolism, and pharmacokinetics of a selective metabotropic glutamate receptor agonist in rats and dogs.

Compound LY354740 [(+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic acid], an analog of glutamic acid, is a selective group 2 metabotropic glutamate receptor agonist in clinical development for the treatment of anxiety. Studies have been conducted to characterize the absorption, disposition, metabolism, and excretion of LY354740 in rats and dogs after intravenous bolus or oral administration. Plasma concentrations of LY354740 were measured using a validated gas chromatography/mass spectrometry assay. In rats, LY354740 demonstrated linear pharmacokinetics after oral administration from 30 to 1000 mg/kg. The oral bioavailability of LY354740 was approximately 10% in rats and 45% in dogs. In the dog, food decreased the mean area under the plasma concentration-time curve value by approximately 34%, hence, decreasing the oral bioavailability of the compound. Excretion studies in both rats and dogs indicate that the absorbed drug is primarily eliminated via renal excretion. In addition, tissue distribution in rats showed that the highest levels of radioactivity were in the kidney and gastrointestinal tract, which is consistent with the excretion studies. Metabolism of LY354740 was evaluated in vitro using rat and dog liver microsomes and rat liver slices. In addition, urine and fecal samples from rat and dog excretion studies were profiled using HPLC with radio-detection. These evaluations indicated that neither rats nor dogs metabolized LY354740. In summary, LY354740 is poorly absorbed in rats, moderately absorbed in dogs, and rapidly excreted as unchanged drug in the urine.     

Long-acting agonist and antagonist activities of naltrexamine bivalent ligands in mice

A series of naltrexamine bivalent ligands, compounds with two naltrexamine pharmacophores separated by a spacer which contains a variable number of glycyl units flanking a succinyl group, were synthesized and evaluated in vivo in mice. These compounds possessed long-acting agonist and especially antagonist activities. The bivalent ligands, 2 and 3 displayed antinociceptive activity that lasted greater than 4 h. Compound 1, a bivalent ligand and 4, the monomer, antagonized the antinociceptive effect of morphine for a week after a single injection i.c.v. The long duration of action may be due to entrapment of these ligands in the central nervous system. These compounds may give future insights into the design of long-acting agonists and antagonists.     

Non-NMDA glutamate receptor antagonist 3H-CNOX binds with equal affinity to two agonist states of quisqualate receptors

Binding of 3H-CNQX to rat cortical membranes is saturable and reversible. Apparently, 3H-CNQX binds to a single site with KD = 39 nM. However, studies using AMPA as inhibitor revealed a biphasic inhibition of 3H-CNQX binding. The results suggest that CNQX binds with the same affinity to two different sites. The molecular target size of 3H-CNQX binding (51.8 +/- 3.4 kD) is equivalent to the size of the high affinity 3H-AMPA binding sites, but different from the high affinity 3H-kainate binding sites. A monoexponential decay curve for the high energy radiation inactivation analysis of 3H-CNQX binding indicates that the two 3H-CNQX binding sites have the same molecular weight and therefore might be two different conformations of the same receptor. The standard excitatory amino acids quisqualate, AMPA and kainate have a different rank order of potency as binding inhibitors at the two conformations of the quisqualate receptor.     

Selective group II glutamate metabotropic receptor agonist LY354740 attenuates pentetrazole- and picrotoxin-induced seizures

There are several data indicating the involvement of metabotropic glutamate receptors (mGluR) in seizures and epileptogenesis. In the present experiments, the selective group II mGluR agonist (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylic acid (LY 354740) at doses from 4 to 16 mg/kg) administered prior to the injection of pentetrazole (80 mg/kg) or picrotoxin (3.2 mg/kg) produced a dose-dependent decrease in the number of mice exhibiting clonic convulsions. Our data suggest that group II mGluR agonist LY 354740 possesses antiseizure activity.     

Age-dependent suppression of hippocampal epileptic afterdischarges by metabotropic glutamate receptor 5 antagonist MTEP

Action of an antagonist of metabotropic glutamate receptors subtype 5 MTEP was studied in a model of complex partial seizures. Dorsal hippocampus of rat pups 12, 18 and 25 days old was stimulated six times with 10-min intervals. MTEP (20 or 40 mg/kg) was injected after the first afterdischarge and duration of afterdischarges was measured.MTEP exhibited marked anticonvulsant action in 12-day-old-rats, the similar effect in 18-day-old rats was observed only with the second stimulation. No anticonvulsant action was seen in 25-day-old animals.Our results may qualify antagonists of mGluR5 as potential antiepileptic drugs for some types of childhood epilepsies.