In vitro activity and in vivo efficacy of icofungipen (PLD-118), a novel oral antifungal agent, against the pathogenic yeast Candida albicans.

Icofungipen (PLD-118) is the representative of a novel class of antifungals, beta amino acids, active against Candida species. It has been taken through phase II clinical trials. The compound actively accumulates in yeast, competitively inhibiting isoleucyl-tRNA synthetase and consequently disrupting protein biosynthesis. As a result, in vitro activity can be studied only in chemically defined growth media without free amino acids that would compete with the uptake of the compound. The MIC of icofungipen was reproducibly measured in a microdilution assay using yeast nitrogen base medium at pH 6 to 7 after 24 h of incubation at 30 to 37 degrees C using an inoculum of 50 to 100 CFU/well. The MICs for 69 Candida albicans strains ranged from 4 to 32 microg/ml. This modest in vitro activity contrasts with the strong in vivo efficacy in C. albicans infection. This was demonstrated in a lethal model of C. albicans infection in mice and rats in which icofungipen showed dose-dependent protection at oral doses of 10 to 20 mg/kg of body weight per day in mice and 2 to 10 mg/kg/day in rats. The in vivo efficacy was also demonstrated against C. albicans isolates with low susceptibility to fluconazole, indicating activity against azole-resistant strains. The efficacy of icofungipen in mice and rats was not influenced by concomitant administration of equimolar amounts of L-isoleucine, which was shown to antagonize its antifungal activity in vitro. Icofungipen shows nearly complete oral bioavailability in a variety of species, and its in vivo efficacy indicates its potential for the oral treatment of yeast infections.

Acamprosate inhibits the binding and neurotoxic effects of trans-ACPD, suggesting a novel site of action at metabotropic glutamate receptors.

BACKGROUND: Several reported effects of acamprosate within the glutamatergic system could result from interactions with metabotropic glutamate receptors (mGluRs). The following experiments were performed to determine whether acamprosate could compete with trnas-ACPD (+/--1-aminocyclopentane-trans-1,3-dicarboxylic acid, an equimolecular mixture of 1S, 3R and 1R, 3S-ACPD and an agonist at both group I and group II mGluRs) sensitive binding sites and protect against trans-ACPD-induced neurotoxicity in organotypic hippocampal slice cultures. METHODS: A P2 membrane preparation of cortices, cerebellums, and hippocampi of adult, male Sprague Dawley rats was used to determine the abilities of N-methyl-D-aspartic acid (NMDA) and trans-ACPD to displace [3H]glutamate in both the absence and the presence of the sodium salt of acamprosate (sodium mono N-acetyl homotaurine or Na-acamprosate). A comparison of the effects of 100 microM guanosine 5'-triphosphate on unlabeled glutamate, trans-ACPD, and Na-acamprosate was performed in the same paradigm. For the neurotoxicity studies, organotypic hippocampal slice cultures from male and female 8-day-old neonatal rats were exposed to either 500 microM -ACPD or 50 microM NMDA for 24 hr in normal culture medium containing serum on day 20 in vitro. The effects of Na-acamprosate and 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893), a noncompetitive antagonist at metabotropic type 5 receptors (mGluR5s), were assessed by determining differences in propidium iodide uptake as compared with neurotoxic challenges alone. RESULTS: Na-acamprosate displaced 31% of [3H]glutamate but did not compete with NMDA for [3H]glutamate binding sites. Na-acamprosate displayed total competition with trans-ACPD. The presence of 100 microM guanosine 5'-triphosphate differentially altered the displacing capabilities of the two mGluR agonists, unlabeled glutamate and trans-ACPD, as compared with Na-acamprosate. Na-acamprosate (200-1000 microM) and SIB-1893 (20-500 microM) both were neuroprotective against trans-ACPD induced neurotoxicity that likely results from mGluR potentiation of NMDARs. In turn, Na-acamprosate and SIB-1893 had no direct effects on NMDA-induced neurotoxicity. CONCLUSIONS: Na-acamprosate demonstrates the binding and functional characteristics that are consistent with a group I mGluR antagonist. The functional similarities between Na-acamprosate and SIB-1893 support an interaction of Na-acamprosate at mGluR5s. The neuroprotective properties of acamprosate and possibly its ability to reduce craving in alcohol-dependent patients may result from its alterations in glutamatergic transmission through mGluRs.

mGluR1-mediated potentiation of NMDA receptors involves a rise in intracellular calcium and activation of protein kinase C.

Potentiation of ionotropic glutamate receptor activity by metabotropic glutamate receptors (mGluRs) is thought to modulate activity at glutamatergic synapses in the hippocampus. However, the precise pathway by which this modulation occurs is not well understood. The present study tests the hypothesis that mGluR1-mediated potentiation of N-methyl-D-aspartate receptors (NMDARs) occurs via a phospholipase C (PLC)-initiated cascade. NMDAR functional activity was examined by whole-cell recording from Xenopus oocytes expressing recombinant NMDARs and mGluR1alpha. The mGluR1 agonist (1S,3R)-1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) significantly potentiated NMDA-elicited currents. mGluR1alpha-mediated potentiation of NMDA responses was eliminated by the PLC inhibitor U-73122. Buffering of intracellular Ca2+ by BAPTA-AM or depletion of intracellular Ca2+ by the Ca2+/ATPase inhibitor thapsigargin greatly reduced ACPD potentiation. ACPD potentiation was reduced by the specific protein kinase C (PKC) inhibitor Ro-32-0432 and eliminated by the broad spectrum kinase inhibitor staurosporine. ACPD produced no further potentiation after potentiation of NMDARs by the PKC-activating phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). Thus, Group I mGluRs potentiate NMDA responses via activation of PLC; at least part of the potentiation is due to rise in intracellular Ca2+ and stimulation of PKC. Cytochalasin D, which disrupts the actin cytoskeleton, blocked ACPD-elicited chloride currents and ACPD-induced potentiation of NMDAR currents, consistent with a role for cytoskeletal protein(s) in the signaling pathway. As Group I mGluRs are localized to the perisynaptic region in juxtaposition to NMDARs at glutamatergic synapses, mGluR-mediated potentiation of NMDAR activity may play a role in synaptic transmission and plasticity including LTP.

Selective blockade of mGlu5 metabotropic glutamate receptors protects rat hepatocytes against hypoxic damage.

Western blot analysis of protein extracts from rat liver revealed the presence of the mGlu5 receptor, one of the G-protein-coupled receptors activated by glutamate (named "metabotropic glutamate receptors" or mGlu receptors). mGlu5 expression was particularly high in extracts from isolated hepatocytes, where levels were comparable with those seen in the rat cerebral cortex. The presence of mGlu5 receptors in hepatocytes was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) analysis, immunohistochemistry in neonate or adult rat liver, as well as by immunocytochemical analysis in HepG2 hepatoma cells, where the receptor appeared to be preferentially distributed in cell membranes. Interestingly, mGlu1 receptors (which are structurally and functionally homologous to mGlu5 receptors) were never found in rat liver or hepatocytes. In hepatocytes exposed to anoxic conditions for 90 minutes, glutamate, (1S,3R)-1-aminocyclopentane-1, 3-dicarboxylic acid (1S,3R-ACPD) and quisqualate, which all activate mGlu5 receptors, accelerated the onset and increased the extent of cell damage, while 4-carboxy-3-hydroxyphenylglycine (4C3HPG), an agonist of mGlu2/3 receptors, was inactive. 2-methyl-6-(2-phenyl-1-ethynyl)-pyridine (MPEP), a novel, noncompetitive, highly selective mGlu5 receptor antagonist, not only abolished the toxic effect of 1S,3R-ACPD, but, unexpectedly, was protective by itself against anoxic damage. This suggests that hepatocytes express mGlu5 receptors and that activation of these receptors by endogenous glutamate facilitates the development of anoxic damage in hepatocytes.

Analysis of the interaction between arachidonic acid and metabotropic glutamate receptor activation reveals that phospholipase C acts as a coincidence detector in the expression of long-term potentiation in the rat dentate gyrus.

We have reported that arachidonic acid and the metabotropic glutamate receptor agonist, trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), act in synergy to increase release of glutamate from synaptosomes prepared from rat dentate gyrus. The observation that prior induction of LTP in perforant path-granule cell synapses occluded this synergism suggested that the interaction between arachidonic acid and ACPD might trigger the increase in glutamate release that accompanies LTP in dentate gyrus. Our objective was to identify the mechanism underlying the synergism between arachidonic acid and ACPD in LTP. The data indicate that both agents activate phospholipase C(PLC); the arachidonic acid-induced increase in phospholipase C activation was inhibited by the tyrosine kinase inhibitor, genistein, suggesting that PLCgamma, which is stimulated by tyrosine phosphorylation may be activated by arachidonic acid. The ACPD-induced increase was inhibited by neomycin, indicating the involvement of a G-protein and suggesting that PLCbeta may be activated by ACPD. We report that arachidonic acid stimulated phosphorylation of the specific tyrosine kinase substrate, poly(Glu80,Tyr20) and direct analysis indicated that arachidonic acid increased phosphorylation of PLCgamma. PLCgamma phosphorylation was assessed in control dentate gyrus and dentate gyrus in which LTP was induced in vivo. We report that the tyrosine kinase inhibitor, genistein, blocked expression of LTP and also blocked the associated increase in phosphorylation of PLCgamma. The data presented here indicate that tyrosine phosphorylation of PLCgamma was significantly enhanced following induction of LTP, but in separate experiments, in which LTP was inhibited by intraventricular injection of genistein, phosphorylation of PLCgamma was inhibited. The evidence presented is consistent with the hypothesis that PLC acts as a coincidence detector in LTP. The data indicate that PLCbeta is activated by ACPD, PLCgamma is activated by arachidonic acid, and coincident activation of both isoforms is necessary to stimulate an increase in glutamate release.

Neuroprotective sigma ligands attenuate NMDA and trans-ACPD-induced calcium signaling in rat primary neurons.

The effect of neuroprotective sigma ligands possessing a range of relative selectivity for sigma and phencyclidine (PCP) binding sites on N-methyl-D-aspartate (NMDA) and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD)-stimulated calcium flux was studied in 12-15-day-old primary cultures of rat cortical neurons. In approximately 80% of the neurons tested, NMDA (80 microM) caused a sustained increase in intracellular calcium ([Ca2+]i). With the exception of R-(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ((+)-3-PPP) (previously shown not to be neuroprotective) all of the sigma ligands studied significantly altered NMDA-induced calcium dynamics. The primary effect of dextromethorphan, (+)-pentazocine, (+)-cyclazocine, (+)-SKF10047, carbetapentane, 1,3-di(2-tolyl) guanidine (DTG), and haloperidol was to shift the NMDA response from a sustained, to either a biphasic or a transient, calcium event. In contrast to NMDA, the primary response observed in 62% of the neurons treated with trans-ACPD (100 microM) was a transient elevation in [Ca2+]i. Here, however, only the highly selective neuroprotective sigma ligands (i.e., those lacking substantial PCP binding affinity) significantly decreased the number of transient responses elicited by trans-ACPD whereas the PCP-related sigma ligands such as dextromethorphan, (+)-SKF10047 and (+)-cyclazocine were ineffective. Unexpectedly, (+)-3-PPP potentiated trans-ACPD activity. These results demonstrating attenuating effects of sigma ligands on NMDA-stimulated neuronal calcium responses agree with earlier studies using glutamate and KCl and identify a sigma receptor modulation of functional NMDA responsiveness. Furthermore, the ability of sigma ligands to attenuate NMDA-, trans-ACPD- and KCl-evoked neuronal calcium dynamics indicates that the receptor mechanisms mediating sigma neuroprotection comprise complex interactions involving ionotropic, metabotropic, and even voltage-gated calcium signaling processes.

Dicarboxyphenylglycines antagonize AMPA- but not kainate-induced depolarizations in neonatal rat motoneurones.

Ionotropic glutamate receptors have been categorized into three main groups according to the selective agonists that activate them, the N-methyl-D-aspartate (NMDA), (S)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propanoic acid (AMPA) and (2S,3S,4S)-3-carboxymethyl-4-isopropenylpyrrolidine-2-carboxylic acid (kainate) receptors. Both AMPA and kainate induce depolarizations in neonatal rat spinal motoneurones. However, selective antagonists capable of discriminating between the effects of these two antagonists are not widely available. As part of a search for such compounds we report the actions of (RS)-3,4-dicarboxyphenylglycine (DCPG) and (RS)-3,5-dicarboxyphenylglycine on agonist-induced motoneuronal depolarizations in the neonatal rat spinal cord preparation. In addition, the actions of (R)- and (S)-3,4-DCPG are also described. (RS)-3,4-DCPG and (RS)-3,5-DCPG antagonized AMPA-induced depolarizations (apparent Kd = 137 microM (n = 3) and 167 microM (n = 5), respectively). However, (RS)-3,5-DCPG (1 mM) potentiated responses due to kainate (n = 5) while (RS)-3,4-DCPG (1 mM) displayed weak antagonism of these responses (apparent Kd > 12 mM, n = 3). (RS)-3,4- and (RS)-3,5-DCPG at 500 microM both displayed antagonism at the NMDA receptor (apparent Kd = 472 microM and 346 microM, respectively) and a postsynaptic subgroup I metabotropic glutamate receptor activated by (1S,3R)-ACPD. The AMPA receptor antagonist activity of (RS)-3,4-DCPG was shown to reside in the (R)-enantiomer (apparent Kd = 77 microM, n = 3). The same isomer was responsible for the NMDA receptor antagonism while showing little or no antagonism of kainate-induced depolarizations (apparent Kd > 3 mM, n = 3), and a weak antagonistic effect at (1S,3R)-ACPD receptors. (S)-3,4-DCPG (500 microM) was unable to antagonize kainate-induced depolarizations, showed weak or no antagonism of NMDA- and AMPA-induced depolarizations, but antagonized (1S,3R)-ACPD-induced depolarizations. Thus (RS)-3,4-, (RS)-3,5- and (R)-3,4-DCPG demonstrate useful discrimination of responses due to AMPA and kainate, strongly suggesting that pharmacologically distinct AMPA and kainate receptors exist in motoneurones in the neonatal rat spinal cord.

Activation of multiple metabotropic glutamate receptor subtypes prevents NMDA-induced excitotoxicity in rat hippocampal slices.

Metabotropic glutamate receptors (mGluRs) belong to a relative large receptor family consisting of multiple members with important roles in a number of brain functions. We report here that activation of mGluRs prevents the neurotoxic effect induced by N-methyl-D-aspartate (NMDA) in slices from the rat hippocampus. Neuroprotection was elicited when slices were simultaneously exposed to both the selective mGluR agonist (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (tACPD) and NMDA. Persisting stimulation of mGluRs after the toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD toxic exposure did not improve the survival of pyramidal or granular cells. The neuroprotection elicited by tACPD was also evoked by its active isomer, (1S, 3R)-ACPD, and was prevented by the selective mGluR antagonist (+)-alpha-methyl-4-carboxyphenyl-glycine (500 microM), confirming that mGluR activation is involved in the mechanism of action of tACPD. The effect of 100 microM tACPD was reproduced by 100 microM quisqualate, an agonist of mGluR2 and mGluR3 subtypes. No neuroprotection was induced by L-2-amino-4-phosphonobutyrate, a selective agonist for mGluR4, mGluR6, mGluR7 and mGluR8, at 500 microM. Since the NMDA-mediated cell death in hippocampal slices is considered relevant to ischaemia-induced brain injury, these results indicate that mGluRs may be important safety devices used by neurons to decrease their sensitivity to excitotoxic stimuli and increase their chance of survival.

Contralateral turning caused by metabotropic glutamate receptor stimulation in the dorsal striatum is reversed by MCPG, TTX, and cis-flupenthixol.

Recent evidence suggests an involvement of metabotropic glutamate receptors in the physiology of the striatum. In this study, rotation was recorded in an automated rotometer for 20 min following dorsal striatal injections (0.5 microliter) in cannulated rats. The metabotropic agonist 1-aminocyclopentane-trans-1, 3-dicarboxylic acid (1S,3R-ACPD) caused dose-dependent contralateral rotation. Turning caused by 500 microM 1S,3R-ACPD was reversed by coinjections of the metabotropic antagonist (+)-alpha-methyl-4-carboxyphenylglycine (MCPG, 1 mM) and by tetrodotoxin (100 microM). Injections of MCPG alone (10 microM, 100 microM, 1 mM) failed to elicit turning. Increasing doses of the dopamine antagonist cis-flupenthixol also reversed 1S,3R-ACPD-induced rotation. Thus unilateral striatal metabotropic glutamate receptor stimulation can cause receptor-specific rotation that may result from an increase in neural activity, and is dependent on intact dopamine neurotransmission.

Characterization of metabotropic glutamate receptor-mediated facilitation of N-methyl-D-aspartate depolarization of neocortical neurones.

1. Facilitation of the N-methyl-D-aspartate (NMDA) receptor-mediated depolarization of cortical neurones induced by metabotropic glutamate receptor (mGluR) agonists in the presence of tetrodotoxin has been examined by use of grease-gap recording. 2. Quisqualate (1-2 microM) and 10 to 100 microM 1S,3R-I-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) facilitated the NMDA-, but not the kainate-induced depolarization with an EC50 of 16 microM for 1S,3R-ACPD. The facilitation induced by quisqualate was reduced, but not blocked, by 4 microM 6-cyano-7-nitroquinoxaline-2,3-dione. 3. D,L-2-Amino-3-phosphonopropionic acid and D,L-2-amino-4-phosphonobutyric acid antagonized the 1S,3R-ACPD facilitation in a non-competitive manner with IC50 values of 0.24 microM and 4.4 microM respectively. 4. Homologous desensitization of the 1S,3R-ACPD induced facilitation was not observed. The facilitation was not altered by 10 nM staurosporine or 3 microM phorbol diacetate. 5. Substitution of 20 microM 8-bromo-cyclic adenosine monophosphate, 20 microM 8-bromo-cyclic guanosine monophosphate, or 10 microM arachidonic acid for 1S,3R-ACPD did not induce facilitation of the NMDA response. However, the 1S,3R-ACPD facilitation was potentiated by 10 mM myo-inositol and exhibited heterologous desensitization following exposure to 100 microM 5-hydroxytryptamine. 6. The 1S,3R-ACPD-induced facilitation persisted in both 10 microM nifedipine and nominally Ca(2+)-free medium and was only gradually eliminated following addition of 100 microM bis-(-o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid in Ca(2+)-free medium. Facilitation of the NMDA response induced by carbachol, but not phenylephrine, was also observed in nominally Ca(2+)-free medium. Perfusing 50 microM bis-(-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid aminoethoxy eliminated the 1S,3R-ACPD facilitation. 7. These experiments have shown that mGluR agonists selectively facilitate the NMDA depolarization of cortical wedges, most likely by activating one or more mGluR subtypes that couple to phospholipase C. We conclude the facilitation results from a Ca(2+)-sensitive mechanism dependent on activation of phospholipase C and release of internal Ca2+. The facilitation is not contingent on activation of protein kinase C or entry of Ca2+ through nifedipine-sensitive Ca2+ channels.

Potent antagonists at the L-AP4- and (1S,3S)-ACPD-sensitive presynaptic metabotropic glutamate receptors in the neonatal rat spinal cord.

In this report we describe the actions of two novel compounds, (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG) and (S)-alpha-ethylglutamate (EGLU), which are potent antagonists at two types of presynaptic metabotropic glutamate (mGlu) receptors in the neonatal rat spinal cord. Selective activation of these receptors by L-2-amino-4-phosphonobutyrate (L-AP4) or (1S,3S)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3S)-ACPD) results in the depression of the monosynaptic component of the dorsal root-evoked ventral root potential (DR-VRP). CPPG produces rightward parallel shifts of the dose-response curves for both L-AP4- and (1S,3S)-ACPD, with Schild slope in each case close to unity, consistent with a competitive mechanism of antagonism. CPPG is the most potent antagonist yet described for both L-AP4- and (1S,3S)-ACPD-sensitive presynaptic mGlu receptors but displays a 30-fold selectivity for the L-AP4-sensitive receptor over the (1S,3S)-ACPD-sensitive receptor (KD values 1.7 microM and 53 microM, respectively). EGLU, on the other hand, is selective for the (1S,3S)-ACPD-sensitive receptor, displaying little or no activity at the L-AP4-sensitive site. EGLU produces a rightward parallel shift of the dose-response curve to (1S,3S)-ACPD, with Schild slope close to unity, again indicative of a competitive mode of antagonism (KD 66 microM). Both CPPG and EGLU displayed only weak or no antagonist activity at postsynaptic metabotropic and ionotropic glutamate receptors.

Metabotropic glutamate receptor modulation of synaptic transmission in corticostriatal co-cultures: role of calcium influx.

Modulation of excitatory glutamatergic transmission at corticostriatal synapses by a metabotropic glutamate receptor (mGluR) was examined using a newly developed cell culture preparation in which small explants of cortical tissue are grown in co-culture with isolated striatal neurons. Electrical stimulation of cortical tissue evoked excitatory postsynaptic currents (eEPSCs) observed during tight-seal, whole-cell recordings from striatal neurons. Transmission was mediated by activation of AMPA/kainate-type glutamate receptors. The mGluR agonists, 1SR,3RS-ACPD and DCG-IV, reduced eEPSC amplitude. The effect of 1SR,3RS-ACPD increased in a concentration-dependent manner. Application of phorbol diacetate (PDAc) potentiated eEPSC amplitude and reduced the inhibitory effect of mGluR activation. Pretreatment with pertussis toxin (PTX) also reduced inhibition by 1SR,3RS-ACPD. Under conditions in which transmission was independent of the function of voltage-gated calcium channels, mGluR activation reduced the frequency of occurrence of miniature EPSCs (mEPSCs), but did not alter mEPSC amplitude. This effect of mGluR activation was reduced by PDAc treatment. mGluR activation modulates glutamatergic transmission via a presynaptic autoreceptor at corticostriatal synapses in this newly-developed corticostriatal co-culture preparation as in striatal slices. Modulation of transmission occurs whether or not transmission involves activation of voltage-gated calcium channels. Furthermore, many of the characteristics of mGluR modulation of eEPSCs are shared by mGluR modulation of mEPSCs. These findings indicate that mechanisms downstream from calcium entry may contribute to modulation of synaptic transmission by mGluR autoreceptors.

Potentiation of excitatory postsynaptic potentials by a metabotropic glutamate receptor agonist (1S,3R-ACPD) in frog spinal motoneurons.

We conducted intracellular recordings of lumbar motoneurons in the arterially-perfused frog spinal cord and investigated the effects of a metabotropic glutamate receptor agonist, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD), on excitatory postsynaptic potentials evoked by stimulation of the descending lateral column fibers (LC-EPSPs). In the absence of Mg2+, ACPD reversibly potentiated the amplitude of monosynaptic LC-EPSPs by more than 15% in 15 of 19 cells with 5 microM ACPD and in 7 of 12 cells with 0.5 microM ACPD. The EPSP amplitudes with 5 and 0.5 microM ACPD were 142 +/- 10% (mean +/- S.E.M., n = 19) and 130 +/- 13% (n = 12) of the controls. The potentiation was seen without a decrease in the input conductance. Glutamate-induced depolarizations in the absence and the presence of 0.5 microM ACPD were not significantly different in cells perfused with the low Ca(2+)-high Mg2+ solution which eliminated chemical transmission. Paired pulse facilitation of LC-EPSPs was reversibly decreased in association with the potentiation. ACPD-induced potentiation of monosynaptic LC-EPSPs was seen in 5 of 6 cells in the presence of D-(-)-2-amino-5-phosphonopentanoic acid (D-AP5), an NMDA receptor antagonist. ACPD occasionally activated polysynaptic components of LC-EPSPs which were mediated mainly via NMDA receptors. On the other hand, ACPD-induced potentiation of EPSPs was inhibited by extracellular Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistent current oscillations produced by activation of metabotropic glutamate receptors in immature rat CA3 hippocampal neurons.

1. The single-electrode voltage-clamp technique was used to study the effects of the metabotropic glutamate receptors (mGluRs) agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD, ACPD, 3-10 microM) on CA3 hippocampal neurons during the 1st 10 days of postnatal (P) life and in adulthood. 2. Repeated applications of 1S,3R-ACPD, in the presence of tetrodotoxin (TTX, 1 microM), tetraethylammonium chloride (TEACl 10 mM), and CsCl (2 mM), induced in immature but not in adult neurons periodic inward currents (PICs) that persisted for several hours after the last application of the agonist. 3. PICs, which were generated by nonspecific cationic currents, reversed polarity at 2.8 +/- 3 (SD) mV. They were reversibly blocked by kynurenic acid (1 mM), suggesting that they were mediated by glutamate acting on ionotropic receptors. They were also abolished in a nominally Ca(2+)-free medium. 4. PICs were irreversibly abolished by thapsigargin (10 microM) but were unaffected by ryanodine (10-40 microM). Caffeine (2 mM) also reversibly blocked PICs; this effect was independent from adenosine 3',5'-cyclic monophosphate (cAMP) accumulation, inhibition of voltage-dependent Ca2+ current, or blockade of adenosine receptors. 5. We suggest that, in neonatal slices, mGluRs-induced PICs are triggered by elevation of [Ca2+]i, after mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive stores. This will lead to a persistent, pulsatile release of glutamate from presynaptic nerve terminals, a phenomenon that is probably maintained via a calcium-induced-calcium release process.

Pharmacological dissociation of glutamatergic metabotropic signal transduction pathways in cortical astrocytes.

Using cultured cortical astrocytes we demonstrate differential activation of metabotropic signal transduction pathways with 1-aminocyclopentane-trans-1S3R-dicarboxylic acid (1S3R-ACPD) and the glutamate transport inhibitor trans-2,4-pyrrolidine dicarboxylic acid (trans-2,4-PDC). Phosphoinositide hydrolysis was more potently stimulated by 1S3R-ACPD than by L-trans-2,4-PDC; however, L-trans-2,4-PDC was far more efficacious than 1S3R-ACPD at inhibiting cyclic AMP accumulation. The metabotropic receptor antagonist (+)-alpha-methyl-4-carboxyphenylglycine ((+)-MCPG) inhibited 1S3R-ACPD stimulation of phosphoinositide hydrolysis but not its ability to inhibit cyclic AMP accumulation thereby demonstrating a means to pharmacologically dissociate these two metabotropic signal transduction pathways in astrocytes. (+)-MCPG produced similar antagonism of the metabotropic agonist properties of L-trans-2,4-PDC. The metabotropic effects of L-trans-2,4-PDC could not be reduced with enzymatic treatment of the cultures to remove extracellular glutamate, suggesting that these effects are not secondary to the ability of this compound to inhibit glutamate uptake. Taken together the findings indicate the presence of multiple glutamatergic signal transduction pathways in astrocytes and suggest a similarity in the pharmacophores for metabotropic receptors and glutamate transporters.

Induction of LTD but not LTP through metabotropic glutamate receptors in visual cortex.

Long-term potentiation (LTP) and long-term depression (LTD), often used as essential components in synaptic models for learning, memory and forgetting, can be produced in cortical tissue by repetitive activation of neural pathways under different stimulus conditions. The involvement of metabotropic glutamate receptors (mGluRs) has been postulated to be necessary for the establishment of either or both forms of synaptic plasticity in hippocampus. The recent introduction of a specific antagonist for mGluRs, (+/-)-alpha-methyl-4-carboxyphenylglycine, prompted the investigation of the respective involvement of this receptor population in the induction of LTP and LTD in visual cortex of the rat in vitro. The results suggest the critical involvement of mGluRs in producing LTD but not LTP.

Competitive antagonism by phenylglycine derivatives at type I metabotropic glutamate receptors.

The metabotropic glutamate receptors (mGluRs) form a family of G-protein-coupled receptors which consists of at least seven members termed mGluR1-mGluR7. These members are classified into subfamilies according to their sequence similarities, signal transduction mechanisms and agonist selectivities. mGluR1 and mGluR5 are coupled to the phosphoinositide hydrolysis/Ca2+ signal transduction and efficiently respond to quisqualate. In this study, we have stably expressed mGluR1 in Chinese hamster ovary cells on which the activation of the phosphoinositide signal transduction pathway was evaluated by means of two methods and their degree of correspondence was analyzed. These two methods involve the Li(+)-dependent accumulation of [3H]inositol-labeled inositol phosphates or the [3H]cytidine-labeled phospholiponucleotide cytidine diphospho (CDP)- diacylglycerol (DAG). The correlation between the two measures was found to be generally uniform for the different agonists evaluated. However, the levels of CDP-DAG were found to be consistently higher. Furthermore, quisqualate showed a differential activity on the two methods behaving as a partial agonist and as a full agonist on the inositol phosphate and the CDP-DAG responses, respectively. On the same cells the activity of a series of carboxyphenylglycines recently described as possible new tools for investigating the role of mGluRs has been evaluated. Three phenylglycine derivatives were tested and found to be competitive antagonists at this mGluR subtype. They inhibited both the phosphoinositide signal transduction pathway and the release of intracellular Ca2+ induced by quisqualate the most potent agonist at mGluR1. The pharmacological nature of these compounds and their relative potencies in antagonizing mGluR1 activation are described.

MCPG antagonizes metabotropic glutamate receptors but not long-term potentiation in the hippocampus.

In the CA1 and CA3 regions of the guinea pig hippocampus, we have tested the ability of the new antagonist (RS)-alpha-methyl-4-carboxyphenylglycine (MCPG) to inhibit the well-known effects of (trans)-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), a specific agonist of glutamate metabotropic receptors. Whole-cell recordings showed that MCPG was able to antagonize the blocking action of ACPD on IAHP in the CA1 region. In addition, we report here that MCPG also antagonized the presynaptic inhibitory actions of ACPD on field excitatory postsynaptic potentials in both areas CA1 and CA3. Thus, MCPG proved to be an effective tool for determining physiological roles of the glutamate metabotropic receptors in synaptic transmission in the hippocampus. We next tested the possible effects of this antagonist on long-term potentiation (LTP). In completely blind experiments MCPG was without effect on LTP in both areas CA1 and CA3. In conclusion, our results suggest that, although MCPG is a valuable antagonist of the ACPD-sensitive receptors, it has no inhibitory effect on LTP.

The function of metabotropic excitatory amino acid receptors in synaptic transmission in the thalamus: studies with novel phenylglycine antagonists.

The phenylglycines 3-hydroxyphenylglycine, 4-carboxy-3-hydroxy-phenylglycine (4C3HPG), 4-carboxyphenylglycine (4CPG) and alpha-methyl-4-carboxyphenylglycine (MCPG) were evaluated as putative selective antagonists of metabotropic glutamate receptors on single neurones of the ventrobasal thalamus of rats, with a view to using these compounds as tools to elucidate synaptic mechanisms in this brain area. The S-isomers of the latter three compounds were found to reduce excitations evoked by iontophoretically applied 1S,3R-ACPD, but not those evoked by ionotropic excitatory amino receptor agonists. When the antagonists were tested against sensory synaptic responses of ventrobasal neurones, it was found that responses evoked by noxious thermal stimulation of the peripheral receptive field were reduced in parallel with responses to 1S,3R-ACPD. In contrast, responses of neurones evoked by non-noxious (air-jet) stimuli were not reduced by the phenylglycine antagonists and 4C3HPG was found to enhance such responses, possibly by a presynaptic action mediated via mGluR2 receptors. The reductions of nociceptive responses are discussed in the context of antagonism of mGluR1 receptors, which are known to be numerous in the thalamus and located on post-synaptic dendrites. The involvement of such receptors in the nociceptive responses of thalamic neurones may be of considerable functional significance.

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). 6. Co-addition of 10 or 100 MicroM NMDA enhanced the initial peak Ins(1,4,5)P3 response to 1S,3RACPD.However, the enhancing effect was only maintained over 10 min in the presence of 1O Micro MNMDA, whilst in contrast, 100 MicroM NMDA ceased to cause a significant enhancement of the metabotropic response by 5 min and completely suppressed lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation at 10 min.7. Both basal and 1S,3R-ACPD-stimulated Ins(1,4,5)P3 accumulations were reduced when slices were incubated in nominally Ca2"-free medium. Under these conditions only a concentration-dependent enhancement of the response was observed (EC50 for NMDA facilitation of lS,3R-ACPD-stimulated Ins(1,4,5)P3 accumulation of 32 MicroM).8. These experiments have revealed that at low concentrations, NMDA can dramatically potentiate1S,3R-ACPD-stimulated phosphoinositide hydrolysis, probably by a Ca2"-dependent facilitation of agonist-stimulated phosphoinositide-specific phospholipase C activity. Higher concentrations of NMDA result in time-dependent inhibition of the metabotropic agonist-stimulated response. We believe the former effect could be fundamental in glutamate receptor 'cross-talk', whereas the latter may reflect a Ca2+-dependent neurotoxic effect of NMDA on the neonatal cerebral cortex slices.