Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors

We have investigated the role of metabotropic glutamate receptors linked to phosphoinositide hydrolysis in the control of glutamate release in cerebrocortical nerve terminals. The activation of these receptors with the agonist 3,5-dihydroxyphenylglycine enhanced intra-synaptosomal diacylglycerol and facilitated both the depolarization-induced increase in the cytosolic free Ca2+ concentration and the release of glutamate. However, 5 min after receptor activation, a second stimulation of the pathway with the agonist failed to produce diacylglycerol and to facilitate glutamate release. Interestingly, during the period in which the diacylglycerol response was desensitized, a strong agonist-induced inhibition of Ca2+ entry and glutamate release was observed. This change in the presynaptic effects of 3,5-dihydroxyphenylglycine is reversible since 30 min after the first stimulation, the agonist-induced inhibition of release disappeared, whereas both the production of diacylglycerol and the facilitation of glutamate release were recovered. The tonic elevation of the extracellular glutamate concentration from basal levels (0.8 microM) up to 5 microM also produced the switch from facilitation to inhibition in the receptor response. The existence of this activity-dependent switch in the presynaptic control of glutamate release suggests that release facilitation is limited to conditions under which an appropriate clearance of synaptic glutamate exists, probably to prevent the neurotoxic accumulation of glutamate in the synapse.     

Group II and group III metabotropic glutamate receptors in the rat retina: distributions and developmental expression patterns

We have studied the distributions of group II metabotropic glutamate receptors, mGluR2 and mGluR3, and a group III metabotropic glutamate receptor, mGluR4, in the adult rat retina and during postnatal development using receptor specific anti-peptide antisera. Of the three receptors examined, mGluR3 was not expressed in the retina. MGluR2 showed a distinct stratification pattern in the inner plexiform layer (IPL). Double-labelling immunocytochemistry revealed that mGluR2 was localized in the processes of cholinergic amacrine cells. MGluR4 was found throughout the entire IPL. At the subcellular level, both mGluR2 and mGluR4 were found to be localized exclusively in processes postsynaptic to bipolar cell synapses in the IPL. During postnatal development, labelling for mGluR2 was detected at around postnatal day five. MGluR4 was already present at postnatal day one, prior to the establishment of synaptic connections in the IPL. The differential expression patterns of individual metabotropic glutamate receptors in the adult and developing rat retina suggest distinct roles for these receptors in retinal synaptic circuitry.     

Free radicals and glutamate uptake in the retina

A 21-year-old man suffered from exertional heat stroke with impaired consciousness and rhabdomyolysis after strenuous physical exercise. Within two weeks the patient recovered completely without any specific therapy. Based on the symptoms and laboratory investigations, this episode suggested a moderate form of malignant hyperthermia. An in vitro contracture test was performed and a predisposition to malignant hyperthermia was diagnosed; other muscular diseases were excluded by histological examination. At present, the in vitro contracture test is the only method used to determine susceptibility to malignant hyperthermia and should be performed when the diagnosis is suggested on clinical grounds.     

Localization and function of five glutamate transporters cloned from the salamander retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.     

Transport-mediated release of endogenous glutamate in the vertebrate retina

In the present study we measured calcium-dependent, vesicular glutamate release, and calcium-independent, transport-mediated glutamate release patterns in the vertebrate retina to better understand the sources of elevated glutamate in neural tissue under ischemic conditions. A potassium concentration of 40 mM, which mimics the extracellular potassium concentration in the central nervous system during ischemia, was applied to the bathing medium of a retinal slice prepared from zebrafish. High external potassium evoked release of endogenous glutamate that was measured using a glutamate-specific fluorometric assay applied to the bath. The slice was visualized under 668 nm light using Normarski optics and fluorescent images were captured using a cooled charge-coupled device (CCD) camera. Following the elevation of external potassium to 40 mM several bands of glutamate fluorescence, reflecting the spatial distribution of glutamate release, were observed. A calcium-dependent cloud of glutamate was observed in the inner plexiform layer, that was antagonized by bath-applied nifedipine. A relatively dense glutamate cloud (1-10 microM) was observed over the ganglion cell layer, which was blocked by dihydrokainate, a glutamate transport antagonist. In contrast, nifedipine, an inhibitor of calcium-dependent neurotransmitter release in the retina, failed to block the cloud of released glutamate in the ganglion cell layer. These data suggest that under pathological conditions in the eye where glutamate levels are elevated surrounding retinal ganglion cells, such as observed in some forms of glaucoma, a possible source of the elevated glutamate is through a glutamate transporter operating in a reversed direction. A likely candidate for mediating this reversed transport of glutamate is the retinal Muller cell.     

Subtype-specific involvement of metabotropic glutamate receptors in two forms of long-term potentiation in the dentate gyrus of freely moving rats

In this study, the role of metabotropic glutamate receptors in N-methyl-D-aspartate receptor-dependent and voltage-gated calcium channel-dependent long-term potentiation in the dentate gyrus of freely moving rats was investigated. Antagonists for group 1 metabotropic glutamate receptors ((S)-4-carboxyphenylglycine), group 1/2 metabotropic glutamate receptors ((RS)-alpha-methyl-4-carboxyphenylglycine) and group 2 metabotropic glutamate receptors ((RS)-alpha-methylserine O-phosphate monophenylester) were used. The N-methyl-D-aspartate receptor antagonist, D(-)-2-amino-5-phosphonopentanoic acid, and the L-type voltage-gated calcium channel antagonist, methoxyverapamil were used to investigate the N-methyl-D-aspartate receptor and voltage-gated calcium channel contribution to the long-term potentiation recorded. Field excitatory postsynaptic potential slope and population spike amplitude were measured. Drugs were applied, prior to tetanus, via a cannula implanted into the lateral cerebral ventricle. 200 Hz tetanization produces a long-term potentiation which is inhibited by application of D(-)-2-amino-5-phosphonopentanoic acid and (RS)-alpha-methyl-4-carboxyphenylglycine. In this study, a dose-dependent inhibition of 200 Hz long-term potentiation expression was obtained with (S)-4-carboxyphenylglycine. Long-term potentiation induced by 400 Hz tetanization was not inhibited by D(-)-2-amino-5-phosphonopentanoic acid, although the amplitude of short-term potentiation was reduced. (RS)-alpha-methyl-4-carboxyphenylglycine and (S)-4-carboxyphenylglycine, both in the presence and absence of D(-)-2-amino-5-phosphonopentanoic acid, inhibited the development of 400 Hz long-term potentiation. (RS)-alpha-methylserine O-phosphate monophenylester had no significant effect on long-term potentiation induced by either 200 or 400 Hz tetanization. Application of methoxyverapamil significantly inhibited 400 Hz long-term potentiation, but had no effect on 200 Hz long-term potentiation. These data suggest that 400 Hz long-term potentiation, induced in the presence of D(-)-2-amino-5-phosphonopentanoic acid, requires activation of L-type calcium channels. Furthermore, these results strongly support a critical role for group 1 metabotropic glutamate receptors in both N-methyl-D-aspartate receptor- and voltage-gated calcium channel-dependent long-term potentiation.     

Characterization of glutamate transporter function in the tiger salamander retina

Glutamate transporters in the tiger salamander retina were studied by autoradiographic and intracellular recording techniques. When the retina was incubated with 15 microM L-[3H]glutamate, photoreceptors and Muller cells were labeled, indicating that these cells had high-affinity glutamate uptake transporters. A much higher dose of glutamate than kainate was required in the bath to produce the same membrane depolarization in horizontal cells (HCs), and the time course of glutamate-induced depolarization was much slower than that of the kainate-induced depolarization. Since glutamate is a substrate of glutamate transporters whereas kainate is not, we attribute these differences to the buffering of extracellular glutamate by glutamate transporters in the retina. D-aspartate (D-asp) increased the efficacy of bath-applied glutamate. Dihydrokainate (DHKA) exerted little effect on glutamate efficacy when applied alone, but it increased glutamate efficacy in the presence of D-asp. These results are consistent with the notion that glutamate transporters in Muller cells are D-asp sensitive and those in photoreceptors are DHKA and D-asp sensitive. Application of DHKA (1-2 mM) did not affect the dark membrane potential or the light responses in rods and cones, but it depolarized the HC dark membrane potential and reduced the HC peak and tail light responses. Our results suggest that DHKA-sensitive glutamate transporters in photoreceptors regulate glutamate levels in rod and cone synaptic clefts. They modulate dark membrane potential and the relative rod cone inputs in retinal HCs.     

Trafficking of molecules and metabolic signals in the retina

Photoreceptors need the support of pigment epithelial (PE) and Müller glial cells in order to maintain visual sensitivity and neurotransmitter resynthesis. In rod outer segments (ROS), all-trans-retinal is transformed to all-trans-retinol by retinol dehydrogenase using NADPH. NADPH is restored in ROS by the pentose phosphate pathway utilizing high amounts of glucose supplied by choriocapillaries. The retinal formed is transported to PE cells where regeneration of 11-cis-retinal occurs. Müller cells take up and metabolize glucose predominantly to lactate which is massively released into the extracellular space (ES). Lactate is taken up by photoreceptors, where it is transformed to pyruvate which, in turn, enters the Krebs cycle in mitochondria of the inner segment. Stimulation of neurotransmitter release by darkness induces 130% rise in the amount of glutamate released into ES. Glutamate is transported into Müller cells where it is predominantly transformed to glutamine. Stimulation of photoreceptors induces an eightfold increase in glutamine formation. It appears, therefore, that there is a signaling function in the transfer of amino acids from Müller cells to photoreceptors. Work on the model-system of the honeybee retina demonstrated that photoreceptors release NH4+ and glutamate in a stimulus-dependent manner which, in turn, contribute to the biosynthesis of alanine in glia. Alanine released into the extracellular space is taken up and used by photoreceptors. Glial cells take glutamate by high-affinity transporters. This uptake induces a transient change in glial cell metabolism. The transformation of glutamate to glutamine is possibly also controlled by the uptake of NH4+ which directly affects cellular metabolism.     

Deduction: moving from the general to the specific

During a 29 month period, 46 patients with chronic hepatitis C virus (HCV) received recombinant human interferon alpha-2a for 6 months and were followed for another 6 months. The dose of interferon was three million units thrice weekly and was increased to six million units if amino transferase levels failed to return to normal after 2 months of therapy. At the end of the treatment 19 patients had a complete response, 6 had a near complete response, 2 patients had breakthrough during treatment, and the remaining 19 did not respond at all. Six months after treatment only 10 of the 19 responders remained in remission. Post-transfusion disease was associated with a significantly higher remission rate than sporadic disease (9/22 vs. 1/24, P < 0.001), as was also found in non-cirrhotic compared to cirrhotic patients (9/27 vs. 1/19, P < 0.001). Age, sex, duration of disease, serum aminotransferase, albumin, bilirubin, alkaline phosphatase, or Child's classification did not correlate with treatment response. Severe side effects necessitating cessation of treatment occurred in six patients, four of whom had major autoimmune phenomena. We conclude that careful selection of HCV patients with favorable response characteristics (post-transfusion etiology and non-cirrhotic liver) and without autoimmune manifestations can improve the remission rate and decrease the complication rate during interferon treatment.     

Metabotropic glutamate receptors in the rat nucleus accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3R)-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCG1 > L-AP4 > (1S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca2+ currents through N- or P-type Ca2+ channels, as inhibition persisted in the presence of omega-conotoxin-GVIA or omega-Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (R,S)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I(AHP)). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

Contributions of GABAA receptors and GABAC receptors to acetylcholine release and directional selectivity in the rabbit retina

GABA is a major inhibitory neurotransmitter in the mammalian retina and it acts at many different sites via a variety of postsynaptic receptors. These include GABAA receptors and bicuculline-resistant GABAC receptors. The release of acetylcholine (ACh) is inhibited by GABA and strongly potentiated by GABA antagonists. In addition, GABA appears to mediate the null inhibition which is responsible for the mechanism of directional selectivity in certain ganglion cells. We have used these two well-known examples of GABA inhibition to compare three GABA antagonists and assess the contributions of GABAA and GABAC receptors. All three GABA antagonists stimulated ACh release by as much as ten-fold. By this measure, the ED50s for SR-95531, bicuculline, and picrotoxin were 0.8, 7.0, and 14 microM, respectively. Muscimol, a potent GABAA agonist, blocked the effects of SR-95531 and bicuculline, but not picrotoxin. This indicates that SR-95531 and bicuculline are competitive antagonists at the GABAA receptor, while picrotoxin blocks GABAA responses by acting at a different, nonreceptor site such as the chloride channel. In the presence of a saturating dose of SR-95531 to completely block GABAA receptors, picrotoxin caused a further increase in the release of ACh. This indicates that picrotoxin potentiates ACh release by a mechanism in addition to the block of GABAA responses, possibly by also blocking GABAC receptors, which have been associated with bipolar cells. All three GABA antagonists abolished directionally selective responses from ON/OFF directional-selective (DS) ganglion cells. In this system, the ED50S for SR-95531, bicuculline, and picrotoxin were 0.7 microM, 8 microM, and 94.6 microM, respectively. The results with SR-95531 and bicuculline indicate that GABAA receptors mediate the inhibition responsible for directional selectivity. The addition of picrotoxin to a high dose of SR-95531 caused no further increase in firing rate. The comparatively high dose required for picrotoxin also suggests that GABAC receptors do not contribute to directional selectivity. This in turn suggests that feedforward GABAA inhibition, as opposed to feedback at bipolar terminals, is responsible for the null inhibition underlying directional selectivity.     

Organization of glutamate receptors at the synapse

Glutamate receptors can be divided in several groups with distinct functional properties. An additional level of complexity has emerged from recent high resolution immunogold analyses which have provided evidence for a differential targeting of glutamate receptors to specific subsynaptic membrane domains. Notably, different types of glutamate receptor may differ in their distance to the release site and in their spatial relation to glutamate transporters. These data imply that the subsynaptic expression of a given glutamate receptor may bias its response to a released quantum of transmitter and suggest that receptor targeting may be implicated in the modulation of glutamatergic neurotransmission.     

Metabotropic glutamate receptors: electrophysiological properties and role in plasticity

Electrophysiological research on mGluRs is now very extensive, and it is clear that activation of mGluRs results in a large number of diverse cellular actions. Studies of mGluRs and on ionic channels has clearly demonstrated that mGluR activation has a widespread and potent inhibitory action on both voltage-gated Ca2+ channels and K+ channels. Inhibition of N-type Ca2+ channels, and inhibition of Ca(++)-dependent K+ current, IAHP, and IM being particularly prominent. Potentiation of activation of both Ca2+ and K+ channels has also been observed, although less prominently than inhibition, but mGluR-mediated activation of non-selective cationic channels is widespread. In a small number of studies, generation of an mGluR-mediated slow excitatory postsynaptic potential has been demonstrated as a consequence of the effect of mGluR activation on ion channels, such as activation of a non-selective cationic channels. Although certain mGluR-modulation of channels is a consequence of direct G-protein-linked action, for example, inhibition of Ca2+ channels, many other effects occur as a result of activation of intracellular messenger pathways, but at present, little progress has been made on the identification of the messengers. The field of study of the involvement of mGluRs in synaptic plasticity is very large. Evidence for the involvement of mGluRs in one form of LTD induction in the cerebellum and hippocampus is now particularly impressive. However, the role of mGluRs in LTP induction continues to be a source of dispute, and resolution of the question of the exact involvement of mGluRs in the induction of LTP will have to await the production of more selective ligands and of selective gene knockouts.     

Metabotropic glutamate receptors in the ventrolateral medulla of rats

We investigated the hypothesis that stimulation of metabotropic excitatory amino acid receptors in the ventrolateral medulla evokes cardiovascular responses. Thus, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD], a selective agonist of metabotropic excitatory amino acid receptors, was microinjected into the rostral or caudal ventrolateral medulla of halothane-anesthetized Sprague-Dawley rats. Microinjections of (1S,3R)-ACPD (100 pmol-1 nmol) into the rostral ventrolateral medulla produced dose-dependent increases in mean arterial pressure (+20 +/- 4 mm Hg by 100 pmol and +35 +/- 2 mm Hg by 1 nmol, p < 0.01 versus artificial cerebrospinal fluid) and integrated splanchnic sympathetic nerve activity (+17 +/- 3% and +46 +/- 4%, respectively, p < 0.01), whereas (1S,3+)-ACPD microinjected into the caudal ventrolateral medulla decreased mean arterial pressure (-28 +/- 2 mm Hg by 100 pmol and -48 +/- 6 mm Hg by 1 nmol, p < 0.01 versus artificial cerebrospinal fluid) and splanchnic sympathetic nerve activity (-24 +/- 4% and -49 +/- 5%, p < 0.01). The blockade of ionotropic excitatory amino acid receptors by the combined injection of 2-amino-7-phosphonoheptanoic acid (200 pmol) and 6,7-dinitroquinoxaline-2,3-dione (200 pmol), which effectively blocked the responses elicited by either N-methyl-D-aspartate (20 pmol) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (5 pmol), failed to affect the responses evoked by either (1S,3R)-ACPD (100 pmol) or L-glutamate (2 nmol) microinjected in the rostral and caudal ventrolateral medulla. These results suggest that metabotropic receptors are present and mediate cardiovascular responses evoked by L-glutamate injections into the rostral and caudal ventrolateral medulla.     

Ionotropic and metabotropic glutamate receptor agonist-induced [Ca2+]i increase in isolated rat supraoptic neurons

Although a number of studies have shown that various free fatty acids (FFAs) and monoacylglycerides (MGs) have bactericidal properties in vitro, the role of these compounds in vivo has not been determined. This study evaluated the antibacterial properties of medium-chain MGs and FFAs for different bacterial enteropathogens with an in-vitro bacterial killing assay and an in-vivo model of intestinal colonisation. Incubation of test bacteria with medium-chain MGs for 4 h led to 100-10,000-fold reductions in numbers of viable cells of Vibrio cholerae, Salmonella typhi, Shigella sonnei and enterotoxigenic Escherichia coli (ETEC). Lauric acid was the only medium-chain FFA to show comparable in-vitro bactericidal activity. The ability of dietary MGs to reduce or eliminate bacterial colonisation of the intestinal tract was evaluated in mice that were predisposed to bacterial colonisation by treatment with streptomycin (STR+). Mice were treated with streptomycin, challenged intragastrically with V. cholerae or ETEC, and given monocaprin (C10:0 MG) either concurrently or as part of the daily diet. Control mice given STR+ without MGs and challenged with V. cholerae or ETEC showed high numbers of challenged bacteria in gastrointestinal contents by 1 h after administration. Concurrent administration of V. cholerae and C10:0 MG (2.5 mg/ml) caused > 1000-fold reduction in numbers of V. cholerae recovered from the gastrointestinal tracts of STR+ mice. Concurrent administration of C10:0 MG with ETEC did not cause a reduction in the number of viable ETEC present in the intestinal tract of STR+ mice. Administration of C10:0 MG in the diet had no effect on the number of viable V. cholerae or ETEC associated with caecal or ileal tissue of STR+ mice when C10:0 MG in the diet was started 1 day before, the same day, or 2 days after bacterial challenge. Collectively, these results suggested that dietary MGs may prevent intestinal colonisation by bacterial enteropathogens if administered at the time of exposure, but have little effect on established intestinal infections.     

The lurcher mutation and ionotropic glutamate receptors: contributions to programmed neuronal death in vivo

Differentiation of trophoblast giant cells in the rodent placenta is accompanied by exit from the mitotic cell cycle and onset of endoreduplication. Commitment to giant cell differentiation is under developmental control, involving down-regulation of Id1 and Id2, concomitant with up-regulation of the basic helix-loop-helix factor Hxt and acquisition of increased adhesiveness. Endoreduplication disrupts the alternation of DNA synthesis and mitosis that maintains euploid DNA content during proliferation. To determine how the mammalian endocycle is regulated, we examined the expression of the cyclins and cyclin-dependent kinases during the transition from replication to endoreduplication in the Rcho-1 rat choriocarcinoma cell line. We cultured these cells under conditions that gave relatively synchronous endoreduplication. This allowed us to study the events that occur during the transition from the mitotic cycle to the first endocycle. With giant cell differentiation, the cells switched cyclin D isoform expression from D3 to D1 and altered several checkpoint functions, acquiring a relative insensitivity to DNA-damaging agents and a coincident serum independence. The initiation of S phase during endocycles appeared to involve cycles of synthesis of cyclins E and A, and termination of S was associated with abrupt loss of cyclin A and E. Both cyclins were absent from gap phase cells, suggesting that their degradation may be necessary to allow reinitiation of the endocycle. The arrest of the mitotic cycle at the onset of endoreduplication was associated with a failure to assemble cyclin B/p34(cdk1) complexes during the first endocycle. In subsequent endocycles, cyclin B expression was suppressed. Together these data suggest several points at which cell cycle regulation could be targeted to shift cells from a mitotic to an endoreduplicative cycle.     

Pharmacological characterization of metabotropic glutamate receptors coupled to phospholipase D in the rat hippocampus

1. Phospholipase D (PLD) is the key enzyme in a signal transduction pathway leading to the formation of the second messengers phosphatidic acid and diacylglycerol. In order to define the pharmacological profile of PLD-coupled metabotropic glutamate receptors (mGluRs), PLD activity was measured in slices of adult rat brain in the presence of mGluR agonists or antagonists. Activation of the phospholipase C (PLC) pathway by the same agents was also examined. 2. The mGluR-selective agonist (1S,3R)-l-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] induced a concentration-dependent (10-300 microM) activation of PLD in the hippocampus, neocortex, and striatum, but not in the cerebellum. The effect was particularly evident in hippocampal slices, which were thus used for all subsequent experiments. 3. The rank order of potencies for agonists stimulating the PLD response was: quisqualate > ibotenate > (2S,3S,4S)-alpha-(carboxycyclopropyl)-glycine > (1S,3R)-ACPD > L-cysteine sulphinic acid > L-aspartate > L-glutamate. L-(+)-2-Amino-4-phosphonobutyric acid and the ionotropic glutamate receptor agonists N-methyl-D-aspartate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, and kainate failed to activate PLD. (RS)-3,5-dihydroxyphenylglycine (100300 microM), an agonist of mGluRs of the first group, stimulated PLC but inhibited the PLD response elicited by 100 microM (1S,3R)-ACPD. 4. (+)-alpha-Methyl-4-carboxyphenylglycine (0.1-1 mM), a competitive antagonist of mGluRs of the first and second group, elicited a significant PLD response. L-(+)-2-Amino-3-phosphonopropionic acid (1 mM), an antagonist of mGluRs of the first group, inhibited the 100 microM (1S,3R)-ACPD-induced PLC response but produced a robust stimulation of PLD. 5. 12-O-Tetradecanoylphorbol 13-acetic acid and phorbol 12,13-dibutyrate (PDBu), activators of protein kinase C, at 1 microM had a stimulatory effect on mGluRs linked to PLD but depressed (1S,3R)-ACPD-induced phosphoinositide hydrolysis. The protein kinase C inhibitor, staurosporine (1 and 10 microM) reduced PLD activation induced by 1 microM PDBu but not by 100 microM (1S,3R)-ACPD. 6. Our results suggest that PLD-linked mGluRs in rat hippocampus may be distinct from any known mGluR subtype coupled to PLC or adenylyl cyclase. Moreover, they indicate that independent mGluRs coupled to the PLC and PLD pathways exist and that mGluR agonists can stimulate PLD through a PKC-independent mechanism.     

Metabotropic glutamate receptors modulate synaptic transmission in the perforant path: pharmacology and localization of two distinct receptors

Metabotropic glutamate receptors (mGluRs) have emerged as an interesting family of eight different receptor subtypes that can be divided into three groups according to their pharmacology and sequence similarity. In the present study, the specific mGluR agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid ((1S,3R)-ACPD) and L(+)-2-amino-4-phosphonobutyric acid (L-AP4) depressed field excitatory postsynaptic potentials (fEPSPs) in the rat dentate gyrus evoked by perforant path stimulation in a concentration-dependent, rapid and reversible manner (EC50: L-AP4 5.9 +/- 1.6 microM, (1S,3R)-ACPD 80 +/- 34 microM). In a 'paired-pulse' stimulation protocol, the first fEPSP showed a stronger reduction, resulting in 'paired-pulse' facilitation. The effects of L-AP4 but not of (1S,3R)-ACPD could be antagonized by the group III mGluR antagonists (S)-2-amino-2-methyl-4-phosphonobutanoic acid (MAP4) and (RS)-alpha-methyl-4-phosphonophenylglycine (MPPG). Moreover, (1S,3R)-ACPD was still potently depressing fEPSPs after preperfusion of near saturating concentrations of L-AP4. Together, the results suggest that both substances act on different mGluRs. The effects of (1S,3R)-ACPD could not be further differentiated by selective group I or group II mGluR agonists. Although (2S,1'S,2'S)-2-carboxycyclopropylglycine (L-CCG-I) blocked fEPSPs at concentrations > 1 microM, these effects, as well as L-AP4 effects, were potently antagonized by MAP4. This suggests that mGluR8 might be responsible for the actions of L-AP4 and L-CCG-I. The two different mGluRs showed a distinct distribution when fEPSPs were recorded simultaneously in the outer and middle molecular layer (OML/MML): The L-AP4 sensitive receptor, possibly mGluR8, seems to be located in the OML while (1S,3R)-ACPD showed its main effect in the MML.     

The metabotropic glutamate receptors of the vestibular organs

This research sought to test the presence and function of metabotropic excitatory amino acid receptors (mGluR) in the frog semicircular canal (SCC). The mGluR agonist +/- 1-aminocyclopentane-trans-1,3-dicarboxylate (ACPD) produced an increase in afferent firing rates of the ampullar nerve of the intact posterior canal. This increase was not due to a stimulation of cholinergic efferent terminals or the acetylcholine (ACh) receptor, since atropine, in concentrations which blocked the response to exogenous acetylcholine, did not affect the response to ACPD. Likewise, ACPD effects were not due to stimulation of postsynaptic NMDA receptors, since the NMDA antagonist D(-)-2-amino-5-phosphonopentanoate (AP-5) did not affect the response to ACPD, reinforcing the reported selectivity of ACPD for mGluRs. When the SCC was superfused with artificial perilymph known to inhibit hair cell transmitter release (i.e. low Ca-high Mg), ACPD failed to increase afferent firing. This suggests that the receptor activated by ACPD is located on the hair cell. Pharmacological evidence suggested that the mGluRs involved in afferent facilitation belong to Group I (i.e. subtypes 1 and 5). In fact, the Group III agonist AP-4 had no effect, and the ACPD facilitatory effect was blocked by the Group I mGluR antagonists (S)-4-carboxyphenylglycine (CPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA). Additional pharmacological evidence supported the presence of Group I mGluRs. Interestingly, the mGluR antagonists, AIDA and 4CPG, by themselves did not affect the resting firing rates of ampullar afferents. This may suggest that the mGluRs are not involved in resting activity but perhaps only in evoked activity (as suggested in Guth et al. (1991) Hear. Res. 56, 69-78). In addition, the mRNA for the mGluR1 has been detected in hair cells of both SCC, utricle, and saccule. In summary, the evidence points to an mGluR localized to the hair cell (i.e. an autoreceptor) which may be activated to produce a positive feedback augmentation of evoked but not resting transmitter release and thus affect afferent activity.