Group I metabotropic glutamate receptors are expressed in the chicken retina and by cultured retinal amacrine cells

Glutamate is well established as an excitatory neurotransmitter in the vertebrate retina. Its role as a modulator of retinal function, however, is poorly understood. We used immunocytochemistry and calcium imaging techniques to investigate whether metabotropic glutamate receptors are expressed in the chicken retina and by identified GABAergic amacrine cells in culture. Antibody labeling for both metabotropic glutamate receptors 1 and 5 in the retina was consistent with their expression by amacrine cells as well as by other retinal cell types. In double-labeling experiments, most metabotropic glutamate receptor 1-positive cell bodies in the inner nuclear layer also label with anti-GABA antibodies. GABAergic amacrine cells in culture were also labeled by metabotropic glutamate receptor 1 and 5 antibodies. Metabotropic glutamate receptor agonists elicited Ca(2+) elevations in cultured amacrine cells, indicating that these receptors were functionally expressed. Cytosolic Ca(2+) elevations were enhanced by metabotropic glutamate receptor 1-selective antagonists, suggesting that metabotropic glutamate receptor 1 activity might normally inhibit the Ca(2+) signaling activity of metabotropic glutamate receptor 5. These results demonstrate expression of group I metabotropic glutamate receptors in the avian retina and suggest that glutamate released from bipolar cells onto amacrine cells might act to modulate the function of these cells.     

Effect of ambient extracellular glutamate on <Emphasis Type="Italic">Drosophila</Emphasis> glutamate receptor trafficking and function

Measurements suggest that the hemolymph glutamate concentrations in Drosophila are relatively high. This raises the possibility that extracellular glutamate could be an important regulator of glutamatergic transmission in vivo. Using voltage clamp electrophysiology, we found that synaptic currents in D. melanogaster larval neuromuscular junctions are reduced by extracellular glutamate (EC50: ~0.4 mM), such that only 10–30% of receptors were functionally available in 1 mM extracellular glutamate. The kinetics of synaptic currents were also slowed in a dose-dependent fashion (EC50: ~1 mM), consistent with the idea that extracellular glutamate preferentially removes the fastest-desensitizing receptors from the functional pool. Prolonged exposure (several hours) to extracellular glutamate also triggers loss of glutamate receptor immunoreactivity from neuromuscular junctions. To determine whether this receptor loss requires that glutamate bind directly to the lost receptors, we examined glutamate-dependent loss of receptor immunoreactivity in larvae with glutamate receptor ligand binding mutations. Our results suggest that glutamate-dependent receptor loss requires binding of glutamate directly to the lost receptors. To determine whether lost receptor protein is degraded or merely redistributed, we used immunoblots. Results suggest that glutamate receptor protein is redistributed, but not degraded, after prolonged exposure to high extracellular glutamate. K. Chen and H. Augustin contributed equally to this work.     

Functional analysis of Caenorhabditis elegans glutamate receptor subunits by domain transplantation

Glutamate receptors are not only abundant and important mediators of fast excitatory synaptic transmission in vertebrates, but they also serve a similar function in invertebrates such as Drosophila and the nematode Caenorhabditis elegans. In C. elegans, an animal with only 302 neurons, 10 different glutamate receptor subunits have been identified and cloned. To study the ion channel properties of these receptor subunits, we recorded glutamate-gated currents from Xenopus oocytes that expressed either C. elegans glutamate receptor subunits or chimeric rat/C. elegans glutamate receptor subunits. The chimeras were constructed between the C. elegans glutamate receptor pore domains and either the rat kainate receptor subunit GluR6, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit GluR1, or the N-methyl-d-aspartate (NMDA) receptor subunit NMDAR1-1a. Although native subunits were nonfunctional, 9 of 10 ion pores were found to conduct current upon transplantation into rat receptor subunits. A provisional classification of the C. elegans glutamate receptor subunits was attempted based on functionality of the chimeras. C. elegans glutamate receptor ion pores, at a position homologous to a highly conserved site critical for ion permeation properties in vertebrate glutamate receptor pores, contain amino acids not found in vertebrate glutamate receptors. We show that the pore-constricting Q/R site, which in vertebrate receptors determines calcium permeability and rectification properties of the ion channel, in C. elegans can be occupied by other amino acids, including, surprisingly, lysine and proline, without loss of these properties.     

Adenosine A 2A receptor antagonists prevent the increase in striatal glutamate levels induced by glutamate uptake inhibitors

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Activation of adenosine A(2A) receptors increases extracellular glutamate levels, while A(2A) receptor antagonists reduce stimulated glutamate outflow. Whether a modulation of the glutamate uptake system is involved in the effects elicited by A(2A) receptor blockers has never been investigated. This study examined the ability of adenosine A(2A) receptor antagonists to prevent the increase in glutamate levels induced by blockade of the glutamate uptake. In rats implanted with a microdialysis probe in the dorsal striatum, perfusion with 4 mm l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), or 10 mm dihydrokainic acid (DHK, a non-transportable competitive inhibitor that mainly blocks the glial glutamate transporter GLT-1), significantly increased extracellular glutamate levels. The effects of PDC and DHK were completely prevented by the adenosine A(2A) receptor antagonists SCH 58261 (0.01 mg/kg i.p.) and/or ZM 241385 (5 nm via probe). Since an impairment in glutamate transporter function is thought to play a major role in neurodegenerative disorders, the regulation of glutamate uptake may be one of the mechanisms of the neuroprotective effects of A(2A) receptor antagonists.     

The regulation of glutamate receptor trafficking and function by TARPs and other transmembrane auxiliary subunits

At excitatory synapses in the brain, glutamate released from nerve terminals binds to glutamate receptors to mediate signaling between neurons. Glutamate receptors expressed in heterologous cells show ion channel activity. Recently, native glutamate receptors were shown to contain auxiliary subunits that modulate the trafficking and/or channel properties. The AMPA receptor (AMPAR) can contain TARP and CNIHs as the auxiliary subunits, whereas kainate receptor (KAR) can contain the Neto auxiliary subunit. Each of these auxiliary subunits uniquely modulates the glutamate receptors, and determines properties of native glutamate receptors. A thorough elucidation of the properties of native glutamate receptor complexes is indispensable for the understanding of the molecular machinery that regulates glutamate receptors and excitatory synaptic transmission in the brain.     

The inhibition of pineal arylalkylamine n-acetyltransferase by glutamic acid and its analogues

Previous studies from this laboratory have identified in bovine pineal gland a glutamate receptor site with a dissociation equilibrium constant (K_D) value of 0.534 μM and a receptor density (B_max) value of 4.84 pmol/mg protein. This pH- and temperature-dependent binding site showed stereospecificity, was activated by Ca²⁺ and displayed affinity for both glutamate receptor agonists and antagonists. The role of this glutamate receptor site was investigated by studying the effects of select glutamate receptor agonists and antagonists and of γ-aminobutyric acid on the basal- and on the norepinephrine-stimulated activity of arylalkylamine N-acetyltransferase in rat pineal glands that were incubated in Dulbecco's Modified Eagle Medium at 37°C for 20 min in an atmosphere of 5% CO₂/95% O₂. l-Glutamate, l-aspartate and glutamate receptor agonists such as γ-amino-3-hydroxy-5-methylisoxazole-4-propinonic acid and quisqualate were all also potent inhibitors of norepinephrine-induced stimulation of N-acetyltransferase. On the other hand, the known glutamate receptor antagonists such as d-glutamylaminomethylsulphonic acid and γ-d-glutamyltaurine stimulated the basal activity of N-acetyltransferase.Evidence of a high concentration of glutamic acid, the presence of glutamate receptors and the inhibition by glutamate receptor agonists of pineal N-acetyltransferase compel one to speculate that, in addition to its well-known metabolic roles, glutamate may modulate in an unknown fashion the activity of melatonin synthesizing enzyme, and the functions of mammalian pineal glands.     

The P2X7 receptor as a route for non‐exocytotic glutamate release: dependence on the carboxyl tail

P2X7 receptors trigger Ca²⁺‐dependent exocytotic glutamate release, but also function as a route for non‐exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full‐length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18‐amino acid cysteine‐rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non‐competitive way by extracellular Mg²⁺, did not require the recruitment of pore‐forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine‐rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca²⁺ elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca²⁺‐dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non‐exocytotic glutamate efflux.     

Glutamate receptor subunit expression in primary enteric glia cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Glutamate Receptor Subunit Expression in Primary Enteric Glia Cultures

Excitotoxicity, which is mediated via glutamate receptors, is also a phenomenon of the enteric nervous system. Whether enteric glial cells (EGCs), which resemble astrocytes of the central nervous system, express glutamate receptors and hence are involved in gut excitotoxicity is not yet known. To investigate glutamate receptor subunit expression in EGCs, primary EGC cultures of the myenteric plexus were analyzed by real-time PCR and Western blotting. These studies indeed showed that in EGC cultures, mRNA of the glutamate receptor subunits NR1, NR2A/B, GluR1, GluR3, and GluR5 and the protein bands of the glutamate receptor subunits NR2A/B, GluR1, GluR3, and GluR5 could be detected. Thus, in the enteric nervous system, glutamate receptor subunits are also expressed by EGCs, indicating that these cells might be involved in gut excitotoxicity.     

Molecular cloning of a cDNA encoding a novel member of the mouse glutamate receptor channel family.

The primary structure of a novel putative subunit of the mouse glutamate receptor channel, designated as delta 1, has been deduced by cloning and sequencing the cDNA. The delta 1 subunit shows 21-25% amino acid sequence identity with previously characterized rodent glutamate receptor channel subunits and thus may represent a new subfamily of the glutamate receptor channel.     

Expression of glutamate receptor subunits in human cancers

Emerging evidence suggests a role for glutamate and its receptors in the biology of cancer. This study was designed to systematically analyze the expression of ionotropic and metabotropic glutamate receptor subunits in various human cancer cell lines, compare expression levels to those in human brain tissue and, using electrophysiological techniques, explore whether cancer cells respond to glutamate receptor agonists and antagonists. Expression analysis of glutamate receptor subunits NR1-NR3B, GluR1-GluR7, KA1, KA2 and mGluR1-mGluR8 was performed by means of RT-PCR in human rhabdomyosarcoma/medulloblastoma (TE671), neuroblastoma (SK-NA-S), thyroid carcinoma (FTC 238), lung carcinoma (SK-LU-1), astrocytoma (MOGGCCM), multiple myeloma (RPMI 8226), glioma (U87-MG and U343), lung carcinoma (A549), colon adenocarcinoma (HT 29), T cell leukemia cells (Jurkat E6.1), breast carcinoma (T47D) and colon adenocarcinoma (LS180). Analysis revealed that all glutamate receptor subunits were differentially expressed in the tumor cell lines. For the majority of tumors, expression levels of NR2B, GluR4, GluR6 and KA2 were lower compared to human brain tissue. Confocal imaging revealed that selected glutamate receptor subunit proteins were expressed in tumor cells. By means of patch-clamp analysis, it was shown that A549 and TE671 cells depolarized in response to application of glutamate agonists and that this effect was reversed by glutamate receptor antagonists. This study reveals that glutamate receptor subunits are differentially expressed in human tumor cell lines at the mRNA and the protein level, and that their expression is associated with the formation of functional channels. The potential role of glutamate receptor antagonists in cancer therapy is a feasible goal to be explored in clinical trials.     

Repeated exposure to cocaine alters the modulation of mesocorticolimbic glutamate transmission by medial prefrontal cortex Group II metabotropic glutamate receptors

Repeated cocaine exposure enhances glutamatergic output from the medial prefrontal cortex to subcortical brain regions. Loss of inhibitory control of cortical pyramidal neurons may partly account for this augmented cortical glutamate output. Recent research indicated that repeated cocaine exposure reduced the ability of cortical Group II metabotropic glutamate receptors to modulate behavioral and neurochemical responses to cocaine. Thus, experiments described below examined whether repeated cocaine exposure alters metabotropic glutamate receptor regulation of mesocorticolimbic glutamatergic transmission using in vivo microdialysis. Infusion of the Group II metabotropic glutamate receptor antagonist LY341495 into the medial prefrontal cortex enhanced glutamate release in this region, the nucleus accumbens and the ventral tegmental area in sensitized animals, compared to controls, following short-term withdrawal but not after long-term withdrawal. Additional studies demonstrated that vesicular (K(+)-evoked) and non-vesicular (cystine-evoked) glutamate release in the medial prefrontal cortex was enhanced in sensitized animals, compared to controls, that resulted in part from a reduction in Group II metabotropic glutamate receptor modulation of these pools of glutamate. In summary, these findings indicate that the expression of sensitization to cocaine is correlated with an altered modulation of mesocorticolimbic glutamatergic transmission via reduction of Group II metabotropic glutamate receptor function.     

A membrane associated glutamate binding protein from Caenorhabditis elegans and Haemonchus contortus

1. A glutamate binding protein has been identified in membrane preparations from the free living nematode, Caenorhabditis elegans, and from the parasitic nematode, Haemonchus contortus. 2. This putative glutamate receptor was solubilized with 30 mM octyl-B-glucoside and partially purified by anion exchange and gel filtration chromatography. 3. An 80-fold purification with recovery of 75% of the glutamate binding activity was achieved. 4. The soluble C. elegans binding protein displayed a Kd for glutamate of 0.1 microM, in close agreement with the findings for the membrane associated binding protein. 5. Quisqualate was capable of displacing glutamate from the soluble C. elegans receptor, again in agreement with previous findings for the membrane bound receptor. 6. The fact that a parasitic nematode, Haemonchus contortus, also possesses this putative glutamate receptor, strengthens the case for using C. elegans as a model system for the study of parasitic nematode neuromuscular physiology.     

Development and characterisation of a glutamate-sensitive motor neurone cell line

Modification of the growth conditions of NSC-34 mouse neuroblastoma x motor neurone cells by serum depletion promotes the expression of functional glutamate receptors as the cells mature into a form that bears the phenotypic characterisation of motor neurones. Immunocytochemical studies demonstrated the presence of the glutamate receptor proteins NMDAR1, NMDAR2A/B, GluR1, GluR2, GluR2/3, GluR4, GluR6/7, and KA2. Toxicity assays using cell counting techniques demonstrated a mild but significant cell death (approximately 30%, p < 0.01) following a 24-h exposure to 1 mM glutamate that could be prevented by the presence of the glutamate receptor antagonists (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (10 microM) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (1 microM). As an indication of glutamate receptor functional activity a novel approach was used to detect the production of free radicals following stimulation with glutamate receptor agonists. The release of superoxide free radicals was detected using a micro-electrochemical sensor following addition of glutamate receptor agonists to the cell bathing solution. Alterations in intracellular calcium concentrations were examined using fura-2 imaging. Exposure of the differentiated NSC-34 cells to glutamate leads to an increase in intracellular calcium concentrations that is prevented by the presence of glutamate receptor antagonists. The motor neurone origin of these cells makes them particularly useful for investigating the potential role of glutamatergic toxicity in motor neurone degeneration.     

Mechanisms Associated with Activation of Intracellular Metabotropic Glutamate Receptor,mGluR5

The group 1 metabotropic glutamate receptor, mGluR5, is found on the cell surface as well as on intracellular membranes where it can mediate both overlapping and unique signaling effects. Previously we have shown that glutamate activates intracellular mGluR5 by entry through sodium-dependent transporters and/or cystine glutamate exchangers. Calibrated antibody labelling suggests that the glutamate concentration within neurons is quite high (~10 mM) raising the question as to whether intracellular mGluR5 is maximally activated at all times or whether a different ligand might be responsible for receptor activation. To address this issue, we used cellular, optical and molecular techniques to show that intracellular glutamate is largely sequestered in mitochondria; that the glutamate concentration necessary to activate intracellular mGluR5 is about ten-fold higher than what is necessary to activate cell surface mGluR5; and uncaging caged glutamate within neurons can directly activate the receptor. Thus these studies further the concept that glutamate itself serves as the ligand for intracellular mGluR5.     

κ Receptor Activation Attenuates l-trans-Pyrrolidine-2,4-Dicarboxylic Acid-Evoked Glutamate Levels in the Striatum

Abstract: The effects of local κ receptor activation and blockade on extracellular striatal glutamate levels evoked by reverse microdialysis of l - trans -pyrrolidine-2,4-dicarboxylic acid ( l - trans -PDC) were investigated. l - trans -PDC elevates extracellular glutamate levels in vivo by acting as a competitive substrate for plasma membrane excitatory amino acid transporters. The selective κ-opioid receptor agonist U-69593 (1-100 n M ) significantly attenuated l - trans -PDC-stimulated glutamate levels in a concentration-dependent manner. The selective κ receptor antagonist nor -binaltorphimine (1-100 n M ) reversed the U-69593-induced decrease in l - trans -PDC-evoked glutamate levels also in a concentration-dependent manner, indicating that the U-69593-induced reduction was mediated by κ receptor activation. In addition, nor -binaltorphimine significantly elevated basal extracellular glutamate levels, implying that κ receptors tonically regulate glutamate efflux in the striatum. Previous data from this laboratory have shown that l - trans -PDC-evoked extracellular glutamate levels are partially calcium-sensitive. The present study demonstrated that the inhibition of l - trans -PDC-evoked glutamate levels by reduced calcium perfusion was not altered by U-69593. Therefore, κ receptors regulate the calcium-dependent component of l - trans -PDC-evoked extracellular glutamate levels in the striatum.     

Excitotoxic Death of a Subset of Embryonic Rat Motor Neurons In Vitro

Abstract : We have used cultures of purified embryonic rat spinal cord motor neurons to study the neurotoxic effects of prolonged ionotropic glutamate receptor activation. NMDA and non-NMDA glutamate receptor agonists kill a maximum of 40% of the motor neurons in a concentration- and time-dependent manner, which can be blocked by receptor subtype-specific antagonists. subunit-specific antibodies stain all of the motor neurons with approximately the same intensity and for the same repertoire of subunits, suggesting that the survival of the nonvulnerable population is unlikely to be due to the lack of glutamate receptor expression. Extracellular Ca²⁺ is required for excitotoxicity, and the route of entry initiated by activation of non-NMDA, but not NMDA, receptors is L-type Ca²⁺ channels. Ca²⁺ imaging of motor neurons after application of specific glutamate receptor agonists reveals a sustained rise in intracellular Ca²⁺ that is present to a similar degree in most motor neurons, and can be blocked by appropriate receptor/channel antagonists. Although the lethal effects of glutamate receptor agonists are seen in only a subset of cultured motor neurons, the basis of this selectivity is unlikely to be simply the glutamate receptor phenotype or the level/pattern of rise in agonist-evoked intracellular Ca²⁺.     

Glutamate receptor subtypes may be classified into two major categories: a study on Xenopus oocytes injected with rat brain mRNA

Three major subtypes of glutamate receptors that are coupled to cation channels are known. Recently an additional subtype that is coupled to G proteins and stimulates inositol phospholipid metabolism (the metabotropic glutamate receptor) has been proposed. The pharmacological characteristics of this receptor have now been examined. Although it shares some agonists with N-methyl-D-aspartate- and quisqualate-subtype receptors, it shares virtually no antagonists with any of the three cation channel-coupled receptor subtypes. Thus the metabotropic glutamate receptor belongs to a receptor category that is completely different from that of the other three receptor subtypes, not only functionally, but also pharmacologically.     

Channel-opening kinetics of GluR6 kainate receptor

GluR6 is an ionotropic glutamate receptor subunit of the kainate subtype. It plays an essential role in synaptic plasticity and epilepsy. We expressed this recombinant receptor in HEK-293 cells and characterized the glutamate-induced channel-opening reaction, using a laser-pulse photolysis technique with the caged glutamate (gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate). This technique permits glutamate to be liberated photolytically from the caged glutamate with a time constant of approximately 30 micros. Prior to laser photolysis, the caged glutamate did not activate the GluR6 channel, nor did it inhibit or potentiate the glutamate response. At the transmembrane voltage of -60 mV, pH 7.4 and 22 degrees C, the channel-opening and -closing rate constants were determined to be (1.1 +/- 0. 4) x 10(4) and (4.2 +/- 0.2) x 10(2) s(-1), respectively. The intrinsic dissociation constant of glutamate and the channel-opening probability were found to be 450 +/- 200 microM and 0.96, respectively. These constants are derived from a minimal kinetic mechanism of the channel activation involving the binding of two glutamate molecules. This mechanism describes the time course of the open-channel form of the receptor as a function of glutamate concentration. On the basis of the channel-opening rate constants obtained, the shortest rise time (20-80% of the receptor current response) or the fastest time by which the GluR6Q channel can open is predicted to be 120 micros. The open-channel form of the receptor determines the transmembrane voltage change, which in turn controls synaptic signal transmission between two neurons. The comparison of the channel-opening kinetic rate constants between GluR6Q and GluR2Q(flip), reported in the companion paper, suggests that at a glutamate concentration of 100 microM, for instance, the integrated neuronal signal will be dominated by a slower GluR6Q receptor response, as compared to the GluR2Q(flip) component.