Metabotropic glutamate receptor 1 acts as a dependence receptor creating a requirement for glutamate to sustain the viability and growth of human melanomas.

Metabotropic glutamate 1 (mGlu) receptor has been proposed as a target for the treatment of metastatic melanoma. Studies have demonstrated that inhibiting the release of glutamate (the natural ligand of mGlu1 receptors), results in a decrease of melanoma tumor growth in mGlu1 receptor-expressing melanomas. Here we demonstrate that mGlu1 receptors, which have been previously characterized as oncogenes, also behave like dependence receptors by creating a dependence on glutamate for sustained cell viability. In the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 (SK2) and SK-MEL-5 (SK5), we show that glutamate is both necessary and sufficient to maintain cell viability, regardless of underlying genetic mutations. Addition of glutamate increased DNA synthesis, whereas removal of glutamate not only suppressed DNA synthesis but also promoted cell death in SK2 and SK5 melanoma cells. Using genetic and pharmacological inhibitors, we established that this effect of glutamate is mediated by the activation of mGlu1 receptors. The stimulatory potential of mGlu1 receptors was further confirmed in vivo in a melanoma cell xenograft model. In this model, subcutaneous injection of SK5 cells with short hairpin RNA-targeted downregulation of mGlu1 receptors resulted in a decrease in the rate of tumor growth relative to control. We also demonstrate for the first time that a selective mGlu1 receptor antagonist JNJ16259685 ((3,4-Dihydro-2H-pyrano[2,3-b]quinolin-7-yl)-(cis-4-methoxycyclohexyl)-methanone) slows SK2 and SK5 melanoma tumor growth in vivo. Taken together, these data suggest that pharmacological inhibition of mGlu1 receptors may be a novel approach for the treatment of metastatic melanoma.

Metabotropic glutamate receptors: from the workbench to the bedside.

Metabotropic glutamate (mGlu) receptors were discovered in the mid 1980s and originally described as glutamate receptors coupled to polyphosphoinositide hydrolysis. Almost 6500 articles have been published since then, and subtype-selective mGlu receptor ligands are now under clinical development for the treatment of a variety of disorders such as Fragile-X syndrome, schizophrenia, Parkinson's disease and L-DOPA-induced dyskinesias, generalized anxiety disorder, chronic pain, and gastroesophageal reflux disorder. Prof. Erminio Costa was linked to the early times of the mGlu receptor history, when a few research groups challenged the general belief that glutamate could only activate ionotropic receptors and all metabolic responses to glutamate were secondary to calcium entry. This review moves from those nostalgic times to the most recent advances in the physiology and pharmacology of mGlu receptors, and highlights the role of individual mGlu receptor subtypes in the pathophysiology of human disorders. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.

Synthesis of N(1)-substituted analogues of (2R,4R)-4-amino-pyrrolidine-2,4-dicarboxylic acid as agonists, partial agonists, and antagonists of group II metabotropic glutamate receptors.

The chemical synthesis of a series of N(1)-substituted derivatives of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid [(2R,4R)-APDC] as constrained analogues of gamma-substituted glutamic acids is described. Appropriate substitution of the N(1)-position results in agonist, partial agonist, or antagonist activity at mGluR2, mGluR3, and/or mGluR6.

1-amino-APDC, a partial agonist of group II metabotropic glutamate receptors with neuroprotective properties.

The synthesis of the 1-amino derivative of (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (1-amino-APDC), a selective metabotropic glutamate ligand, is disclosed. This compound acts as a partial agonist of the group II mGluRs and shows pronounced neuroprotective properties in the NMDA model of cell toxicity.

Neuroprotective activity of N-acetylaspartylglutamate in cultured cortical cells.

The endogenous dipeptide, alpha-N-acetylaspartylglutamate behaves as a partial agonist of N-methyl-D-aspartate receptors, but can also activate metabotropic glutamate receptors, with a high degree of selectivity for the metabotropic glutamate receptor 3 subtype. Knowing that agonists of group-II metabotropic glutamate receptors (i.e. of mGlu2 and -3 receptors) are neuroprotective, we have examined the neuroprotective activity of alpha-N-acetylaspartylglutamate in mixed cultures of mouse cortical cells exposed to a toxic pulse with N-methyl-D-aspartate. Alpha-N-acetylaspartylglutamate co-applied with N-methyl-D-aspartate was neuroprotective, but its action was insensitive to the selective group-II metabotropic glutamate receptor antagonist, ethylglutamate. Protection was instead antagonized by ethylglutamate when alpha-N-acetylaspartylglutamate was applied to the cultures immediately after the N-methyl-D-aspartate pulse, a condition in which there was no direct competition between alpha-N-acetylaspartylglutamate and N-methyl-D-aspartate at the level of N-methyl-D-aspartate receptors. alpha-N-acetylaspartylglutamate was highly neuroprotective when transiently applied to pure cultures of cortical astrocytes and the conditioned medium, collected 20 h later, was transferred to sister mixed cultures challenged with N-methyl-D-aspartate. This particular form of neuroprotection was attenuated or abolished when astrocytes where exposed to alpha-N-acetylaspartylglutamate in the presence of the group-II metabotropic glutamate receptor antagonists ethylglutamate or (2S, 1'S,2'S,3'R)-2-(2'-carboxy-3'-phenylcyclopropyl)glycine, but not in the presence of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonopentanoate. These results indicate that alpha-N-acetylaspartylglutamate induces neuroprotective effects in culture, which are mediated, at least in part, by the activation of glial metabotropic glutamate receptor 3 receptors.

Synthesis and biology of the conformationally restricted ACPD analogue, 2-aminobicyclo[2.1.1]hexane-2,5-dicarboxylic acid-I, a potent mGluR agonist.

To better characterize the roles of metabotropic glutamate receptors (mGluRs) in physiological and pathophysiological processes, there is an important need to learn more about the structural features relevant to the design of novel, high-affinity ligands that are family and subtype specific. To date, many of the biological studies that have been conducted in the area of mGluR research have made use of the agonist (1S,3R)-ACPD. This compound has been shown to act as an agonist at both the group I and group II receptors while showing little selectivity among the four subtypes belonging to these two groups. Moreover, (1S,3S)-ACPD, the cis isomer, shows negligible activity at group I receptors and is a good agonist of mGluR2. Since ACPD is itself somewhat flexible, with four distinctive conformations being identified from molecular modeling studies for the trans isomer and five conformations for the cis isomer, we believed that it would be of interest to examine the activity of an ACPD analogue that has been constrained through the introduction of a single carbon atom bridge. Accordingly, we have prepared an aminobicyclo[2.1.1]hexanedicarboxylic acid (ABHxD-I) analogue of ACPD. The synthesis of this compound was accomplished by use of an intramolecular [2 + 2] photocycloaddition reaction, in which four distinct isomers were isolated. Of these four compounds, only a single isomer, ABHxD-I (6a), was found to be a potent agonist of the mGluRs. This compound, which expresses the fully extended glutamate conformation, was found to be more potent than ACPD at all six of the eight mGluR subtypes that were investigated and to be comparable to or more potent than the endogenous ligand, glutamate, for these receptors. Interestingly, despite its fixed conformation, ABHxD-I, like glutamate, shows little subtype selectivity. Through modeling studies of ABHxD-I (6a), ABHD-VI, LY354740, (1S,3R)-ACPD, (1S, 3S)-ACPD, and l-glutamate, we conclude that the aa conformation of l-glutamate is the active conformation for both group I and group II mGluRs. Moreover, the modeling-based comparisons of these ligands suggest that the selectivity exhibited by LY354740 between the group I and group II mGluRs is not a consequence of different conformations of L-glutamate being required for recognition at these mGluRs but rather is related to certain structural elements within certain regions having a very different impact on the group I and group II mGluR activity. The enhanced potency of ABHxD-I relative to trans-ACPD commends it as a useful starting point in the design of subtype selective mGluR ligands.

alpha-substituted quisqualic acid analogs: new metabotropic glutamate receptor group II selective antagonists.

Syntheses of both the alpha-methyl and benzyl analogs of quisqualic acid are described. Testing of these compounds for their activity at excitatory amino acid receptors revealed a striking change in activity in comparison to quisqualic acid. This structural modification results in the loss of quisqualate's potent agonist action at both non-NMDA ionotropic glutamate receptors as well as at group I mGluRs, while allowing these analogs to acquire antagonist properties with relative selectivity for group II metabotropic glutamate receptors.

Synthesis and biological evaluation of two analogues of (S)-alpha-methyl-3-carboxyphenylalanine.

Two analogues 2, 3 of (S)-alpha-methyl-3-carboxylphenylalanine 1 were synthesized to test their activity for metabotropic glutamate receptors (mGluRs). Both compounds 2 and 3 were inactive as antagonist for mGluRs, but 2 showed weak agonistic activity for GluR6 in contrast to that reported by Kemp and co-workers.

Apoptosis induced by hyperthermia and verapamil in vitro in a human colon cancer cell line.

The aim of this study was to determine the mechanisms responsible for the growth inhibitory effect of hyperthermia and verapamil in human colon cancer cell line HT-29. Apoptotic cell death was verified by flow cytometry analysis. The effect of treatment with hyperthermia and verapamil on the expression of apoptosis-associated proteins including Bcl-2, p53, bax, and c-Myc was studied by Western blot analysis. Changes in intracellular calcium homeostasis was analysed by fluorescence microscopy. The combination of 42 degrees C hyperthermia and verapamil caused a significant delay of human colon cancer cell proliferation as a result of apoptosis. Administration of these agents alone did not cause any cell inhibitory effect. Our experiments have shown that HT-29 cells constitutively express apoptosis-promoting proteins, such as Bax and c-Myc, while they fail to produce Bcl-2. Therefore, we hypothesize that HT-29 cells must have Bcl-2 independent pathways to protect cells against death-inducing signals. Also, apoptosis of HT-29 cells produced by hyperthermia in the presence of verapamil is a p53-independent process. Verapamil, when it did not act as a calcium channel blocker or inhibitor of release from intracellular storages under hyperthermic conditions, accelerated the increase of [Ca2+]i in HT-29 cells which resulted in programmed cell death (apoptosis).

N-acetylaspartylglutamate selectively activates mGluR3 receptors in transfected cells.

In previous studies, we demonstrated that the neuropeptide, N-acetylaspartylglutamate (NAAG), meets the traditional criteria for a neurotransmitter and selectively activates metabotropic glutamate receptor mGluR2 or mGluR3 in cultured cerebellar granule cells and glia. Sequence homology and pharmacological data suggest that these two receptors are highly related structurally and functionally. To define more rigorously the receptor specificity of NAAG, cloned rat cDNAs for mGluR1-6 were transiently or stably transfected into Chinese hamster ovary cells and human embryonic kidney cells and assayed for their second messenger responses to the two endogenous neurotransmitters, glutamate and NAAG, as well as to metabotropic receptor agonists, trans-1-aminocyclopentane-1,3-dicarboxylate (trans-ACPD) and L-2-amino-4-phosphonobutyrate (L-AP4). Despite the high degree of relatedness of mGluR2 and mGluR3, NAAG selectively activated the mGluR3 receptor. NAAG activated neither mGluR2 nor mGluR1, mGluR4, mGluR5, or mGluR6. The mGluR agonist, trans-ACPD, activated each of the transfected receptors, whereas L-AP4 activated mGluR4 and mGluR6, consistent with the published selectivity of these agonists. Hybrid cDNA constructs of the extracellular domains of mGluR2 and mGluR3 were independently fused with the transmembrane and cytoplasmic domain of mGluR1a. This latter receptor domain is coupled to phosphoinositol turnover, and its activation increases intracellular calcium. The cells transfected with these chimeric receptors responded to activation by glutamate and trans-ACPD with increases in intracellular calcium. NAAG activated the chimeric receptor that contained the extracellular domain of mGluR3 and did not activate the mGluR2 chimera.

Concanavalin A enhances quisqualate-induced calcium influx in cerebellar granule cells in culture.

In primary cultures of cerebellar granule cells kainate produced marked influx of 45Ca2+, partially sensitive to the N-methyl-D-aspartate (NMDA) antagonist, 3-(+/-)-2-carboxypiperazin-4-yl)- propyl-1-phosphonic acid (CPP), indicating involvement of an NMDA receptor-sensitive component that may be secondary to kainate-induced glutamate release. Sodium removal partially inhibited kainate's effect. Quisqualate also produced influx of 45Ca2+, but with lower efficacy and higher potency than kainate. This action of quisqualate was unaffected by CPP and by sodium removal. Preincubation of cells with the plant lectin concanavalin A (Con A), but not with its succinyl derivative, enhanced quisqualate-induced calcium influx, and to a lesser extent kainate's effect. Inclusion of quisqualate in preincubation medium antagonized Con A potentiation of quisqualate response. Also Con A was ineffective when included in the incubation medium only, without preincubation. Preincubation of rat brain cortical membranes with Con A but not with succinyl Con A increased the binding of the AMPA receptor agonist, [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA). The results suggest that Con A enhancement of quisqualate response possibly involves the modification of an AMPA recognition site and requires preincubation in the absence of an agonist (here quisqualate).

Concanavalin A increases N-methyl-D-aspartate-induced Ca2+ influx in cerebellar granule cells in culture.

Desensitization of glutamatergic receptors can be inhibited by various endogenous and/or exogenous agents e.g. by lectins. Effect of lectin, concanavalin A (con A) on N-methyl-D-aspartate (NMDA)-induced Ca2+ influx was studied in cultured cerebellar granule cells. Additionally the effect of con A on 3H-(+)-5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (3H-(+)MK-801) binding to NMDA receptors was investigated. Preincubation with con A enhanced NMDA efficacy (but not potency) to stimulate Ca2+ influx. The potency of MK-801 to inhibit NMDA effect was similar in both control and con A pretreated cells. Preincubation of cortical membranes with con A resulted in a robust decrease of Bmax 3H-MK-801 binding and modest decrease in affinity. This contradiction between Ca2+ influx and binding experiments may result from the difference in the subtype of NMDA receptors or different expression of con A action in both assays.

Temporal and depolarization-induced changes in the absolute amounts of mRNAs encoding metabotropic glutamate receptors in cerebellar granule neurons in vitro.

Cerebellar granule neurons in primary culture express metabotropic glutamate receptors (mGluRs) coupled to the stimulation of phosphoinositide hydrolysis and to the inhibition of cyclic AMP (cAMP) formation. To evaluate which mGluR mRNAs are expressed in granule neurons under different depolarizing conditions, we measured the absolute amounts of selected receptor mRNAs in neurons cultured for 3-13 days in the presence of either 10 or 25 mM KCl. mGluR-specific primer pairs and internal standards, corresponding to unique regions of mGluR1a, mGluR2, mGluR3, mGluR4, and mGluR5, were constructed and used in a competitive PCR-derived assay to quantify the corresponding mRNA levels. For phosphoinositide-coupled receptors, the absolute content of mGluR1a mRNA was three to 10 times higher than the content of mGluR5 mRNA. The expression of mGluR5 mRNA increased up to 9 days in vitro and was much higher in 10 mM than in 25 mM KCl. For the cAMP-coupled receptors, there was a large amount of mGluR4 mRNA and a much smaller content of the mGluR3 and mGluR2 mRNAs. Maintaining the granule neurons in vitro in 10 mM KCl increased the absolute amount of mRNAs encoding mGluR2 and mGluR4 at 9 and 13 days in vitro. In contrast, the content of the mGluR3 mRNA was consistently higher in neurons cultured in 25 mM KCl. These data are consistent with the possibility that in primary cultures of cerebellar neurons, phosphoinositide responses may be predominantly mediated by mGluR1a, rather than mGluR5, and that cAMP inhibition involves preferentially mGluR4 and mGluR3.

Intracellular sodium concentration in cultured cerebellar granule cells challenged with glutamate.

We monitored simultaneously the changes in the intracellular sodium concentration ([Na+]i) and intracellular calcium concentration ([Ca2+]i) in individual neurons from primary cultures of cerebellar granule cells loaded with sodium-binding benzofuran isophthalate and fluo-3. An application of glutamate (50 microM) in Mg(2+)-free medium containing 10 microM glycine evoked [Na+]i and [Ca2+]i increases that exceeded 60 mM and 1 microM, respectively. The kinetics of [Na+]i and [Ca2+]i decreases after the termination of the glutamate pulse were different. [Na+]i failed to decrease immediately after glutamate withdrawal and the delay in the onset of [Na+]i decrease after the glutamate pulse termination was proportional to the glutamate dose, the glutamate pulse duration, and the extent of [Ca2+]i elevation elicited by glutamate. The kinetics of [Ca2+]i decrease were biphasic, with the first phase occurring immediately after glutamate withdrawal and the second phase being correlated in time with a [Na+]i value lower than 15-20 mM. These results were interpreted to indicate that the glutamate-evoked calcium influx may lead to sodium homeostasis destabilization. The delay in the restoration of the sodium gradient may in turn prolong the neuronal exposure to toxic [Ca2+]i values, due to the decrease in the efficiency of the Na+/Ca2+ exchanger to extrude calcium. The glutamate effects on [Na+]i and [Ca2+]i were potentiated by glycine. Glycine (10 microM) added alone also evoked [Na+]i and [Ca2+]i increases; this effect was inhibited by a competitive inhibitor of the N-methyl-D-aspartate receptor, 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, indicating an involvement of endogenous glutamate.

Acute and long-term inhibition of agonist-stimulated phosphoinositide hydrolysis by pulse treatment of cerebellar granule cells with TPA.

Acute pretreatment (30 min) of primary cultures of cerebellar granule cells with TPA (10 nM) resulted in a decrease in carbachol-and glutamate-stimulated phosphoinositide hydrolysis, but not in basal levels of PI hydrolysis. To investigate the mechanism of TPA action, phospholipase C was assayed in membranes prepared from cerebellar granule cells acutely treated with TPA. TPA had no effect on basal, GTP gamma S-, NaF-, and calcium-stimulated phospholipase C when compared with membranes prepared from vehicle-treated cells. The effects of pulsing with TPA (30-min pulse, 10 nM) on agonist-stimulated PI hydrolysis were studied 1, 3, and 5 or 6 d after TPA treatment. TPA treatment results in a statistically significant decrease in glutamate-stimulated PI hydrolysis, and a slight reduction of carbachol-stimulated PI hydrolysis when compared to temporally matched controls. Measurements in membranes prepared from TPA-treated vs control cells 1, 3, and 5 d after treatment showed that calcium- and NaF-stimulated phospholipase C activity was significantly decreased at all days tested, whereas GTP gamma S-stimulated phospholipase C activity was significantly decreased only at d 3. These data demonstrate differences in the acute vs long-term effects of TPA treatment on agonist-stimulated PH hydrolysis, and suggest that the acute effects may be mediated at the level of the receptor, whereas long-term effects of TPA on PI hydrolysis may be mediated by deficits in effector function.

N-methyl-D-aspartate receptor-induced translocation of protein kinase C to the nucleus in rat cerebellar slices.

Rat cerebellar slices were incubated in absence and presence of N-methyl-D-aspartate and then used to prepare a purified nuclear fraction. The purity of the nuclear fraction was assessed by electron microscopy and measurements of Na+, K(+)-ATPase activity. The presence of protein kinase C in nuclear fractions was measured by [3H]phorbol dibutyrate binding. Treatment of cerebellar slices with N-methyl-D-aspartate caused a significant, two-fold increase in the density of nuclear [3H]phorbol dibutyrate binding sites, indicating the translocation of protein kinase C to the nuclear fraction. The effect of N-methyl-D-aspartate was prevented by the presence of the N-methyl-D-aspartate receptor antagonist (+)5-methyl-10,11-dihydro-5H-dibenzocyclohepten-5,10-imine maleate (MK-801). These results suggest a possible role for protein kinase C in mediating N-methyl-D-aspartate-induced nuclear events.

Glutamate impairs neuronal calcium extrusion while reducing sodium gradient.

The rate of decrease of neuronal [Ca2+]i after an elevation induced by a glutamate pulse is much slower than that after a comparable [Ca2+]i elevation induced by a K+ depolarization. To investigate whether the [Na+]i increase taking place during the glutamate pulse reduces the rate of Ca2+ extrusion, we monitored simultaneously [Na+]i and [Ca2+]i during a K+ depolarization and a glutamate pulse lasting 1 min. The K+ depolarization evoked only a transient increase of [Na+]i from 4 mM to 13 mM, whereas the glutamate pulse increased [Na+]i to 60 mM, and this increase persisted after glutamate removal. An application of bepridil immediately after glutamate pulse when [Na+]i was greatly elevated, but not 14 min after glutamate removal when a basal [Na+]i was restored, evoked a [Ca2+]i increase accompanied by a decrease of [Na+]i, indicating a reverse mode of operation of the Na+/Ca2+ exchanger. These data suggest that the glutamate-evoked increase in [Na+]i may play a role in Ca2+ homeostasis destabilization.