6-methyl-2-(phenylethynyl)pyridine and 1-amino-1-3-dicarboxycyclopentane

Astrocytes express mainly metabotropic glutamate receptor 3 and metabotropic glutamate receptor 5 receptor subtypes, which show opposing effects on cellular proliferation upon activation. In this study, we investigated the mechanisms by which activation of these receptors modulates astrocyte proliferation. Activation of metabotropic glutamate receptor 5 with (S)-3,5-dihydroxyphenylglycine increased phospholipase D activity in astrocytes as well as astrocyte proliferation. The 3,5-dihydroxyphenylglycine-induced proliferation was inhibited in the presence of the metabotropic glutamate receptor 5 antagonist (2-methyl-6-(phenylethynyl)pyridine), the protein kinase C inhibitor GF109203X, brefeldin A and 1-butanol. Activation of metabotropic glutamate receptor 3 with (2'S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine-IV (DCG-IV) inhibited astrocyte proliferation without affecting metabotropic glutamate receptor 5-mediated phospholipase D activity. Metabotropic glutamate receptor 3 activation, however, only partially inhibited metabotropic glutamate receptor 5-mediated proliferation. In conclusion, metabotropic glutamate receptor 5 stimulates astrocyte proliferation via a protein kinase C-phospholipase D-phosphatidic acid-dependent pathway, whereas metabotropic glutamate receptor 3-mediated inhibition of astrocyte proliferation does not involve phospholipase D, and is independent of metabotropic glutamate receptor 5-mediated effects.

Topics: Animals; Animals, Newborn; Astrocytes; Cell Count; Cell Proliferation; Cells, Cultured; Cerebral Cortex; Cycloleucine; Cyclopropanes; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycerophospholipids; Glycine; Indoles; Maleimides; Methoxyhydroxyphenylglycol; Phospholipase D; Pyridines; Rats; Rats, Wistar; Receptors, Glutamate; Tritium

Neuronal activity in the brain is thought to be coupled to cerebral arterioles (functional hyperemia) through Ca2+ signals in astrocytes. Although functional hyperemia occurs rapidly, within seconds, such rapid signaling has not been demonstrated in situ, and Ca2+ measurements in parenchymal arterioles are still lacking. Using a laser scanning confocal microscope and fluorescence Ca2+ indicators, we provide the first evidence that in a brain slice preparation, increased neuronal activity by electrical stimulation (ES) is rapidly signaled, within seconds, to cerebral arterioles and is associated with astrocytic Ca2+ waves. Smooth muscle cells in parenchymal arterioles exhibited Ca2+ and diameter oscillations ("vasomotion") that were rapidly suppressed by ES. The neuronal-mediated Ca2+ rise in cortical astrocytes was dependent on intracellular (inositol trisphosphate [IP3]) and extracellular voltage-dependent Ca2+ channel sources. The Na+ channel blocker tetrodotoxin prevented the rise in astrocytic [Ca2+]i and the suppression of Ca2+ oscillations in parenchymal arterioles to ES, indicating that neuronal activity was necessary for both events. Activation of metabotropic glutamate receptors in astrocytes significantly decreased the frequency of Ca2+ oscillations in parenchymal arterioles. This study supports the concept that astrocytic Ca2+ changes signal the cerebral microvasculature and indicate the novel concept that this communication occurs through the suppression of arteriolar [Ca2+]i oscillations and corresponding vasomotion. The full text of this article is available online at http://circres.ahajournals.org.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arterioles; Astrocytes; Boron Compounds; Calcium Channels; Calcium Signaling; Cerebral Cortex; Cerebrovascular Circulation; Cycloleucine; Electric Stimulation; Hyperemia; In Vitro Techniques; Indans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Microscopy, Video; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Neurons; Nifedipine; Pyridines; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Receptors, Metabotropic Glutamate; Sodium Channel Blockers; Sodium Channels; Synaptic Transmission; Tetrodotoxin

We have previously shown that the release of central neurotransmitters can be modulated by the activation of Group I and Group II subtypes of G-protein-linked metabotropic glutamate (mGlu) receptors. To date, however, very little is known about the regulation of serotonergic neurotransmission by these receptor subtypes. In the present study, we have utilized in vivo intracerebral microdialysis to elucidate the roles of Group I and Group II mGlu receptors in the regulation of neuronal 5-hydroxytryptamine (5-HT) release in the frontal cortex of conscious, freely moving rats. Dialysate 5-HT was of neuronal origin with basal release showing strong calcium dependency and tetrodotoxin sensitivity and marked elevation following K(+)-induced depolarization. The broad-spectrum mGlu receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD; 1-3 mM] did not significantly modify basal cerebrocortical 5-HT release. Similarly, the Group I mGlu receptor-specific agonist (RS)-3,5-dihydroxyphenylglycine [(RS)-3,5-DHPG; 1-3 mM] showed no marked effect on cortical dialysate 5-HT levels. To eliminate the possibility that these findings were the result of receptor desensitization, the effects of lower concentrations of (RS)-DHPG (100-300 microM) and shorter ligand exposure time (15 min) were also evaluated. Dialysate 5-HT levels remained unmodified by these manipulations. In comparison, the Group II mGlu receptor agonist, (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-1; 500 microM), evoked a marked facilitation of release (approximately 150% of basal) which was fully reversed by the Group I/II antagonist, (S)-alpha-methyl-4-carboxyphenylglycine [(S)-MCPG; 3 mM]. The modulatory action of L-CCG-1 showed a bell-shaped concentration-response relationship. (S)-MCPG (3 mM) and the potent and selective mGlu(5) receptor antagonist, 2-methyl-6-(phenylethynyl)pyridine (MPEP; 100 microM), when given alone, did not significantly modify 5-HT levels.The current data provide strong evidence to suggest that while the release of neuronal 5-HT in the rat frontal cortex is not subject to regulation by facilitatory Group I mGlu receptors, it may be positively modulated by activation of Group II mGlu receptors. Taken together with data from other studies, the present investigation lends emphasis to the notion that neuromodulation by mGlu receptors is a region-specific phenomenon and also proposes that the heterogeneous distribution of these receptors is neurone-specific

Topics: Amino Acids, Dicarboxylic; Analysis of Variance; Anesthetics, Local; Animals; Benzoates; Calcium; Cerebral Cortex; Cycloleucine; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Ligands; Male; Microdialysis; Potassium Chloride; Pyridines; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Serotonin; Synaptic Transmission; Tetrodotoxin

From previous work, it appears that glutamate can activate the hypothalamic-pituitary-adrenocortical (HPA) axis by an interaction at either ionotopic or metabotropic (G-protein coupled) receptors. For example, (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate (ACPD), a metabotropic glutamate (mGlu) receptor agonist, has been shown to increase the levels of serum corticosterone in rats. The present study was undertaken to further characterize which of the mGlu receptors are substantially involved in control of the HPA axis. The group I mGlu receptor agonists, 3,5-dihydroxyphenylglycine (DHPG), 1S,3R-ACPD, and 2-chloro-5-hydroxyphenylglycine (CHPG) but not the inactive isomer 1R,3S-ACPD were found to dose-dependently increase serum corticosterone 1 h after intracerebroventricular (i.c.v.) injection in male rats. The relative potency, DHPG (EC50 = 520 nmol) > 1S,3R-ACPD (1.4 micromol) = CHPG (2.7 micromol) >> 1R,3S-ACPD (>> 3 micromol) is consistent with activation of group I (mGlu1/5) receptors. The effects of DHPG were long lasting with substantial elevations in corticosterone remaining for at least 3 h. In a similar manner, the group III mGlu receptor agonists, L-AP4 (4-phosphono-2-aminobutyric acid) and L-SOP (serine-O-phosphate), were found to increase serum corticosterone levels at 1 h. In contrast, the mGlu group II selective agonists LY354740 (10 mg/kg, i.p.) and subtype-selective doses of the group II antagonist LY341495 (1 mg/kg, i.p.) did not significantly elevate serum corticosterone. Given the group I agonists results, it was surprising to find that group I selective and mGlu1 selective antagonists given alone also increased serum corticosterone. As with the agonists, the rise in serum corticosterone with LY393675 (an mGlu1/5 antagonist, EC50 = 20 nmol, i.c.v.) and LY367385 (an mGlu1 antagonist, 325 nmol, i.c.v.) were dose-dependent and consistent with their relative affinity for the group I mGlu receptors. The selective mGlu5 antagonist MPEP [2-methyl-6-(phenylethylnyl)pyridine] increased serum corticosterone but only at high doses (> 30 mg/kg, i.p.). A model involving the high glutamatergic tone on GABAergic interneurons in the paraventricular nucleus of the hypothalamus is discussed as a possible explanation for these results.

Topics: Adrenalectomy; Adrenocorticotropic Hormone; Amino Acids; Animals; Benzoates; Bridged Bicyclo Compounds; Corticosterone; Cycloleucine; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; Glycine; Male; Neuroprotective Agents; Paraventricular Hypothalamic Nucleus; Phenylacetates; Propionates; Pyridines; Rats; Rats, Sprague-Dawley; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Resorcinols; Xanthenes

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.

Topics: Animals; Animals, Newborn; Blotting, Western; Cell Hypoxia; Cells, Cultured; Cycloleucine; Excitatory Amino Acid Agonists; Glycine; Immunohistochemistry; Liver; Male; Neuroprotective Agents; Pyridines; Quisqualic Acid; Rats; Rats, Wistar; Receptor, Metabotropic Glutamate 5; Receptors, Metabotropic Glutamate; Reverse Transcriptase Polymerase Chain Reaction; Tumor Cells, Cultured

Our previous work has shown that Group I mGlu receptors participate in thalamic sensory processing in vivo. However, unequivocal demonstration of mGlu5 participation has not been possible due to the lack of specific ligands. We have therefore made a preliminary study of the in vivo actions of the agonist (R,S)-2-Chloro-5-hydroxyphenylglycine [CHPG] and the novel mGlu5 antagonist 6-methyl-2-(phenylethynyl)-pyridine [MPEP] in order to characterize their suitability for functional studies. Iontophoretically administered MPEP selectively antagonized excitatory responses of single rat thalamic neurones to CHPG compared to the broad-spectrum mGlu agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate. In contrast, the established mGlu1 and mGlu5 antagonist (S)-4-carboxyphenylglycine reduced responses to both agonists. These findings are the first demonstration of an in vivo action of CHPG and its antagonism by a selective mGlu5 antagonist. Furthermore MPEP appears to be a good tool for functional studies of mGlu5.

Topics: Animals; Cycloleucine; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glycine; Phenylacetates; Pyridines; Rats; Rats, Wistar; Receptors, Metabotropic Glutamate; Thalamus