ABSTRACT
Acute treatment with positive allosteric modulators (PAMs) of mGlu1 and mGlu5 metabotropic glutamate receptors (RO0711401 and VU0360172, respectively) reduces the incidence of spike-and wave discharges in the WAG/Rij rat model of absence epilepsy. However, from the therapeutic standpoint, it was important to establish whether tolerance developed to the action of these drugs. We administered either VU0360172 (3 mg/kg, s.c.) or RO0711401 (10 mg/kg, s.c.) to WAG/Rij rats twice daily for ten days. VU0360172 maintained its activity during the treatment, whereas rats developed tolerance to RO0711401 since the 3rd day of treatment and were still refractory to the drug two days after treatment withdrawal. In response to VU0360172, expression of mGlu5 receptors increased in the thalamus of WAG/Rij rats after 1 day of treatment, and remained elevated afterwards. VU0360172 also enhanced mGlu5 receptor expression in the cortex after 8 days of treatment without changing the expression of mGlu1a receptors. Treatment with RO0711401 enhanced the expression of both mGlu1a and mGlu5 receptors in the thalamus and cortex of WAG/Rij rats after 3-8 days of treatment. These data were different from those obtained in non-epileptic rats, in which repeated injections of RO0711401 and VU0360172 down-regulated the expression of mGlu1a and mGlu5 receptors. Levels of VU0360172 in the thalamus and cortex remained unaltered during the treatment, whereas levels of RO0711401 were reduced in the cortex at day 8 of treatment. These findings suggest that mGlu5 receptor PAMs are potential candidates for the treatment of absence epilepsy in humans.
Subject(s)
Anticonvulsants/pharmacology , Epilepsy, Absence/drug therapy , Epilepsy, Absence/physiopathology , Excitatory Amino Acid Agents/pharmacology , Receptor, Metabotropic Glutamate 5/metabolism , Receptors, Metabotropic Glutamate/metabolism , Animals , Blotting, Western , Cerebral Cortex/drug effects , Cerebral Cortex/physiopathology , Disease Models, Animal , Drug Tolerance , Electrodes, Implanted , Electroencephalography , Male , Mice, Transgenic , Niacinamide/analogs & derivatives , Niacinamide/pharmacology , Rats , Rats, Inbred ACI , Rats, Wistar , Receptor, Metabotropic Glutamate 5/genetics , Receptors, Metabotropic Glutamate/genetics , Thalamus/drug effects , Thalamus/physiopathology , Time FactorsABSTRACT
Drug treatment of malignant gliomas is limited by the intrinsic resistance of glioma stem cells (GSCs) to chemotherapy. GSCs isolated from human glioblastoma multiforme (GBM) expressed metabotropic glutamate receptors (mGlu3 receptors). The DNA-alkylating agent, temozolomide, killed GSCs only if mGlu3 receptors were knocked down or pharmacologically inhibited. In contrast, mGlu3 receptor blockade did not affect the action of paclitaxel, etoposide, cis-platinum, and irinotecan. mGlu3 receptor blockade enabled temozolomide toxicity by inhibiting a phosphatidylinositol-3-kinase/nuclear factor-κB pathway that supports the expression of O(6)-methylguanine-DNA methyltransferase (MGMT), an enzyme that confers resistance against DNA-alkylating agents. In mice implanted with GSCs into the brain, temozolomide combined with mGlu3 receptor blockade substantially reduced tumor growth. Finally, 87 patients with GBM undergoing surgery followed by adjuvant chemotherapy with temozolomide survived for longer time if tumor cells expressed low levels of mGlu3 receptors. In addition, the methylation state of the MGMT gene promoter in tumor extracts influenced survival only in those patients with low expression of mGlu3 receptors in the tumor. These data encourage the use of mGlu3 receptor antagonists as add-on drugs in the treatment of GBM, and suggest that the transcript of mGlu3 receptors should be measured in tumor specimens for a correct prediction of patients' survival in response to temozolomide treatment.
Subject(s)
Glioblastoma/metabolism , Neoplastic Stem Cells/metabolism , Receptors, Metabotropic Glutamate/metabolism , Amino Acids/toxicity , Animals , Antineoplastic Agents, Alkylating/pharmacology , Antineoplastic Agents, Alkylating/therapeutic use , Chemotherapy, Adjuvant , Combined Modality Therapy , DNA Methylation/drug effects , Dacarbazine/analogs & derivatives , Dacarbazine/pharmacology , Dacarbazine/therapeutic use , Drug Resistance, Neoplasm/drug effects , Glioblastoma/drug therapy , Glioblastoma/mortality , Humans , Mice , NF-kappa B/metabolism , Neoplastic Stem Cells/cytology , O(6)-Methylguanine-DNA Methyltransferase/genetics , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Promoter Regions, Genetic , RNA, Messenger/metabolism , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/genetics , Signal Transduction , Survival Rate , Temozolomide , Transplantation, Heterologous , Tumor Cells, Cultured , Xanthenes/toxicityABSTRACT
Carbon monoxide (CO) is produced during the catabolism of free haem, catalyzed by haem oxygenase (HO) enzymes, and its physiological roles include vasodilation, neurotransmission, inhibition of platelet aggregation and anti-proliferative effects on smooth muscle. In vivo preclinical studies have shown that exogenously administered quantities of CO may represent an effective treatment for conditions characterized by a dysregulated immune response. The carbon monoxide-releasing molecules (CORMs) represent a group of compounds capable of carrying and liberating controlled quantities of CO in the cellular systems. This review covers the physiological and anti-inflammatory properties of the HO/CO pathway in the central nervous system. It also discusses the effects of CORMs in preclinical models of inflammation. The accumulating data discussed herein support the possibility that CORMs may represent a novel class of drugs with disease-modifying properties in multiple sclerosis.
Subject(s)
Boranes/therapeutic use , Carbon Monoxide/therapeutic use , Carbonates/therapeutic use , Multiple Sclerosis/drug therapy , Organometallic Compounds/therapeutic use , Animals , Anti-Inflammatory Agents/administration & dosage , Anti-Inflammatory Agents/therapeutic use , Autoimmunity/drug effects , Boranes/administration & dosage , Carbon Monoxide/administration & dosage , Carbon Monoxide/metabolism , Carbonates/administration & dosage , Cardiotonic Agents/administration & dosage , Cardiotonic Agents/therapeutic use , Cytokines/biosynthesis , Drug Evaluation, Preclinical , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Encephalomyelitis, Autoimmune, Experimental/immunology , Guanylate Cyclase/metabolism , Heme/metabolism , Heme Oxygenase (Decyclizing)/physiology , Heme Oxygenase-1/deficiency , Heme Oxygenase-1/physiology , Humans , Inflammation/drug therapy , Multiple Sclerosis/immunology , Neuroimmunomodulation/drug effects , Neuroimmunomodulation/physiology , Organometallic Compounds/administration & dosage , Oxidation-Reduction , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/drug effects , Soluble Guanylyl Cyclase , Vasodilator Agents/administration & dosage , Vasodilator Agents/therapeutic useABSTRACT
Eight-month old WAG/Rij rats, which developed spontaneous occurring absence seizures, showed a reduced function of mGlu1 metabotropic glutamate receptors in the thalamus, as assessed by in vivo measurements of DHPG-stimulated polyphosphoinositide hydrolysis, in the presence of the mGlu5 antagonist MPEP as compared to age-matched non-epileptic control rats. These symptomatic 8-month old WAG/Rij rats also showed lower levels of thalamic mGlu1α receptors than age-matched controls and 2-month old (pre-symptomatic) WAG/Rij rats, as detected by immunoblotting. Immunohistochemical and in situ hybridization analysis indicated that the reduced expression of mGlu1 receptors found in symptomatic WAG/Rij rats was confined to an area of the thalamus that excluded the ventroposterolateral nucleus. No mGlu1 receptor mRNA was detected in the reticular thalamic nucleus. Pharmacological manipulation of mGlu1 receptors had a strong impact on absence seizures in WAG/Rij rats. Systemic treatment with the mGlu1 receptor enhancer SYN119, corresponding to compound RO0711401, reduced spontaneous spike and wave discharges spike-wave discharges (SWDs) in epileptic rats. Subcutaneous doses of 10 mg/kg of SYN119 only reduced the incidence of SWDs, whereas higher doses (30 mg/kg) also reduced the mean duration of SWDs. In contrast, treatment with the non-competitive mGlu1 receptor antagonist, JNJ16259685 (2.5 and 5 mg/kg, i.p.) increased the incidence of SWDs. These data suggest that absence epilepsy might be associated with a reduction of mGlu1 receptors in the thalamus, and that compounds that amplify the activity of mGlu1 receptors might be developed as novel anti-absence drugs. This article is part of a Special Issue entitled 'Trends in neuropharmacology: in memory of Erminio Costa'.
Subject(s)
Epilepsy, Absence/metabolism , Receptors, Metabotropic Glutamate/metabolism , Allosteric Regulation , Animals , Ciprofloxacin/analogs & derivatives , Ciprofloxacin/pharmacology , Disease Models, Animal , Dose-Response Relationship, Drug , Electroencephalography/drug effects , Epilepsy, Absence/drug therapy , Epilepsy, Absence/genetics , Excitatory Amino Acid Antagonists/pharmacology , Male , Motor Activity/drug effects , Motor Activity/physiology , Nucleic Acid Synthesis Inhibitors/pharmacology , Quinolines/pharmacology , RNA, Messenger/metabolism , Rats , Rats, Inbred ACI , Rats, Inbred Strains , Receptors, Metabotropic Glutamate/genetics , Signal Transduction/drug effects , Thalamic Nuclei/metabolism , Thalamic Nuclei/physiopathology , Thalamus/metabolism , Thalamus/physiopathologyABSTRACT
We have evaluated the effects of the carbon monoxide-releasing molecule CORM-A1 [Na(2) (BH(3) CO(2) ); ALF421] on the development of relapsing-remitting experimental allergic encephalomyelitis (EAE) in SJL mice, an established model of multiple sclerosis (MS). The data show that the prolonged prophylactic administration of CORM-A1 improves the clinical and histopathological signs of EAE, as shown by a reduced cumulative score, shorter duration and a lower cumulative incidence of the disease as well as milder inflammatory infiltrations of the spinal cords. This study suggests that the use of CORM-A1 might represent a novel therapeutic strategy for the treatment of multiple sclerosis.
Subject(s)
Boranes/therapeutic use , Carbon Monoxide/therapeutic use , Carbonates/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Myelin Proteolipid Protein/immunology , Peptide Fragments/immunology , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/blood , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Body Weight/drug effects , Boranes/pharmacokinetics , Carbon Monoxide/administration & dosage , Carbon Monoxide/blood , Carbon Monoxide/pharmacology , Carbonates/pharmacokinetics , Dexamethasone/pharmacology , Dexamethasone/therapeutic use , Encephalomyelitis, Autoimmune, Experimental/diagnosis , Encephalomyelitis, Autoimmune, Experimental/immunology , Encephalomyelitis, Autoimmune, Experimental/pathology , Female , Mice , Neutrophils/pathology , Spinal Cord/drug effects , Spinal Cord/pathologyABSTRACT
HE3286 (17alpha-ethynyl-5-androstene-3beta, 7beta, 17beta-triol) is a synthetic derivative of a natural anti-inflammatory steroid, beta-AET (5-androstene-3beta, 7beta, 17beta-triol). HE3286 is orally bioavailable and treats established disease in models of ulcerative colitis, collagen-induced arthritis, and collagen antibody-induced arthritis, reducing clinical signs of disease and proinflammatory signals, including IL-6 and matrix metallopeptidase 3. HE3286 modulates nuclear factor kappaB through an unknown mechanism but does not interact with any of the steroid-binding nuclear hormone receptors and is not immune suppressive. HE3286 was safe and well tolerated in phase I studies and is under evaluation in multicenter phase I/II clinical trials for ulcerative colitis and arthritis. HE3286 may provide a new treatment option for patients with inflammatory and autoimmune diseases.
Subject(s)
Autoimmune Diseases/drug therapy , Dehydroepiandrosterone/analogs & derivatives , Administration, Oral , Androstenols/administration & dosage , Androstenols/therapeutic use , Animals , Clinical Trials as Topic , Colitis, Ulcerative/drug therapy , Dehydroepiandrosterone/administration & dosage , Dehydroepiandrosterone/therapeutic use , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Encephalomyelitis, Autoimmune, Experimental/drug therapy , Estradiol/administration & dosage , Estradiol/analogs & derivatives , Estradiol/therapeutic use , Estrogen Antagonists/administration & dosage , Estrogen Antagonists/therapeutic use , Female , Fulvestrant , Humans , Male , Mice , Mice, Inbred Strains , Rats , Rats, Wistar , Treatment OutcomeABSTRACT
The interaction between 5-hydroxytryptamine(2A) (5-HT(2A)) serotonin receptors and metabotropic glutamate (mGlu) 2/3 receptors underlies the antipsychotic activity of mGlu2/3 receptor agonists in experimental animals and humans. The molecular nature of this interaction is only partially known. We here report for the first time that pharmacological activation of mGlu2/3 receptors attenuates the stimulation of polyphosphoinositide (PI) hydrolysis mediated by 5-HT(2A) receptors in the frontal cortex of living mice. Mice were injected intracerebroventricularly with [myo-(3)H]inositol and treated with drugs 1 h after a pretreatment with lithium, which blocks the conversion of inositol monophosphate into free inositol. Systemic injection of the mGlu2/3 receptor agonist (-)-2-oxa-4-aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY379268) inhibited the stimulation of PI hydrolysis induced by the hallucinogenic 5-HT(2A) receptor agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) without affecting the stimulation by mGlu1/5 or muscarinic receptors. The action of LY379268 was prevented by the preferential mGlu2/3 receptor antagonist (2S,1'S,2'S)-2-(9-xanthylmethyl)-2-(2'-carboxycyclopropyl)glycine (LY341495). N-(4'-cyano-biphenyl-3-yl)-N-(3-pyridinylmethyl)-ethanesulfonamide hydrochloride (LY566332), a selective mGlu2 receptor enhancer, also reduced DOI-stimulated PI hydrolysis when combined with subthreshold doses of LY379268. Systemic LY379268 inhibited DOI-stimulated PI hydrolysis in mice lacking either mGlu2 or mGlu3 receptors but was inactive in double mGlu2/mGlu3 receptor knockout mice, suggesting that both mGlu2 and mGlu3 receptors interact with 5-HT(2A) receptors. Surprisingly, contrasting results were obtained in cortical slice preparations, where LY379268 amplified both DOI- and 3,5-dihydroxyphenylglycine-stimulated PI hydrolysis. Amplification was abrogated by the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl)pyridine, suggesting that experiments in brain slices are biased by an additional component of receptor-stimulated PI hydrolysis. This highlights the importance of in vivo models for the study of the interaction between 5-HT(2A) and mGlu2/3 receptors.
Subject(s)
Frontal Lobe/drug effects , Phosphatidylinositols/pharmacology , Receptor, Serotonin, 5-HT2A/metabolism , Receptors, Metabotropic Glutamate/metabolism , Amino Acids/pharmacology , Amphetamines/pharmacology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Dose-Response Relationship, Drug , Excitatory Amino Acid Antagonists/pharmacology , Hydrolysis , Male , Mice , Mice, Inbred Strains , Mice, Knockout , Pyridines/pharmacology , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Receptors, Metabotropic Glutamate/physiology , Sulfonamides/pharmacology , Xanthenes/pharmacologyABSTRACT
Alzheimer's disease (AD), the leading cause of senile dementia, has become a considerable social and economical problem. Current AD therapeutics provide mainly symptomatic short-term benefit, rather than targeting disease mechanisms. The hallmarks for AD are beta-amyloid plaques, neurofibrillary tangles, and regionalized neuronal loss. Additional neuropathological features have been described that may provide some clues to the mechanism by which neurons die in AD. Specifically, the aberrant expression of cell cycle proteins and the presence of de novo-replicated DNA in neurons have been described both in AD brain and in culture models of the disease. The unscheduled cell cycle events are deleterious to neurons, which undergo death rather than complete the cell cycle. Although our understanding of the neuronal cell cycle is not complete, experimental evidence suggests that compounds able of arresting the aberrant cell cycle will yield neuroprotection. This review focuses on drug development centered on the cell cycle hypothesis of AD.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Cell Cycle/drug effects , Nerve Degeneration/drug therapy , Nerve Degeneration/metabolism , Alzheimer Disease/metabolism , Animals , Drug Evaluation, Preclinical , Humans , Models, Neurological , Nerve Degeneration/pathology , Neuroprotective Agents/therapeutic useABSTRACT
Mouse embryonic stem (ES) cells were stimulated to differentiate either as adherent monolayer cultures in DMEM/F12 supplemented with N2/B27, or as floating embryoid bodies (EBs) exposed to 1 microM retinoic acid (RA) for 4 days, starting from 4 DIV, and subsequently re-plated in DMEM/F12 medium. Adherent monolayer cultures of ES cells expressed mGlu5 receptors throughout the entire differentiation period. Selective pharmacological blockade of mGlu5 receptors with methyl-6-(phenylethynyl)-pyridine (MPEP) (1 microM, added once a day) accelerated the appearance of the neuronal marker, beta-tubulin. In addition, treatment with MPEP increased the number of cells expressing glutamate decarboxylase-65/67 (GAD(65/67)), a marker of GABAergic neurons. In floating EBs, mGlu5 receptors are progressively replaced by mGlu4 receptors. The orthosteric mGlu4/6/7/8 receptor agonist, L-2-amino-4-phosphonobutanoate (L-AP4), or the selective mGlu4 receptor enhancer, PHCCC,--both combined with RA at concentrations of 30 microM--increased the expression of both beta-tubulin and GAD(65/67), inducing the appearance of fully differentiated neurons that released GABA in response to membrane depolarization. We conclude that mGlu receptor subtypes regulate neuronal differentiation of ES cells in a context-dependent manner, and that subtype-selective ligands of these receptors might be used for the optimization of in vitro protocols aimed at producing GABAergic neurons from ES cells.
Subject(s)
Cell Differentiation , Embryonic Stem Cells/metabolism , Neurons/metabolism , Receptors, Metabotropic Glutamate/metabolism , gamma-Aminobutyric Acid/metabolism , Aminobutyrates/pharmacology , Animals , Benzopyrans/pharmacology , Cell Adhesion , Cell Differentiation/drug effects , Cell Line , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/enzymology , Excitatory Amino Acid Antagonists/pharmacology , Glutamate Decarboxylase/metabolism , Membrane Potentials , Mice , Neurons/drug effects , Neurons/enzymology , Phenotype , Pyridines/pharmacology , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/drug effects , Time Factors , Tretinoin/pharmacology , Tubulin/metabolismABSTRACT
We examined the interaction between the selective serotonin reuptake inhibitor, fluoxetine, and group-II metabotropic glutamate (mGlu) receptors using progenitor cells isolated from cultured cerebellar granule cells, considered as an in vitro model of antidepressant-drug induced neurogenesis. These cells expressed mGlu3 receptors negatively coupled to adenylyl cyclase. A 72-h treatment with either fluoxetine or low concentrations of mGlu2/3 receptor agonists (LY379268 or 2R,4R-APDC) enhanced cell proliferation. The action of fluoxetine was mediated by the activation of 5-HT(1A) receptors. We found a strong synergism between fluoxetine and LY379268 in enhancing cell proliferation and inhibiting cAMP formation. The increased cell proliferation induced by fluoxetine+LY379268 was abrogated by the cAMP analogue, 8-Br-cAMP, as well as by drugs that inhibit the mitogen-activated protein kinase and phosphatidyilinositol-3-kinase pathways. Interestingly, fluoxetine and LY379268 also acted synergistically in promoting neuronal differentiation when progenitor cells were incubated in the presence of serum. These data support the hypothesis that a combination between classical antidepressants and mGlu2/3 receptor agonists may be helpful in the experimental treatment of depression.
Subject(s)
Amino Acids/pharmacology , Antidepressive Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Fluoxetine/pharmacology , Neurons/drug effects , Receptors, Metabotropic Glutamate/agonists , Selective Serotonin Reuptake Inhibitors/pharmacology , Animals , Animals, Newborn , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Cerebellum/cytology , Cerebellum/drug effects , Cyclic AMP/metabolism , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Drug Synergism , Immunohistochemistry , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Stem Cells/drug effectsSubject(s)
Adjuvants, Pharmaceutic/administration & dosage , Antidepressive Agents, Tricyclic/administration & dosage , Excitatory Amino Acid Agents/administration & dosage , Hippocampus/drug effects , Imipramine/administration & dosage , Receptors, Metabotropic Glutamate/drug effects , Amino Acids/administration & dosage , Animals , Bridged Bicyclo Compounds, Heterocyclic/administration & dosage , Depressive Disorder/drug therapy , Disease Models, Animal , Drug Interactions , Drug Therapy, Combination , Hippocampus/cytology , Hippocampus/metabolism , In Vitro Techniques , Mice , Neurons/drug effects , Rats , Rats, Inbred Strains , Receptor Cross-Talk/drug effects , Receptors, Metabotropic Glutamate/metabolism , Time Factors , Xanthenes/administration & dosageABSTRACT
Systemic injection of the mGlu2/3 receptor antagonist, LY341495 (1 mg/kg, i.p.), increased plasma corticosterone in mice to an extent similar to that induced by the despair test. Treatment with the mGlu2/3 receptor agonist, LY379268 (1 mg/kg, i.p.), or the non-competitive mGlu5 receptor antagonist, MPEP (5 mg/kg, i.p.), failed to induce significant changes in corticosterone levels. Searching for a site of action of LY341495, we examined the expression of mGlu receptor subtypes in the various anatomical regions of the mouse hypothalamic-pituitary-adrenal (HPA) axis. Only mGlu5 and -7 receptor mRNAs were detected in the adrenal gland by RT-PCR, whereas mGlu -1, -3, -4, -5, -7 and -8 receptor mRNAs were detected in the anterior pituitary. All transcripts (with the exception of mGlu5 and mGlu6 receptor mRNAs) were detected in the hypothalamus. However, Western blot analysis showed the presence of mGlu2/3 receptor proteins only in the hypothalamus and not in the anterior pituitary. This was consistent with functional data showing that LY341495 (0.1 and 1 microM) failed to affect ACTH secretion from isolated mouse anterior pituitaries. Moving from these observations, we examined whether LY341495 could activate the HPA axis by inhibiting mGlu2/3 receptors at hypothalamic level. We measured the release of corticotropin releasing hormone (CRH) in isolated mouse hypothalami incubated in the presence of subtype-selective mGlu receptor agonists or antagonists. Among all the drugs we have tested, only LY341495 was able to increase CRH secretion. With high concentrations of LY341495 (1 microM) this increase was similar to that induced by 50 mM K(+). The action of LY341495 was prevented by the combined application of the mGlu2/3 receptor agonist, LY379268. We conclude that group-II mGlu receptors tonically regulate the HPA axis by controlling CRH secretion at hypothalamic level.
Subject(s)
Hypothalamo-Hypophyseal System/metabolism , Pituitary-Adrenal System/metabolism , Receptors, Metabotropic Glutamate/metabolism , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Animals , Corticosterone/blood , Corticotropin-Releasing Hormone/metabolism , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Hypothalamo-Hypophyseal System/drug effects , Hypothalamus/drug effects , Hypothalamus/metabolism , In Vitro Techniques , Male , Mice , Pituitary-Adrenal System/drug effects , Rats , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/antagonists & inhibitorsABSTRACT
The role of group I metabotropic glutamate (mGlu) receptors in neurodegeneration is controversial because of the contradictory effects of mGlu1/5 agonists in in vitro models of neuronal cell death. In this study, novel and selective antagonists of mGlu1 and mGlu5: LY367385 and LY367366 were found to show consistent neuroprotective effects against N-methyl-D-aspartate (NMDA)-induced excitotoxicity in vitro and in vivo. Furthermore, intraventricular administration of LY367385 reduced hippocampal cell death in gerbils subjected to transient global ischemia. Previous studies have also shown that activation of group II mGlu receptors may contribute to neuroprotective mechanisms in vitro and in vivo. Three potent group II mGlu agonists--LY354740, LY379268 and LY389795--were found to attenuate both NMDA excitotoxicity and staurosporine-induced neuronal cell death. LY354740 and LY379268 were protective against transient global ischemia in gerbils when dosed intraperitoneally. These results support the view that antagonists of mGlu1 and mGlu5 and agonists of group II mGlu receptors may be useful agents in the therapeutic treatment of neurodegenerative disease.
Subject(s)
Benzoates , Excitatory Amino Acid Antagonists/therapeutic use , Ischemia/drug therapy , Nerve Degeneration/drug therapy , Neurons/drug effects , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Animals , Bridged Bicyclo Compounds/pharmacology , Cells, Cultured , Cerebral Cortex/drug effects , Cerebral Cortex/embryology , Drug Evaluation, Preclinical , Excitatory Amino Acid Agonists/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Gerbillinae , Glycine/analogs & derivatives , Glycine/therapeutic use , Male , N-Methylaspartate/pharmacology , Rats , Rats, Sprague-Dawley , Thiophenes/therapeutic useABSTRACT
We investigated the expression and coupling to the phospholipase C signal transduction pathway of metabotropic glutamate receptor (mGluR) subtypes by Western blot analysis and agonist-stimulated inositol monophosphate formation in several brain regions of postnatal day 9 (P9) and adult rats. In the cerebral cortex, hippocampus, corpus striatum, olfactory bulb, cerebellum and hypothalamus, the expression level of mGluR5 was greater at P9 than in adulthood. The mGluR5 signal was very low or absent in the adult cerebellum and hypothalamus. The expression of mGluR1a was slightly greater at P9 in the hypothalamus, hippocampus and olfactory bulb, whereas it substantially increased with age in the cerebellum, and did not change in the cerebral cortex and corpus striatum. mGluR1b and -1c were nearly undetectable by Western blot analysis. The expression level of mGluR5, but not that of mGluR1a, was significantly correlated with the extent of phosphoinositide hydrolysis stimulated by mGluR agonists in slices prepared from these brain regions. The mGluR antagonist cyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt), potently antagonized responses mediated by mGluR1, but much less potently those mediated by mGluR5a in recombinant cells. CPCCOEt, at a concentration which efficiently blocks mGluR1 responses, did not substantially affect the polyphosphoinositide response in hippocampal or cerebellar slices from newborn animals, and antagonized only a minor component of the polyphosphoinositide response in adult hippocampal slices. CPCCOEt, however, prevented the small stimulation of polyphosphoinositide hydrolysis by mGluR agonists in adult cerebellar slices. We conclude that (i) the efficient mGluR-mediated polyphosphoinositide hydrolysis in 9-day-old rats is mediated by mGluR5; (ii) the increased expression of mGluR1 in the adult cerebellum does not substitute for the decline of mGluR5 expression in the ability to mediate polyphosphoinositide hydrolysis; and therefore (iii) mGluR1a might couple less efficiently than mGluR5 to polyphosphoinositide hydrolysis.
Subject(s)
Animals, Newborn/metabolism , Brain Chemistry/physiology , Phosphatidylinositols/metabolism , Receptors, Metabotropic Glutamate/metabolism , Aging/metabolism , Amino Acid Sequence , Animals , Blotting, Western , Brain Chemistry/drug effects , DNA, Complementary/biosynthesis , Excitatory Amino Acid Agonists/pharmacology , Humans , Hydrolysis , In Vitro Techniques , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Receptors, Metabotropic Glutamate/antagonists & inhibitorsABSTRACT
We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S,3R)-cyclopentanedicarboxylic acid (1S,3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GT1-7 cells stained positively for the subtypes mGluR1a, -1b and -5 (belonging to group I mGluR2/3 (group II) and mGluR7 (group III). Agonists of group I mGluRs, including 1S,3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S,3R-ACPD effectively released GnRH in confluent cultures. We then focused on group II and III mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonist which preferentially activate group II and III mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of cAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as beta-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a Gs-type of GTP-binding protein.
Subject(s)
Cyclic AMP/metabolism , Cycloleucine/analogs & derivatives , Gonadotropin-Releasing Hormone/biosynthesis , Hypothalamus/metabolism , Neurons/metabolism , Receptors, Metabotropic Glutamate/biosynthesis , Animals , Cell Division , Cell Line, Transformed , Colforsin/pharmacology , Cycloleucine/pharmacology , Dinoprostone/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Gonadotropin-Releasing Hormone/metabolism , Mice , Neurons/cytology , Neurons/drug effects , Norepinephrine/pharmacology , Phosphatidylinositols/metabolism , Quisqualic Acid/pharmacology , Receptors, Metabotropic Glutamate/agonistsABSTRACT
Glutamate receptors coupled to polyphosphoinositide (PPI) hydrolysis (metabotropic glutamate receptors, mGluR), are highly efficient during the early stages of postnatal life and are thought to be involved in developmental plasticity. The dramatic decrease with age in mGluR activity suggests the existence of mechanisms that down-regulate this receptor after a certain stage of neuronal maturation. In cultured cerebellar granule neurons grown under conditions that promote the survival and maturation of cells (serum-containing medium with 25 mM K+), enzymatic depletion of extracellular glutamate prevented the age-dependent decrease in mGluR agonist-stimulated PPI hydrolysis that normally occurs after 4 days of maturation in vitro, suggesting that mGluR activity declines as a result of developmental changes affecting homologous desensitization. This was borne out by the observation that glutamate at low concentrations (1-10 microM) readily desensitized mGluR at 7 days but not at 4 days in culture. Furthermore, the critical period during which the high sensitivity to agonist-induced desensitization of mGluR developed coincided with the period when phorbol ester-activated protein kinase C acquired the ability to suppress mGluR activity. The developmental pattern of mGluR agonist-induced PPI hydrolysis was similar in granule cells grown under "trophic" and "nontrophic" conditions (in cultures in 25 mM K+ and in a medium containing "low" K+, in this study, 10 mM, respectively). However, the developmental decline in the response to mGluR stimulation after 4 days in vitro was not prevented in cells grown in 10 mM K+ by the removal of extracellular glutamate; rather, it could be counteracted by treatment with N-methyl-D-aspartate (NMDA) (EC50, approximately 4 microM), which blocked the development of mGluR desensitization. The effect was NMDA receptor mediated and required DNA transcription and protein synthesis. However, NMDA exerted a different effect in cells grown in 25 mM K+, inducing a substantial decrease rather than an increase in mGluR activity. The effect of growth conditions was also examined on mGluR mRNA levels, which were not always correlated with mGluR activity. In general, either increases in the medium K+ concentrations or NMDA supplementation of the cultures resulted in a decrease in mGluR mRNA levels. It is noteworthy that NMDA could also restore mGluR activity after the metabotropic response had reached its peak. This implies that NMDA receptor activation may be involved in the increase in mGluR activity in adult life under conditions that elicit plastic changes in the nervous system.
Subject(s)
Cerebellum/metabolism , Receptors, Metabotropic Glutamate/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Aging/metabolism , Animals , Carbachol/pharmacology , Cells, Cultured , Cerebellum/cytology , Glutamates/metabolism , Glutamic Acid , Hydrolysis , N-Methylaspartate/pharmacology , Phosphatidylinositol Phosphates/metabolism , Potassium/metabolism , Quisqualic Acid/pharmacology , RNA, Messenger/metabolism , Rats , Receptors, Metabotropic Glutamate/drug effects , Receptors, N-Methyl-D-Aspartate/drug effectsABSTRACT
Metabotropic glutamate receptors (mGluRs) have been recently described as a family of guanine nucleotide-binding regulatory protein-coupled receptors with multiple signal transduction pathways. At least one of these receptors appears to be negatively coupled to adenylyl cyclase when stably expressed in transfected cells. We have studied how activation of native mGluRs modulates cyclic AMP (cAMP) formation in brain slices prepared from rats at different ages. 1S,3R-1-Aminocyclopentane-1,3-dicarboxylic acid (1S,1R-ACPD), a selective agonist of mGluRs, slightly increased basal cAMP formation but reduced forskolin-stimulated cAMP formation in adult hippocampal slices, in agreement with previous results. The action of 1S,3R-ACPD on basal cAMP formation was not reproduced by the ionotropic receptor agonists N-methyl-D-aspartate, kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate and was antagonised by L-2-amino-3-phosphonopropionate (L-AP-3). L-AP-3, however, did not prevent but rather mimicked the inhibitory action of 1S,3R-ACPD on forskolin-stimulated cAMP formation. In hippocampal slices from 1-, 8-, or 15-day-old rats, 1S,3R-ACPD increased basal cAMP formation but failed to reduce the action of forskolin. A similar development pattern of modulation was observed in hypothalamic slices with the difference that 1S,3R-ACPD did not stimulate basal cAMP formation in the hypothalamus of adult animals. These results suggest that inhibition of forskolin-stimulated cAMP formation by 1S,3R-ACPD is mediated by a specific mGluR subtype that is preferentially expressed in the adult.
Subject(s)
Brain/metabolism , Cyclic AMP/metabolism , Cycloleucine/analogs & derivatives , Receptors, Neurotransmitter/physiology , Aging/metabolism , Animals , Animals, Newborn/growth & development , Animals, Newborn/metabolism , Colforsin/pharmacology , Cycloleucine/pharmacology , Dose-Response Relationship, Drug , Hippocampus/metabolism , Hypothalamus/metabolism , In Vitro Techniques , Male , Rats , Rats, Inbred Strains , Receptors, GlutamateABSTRACT
Wistar rats infected with Streptococcus pneumoniae (type III ATCC) rapidly develop an acute form of experimental lobar pneumonia (ELP) with death of 80-90% of the animals by 6 days after the infection. Prophylactic treatment of these animals with the novel immunomodulator Pidotimod, at the dose of 25 mg/kg bw, significantly increased their rate of survival as compared to the control group (50 vs. 90% respectively). Recovery from the infection appeared definitive since all the Pidotimod-treated survivors were alive and in good condition at the end of the observation period (45 days post infection). Prophylactic treatment with higher or lower doses of the drug was ineffective. Therapy with Pidotimod was not effective. This preliminary study suggests that Pidotimod may have contributed to activation of specific and non-specific immune effectors involved in the host response to S. pneumoniae infection.
Subject(s)
Adjuvants, Immunologic/therapeutic use , Pneumonia, Pneumococcal/therapy , Pyrrolidonecarboxylic Acid/analogs & derivatives , Thiazoles/therapeutic use , Animals , Male , Pneumonia, Pneumococcal/mortality , Pyrrolidonecarboxylic Acid/therapeutic use , Rats , Rats, Wistar , ThiazolidinesABSTRACT
Excitatory amino acids (EAAs) are known to stimulate neurohormone release through the activation of ion-channel-linked receptors (ionotropic receptors). Here we report that a receptor for EAAs linked to polyphosphoinositide hydrolysis (metabotropic receptor) is also present at the hypothalamus where its expression is developmentally regulated. Stimulation of [3H]inositolmonophosphate ([3H]InsP) formation by quisqualate (EC50 = 1.5 microM), ibotenate (EC50 = 100 microM) and trans-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD; EC50 = 30 microM) is extremely high (up to 50-fold) in the first 10 days of postnatal life, progressively declines during maturation and is virtually absent in the adult. Stimulation of phosphoinositide hydrolysis by quisqualate, ibotenate and t-ACPD is more pronounced than that induced by classical neurotransmitters that stimulate inositol phosphate formation such as norepinephrine and carbamylcholine. Agonists of the ionotropic glutamate receptor such as kainate, NMDA and alpha-amino-3-hydroxy-5-methyl-5-isoxazolpropionate (AMPA), do not modify inositol phosphate accumulation in hypothalamic slices. The selective antagonist of quisqualate metabotropic receptor, D,L-2-amino-3-phosphonopropionate (AP3), produces a slight stimulation of phosphoinositide hydrolysis, but potently antagonizes the stimulation produced by quisqualate and t-ACPD.
Subject(s)
Hypothalamus/metabolism , Receptors, Neurotransmitter/metabolism , Aging/metabolism , Amino Acids/pharmacology , Animals , Hypothalamus/growth & development , In Vitro Techniques , Inosine Monophosphate/metabolism , Male , Neurons/metabolism , Neurotransmitter Agents/metabolism , Rats , Rats, Inbred Strains , Receptors, GlutamateABSTRACT
Repeated (once a day for 8 days) but not single administration of estradiol benzoate (10 micrograms/kg, s.c.) induced a sevenfold increase in anterior pituitary gamma-aminobutyric acid (GABA) concentration in male rats. GABA concentration also increased in the median eminence whereas no changes or decreases were observed in other brain regions including hypothalamic arcuate nucleus, lateral septum, hippocampus, caudate nucleus, and substantia nigra. Eight-day estradiol benzoate injection also enhanced the Vmax of median eminence glutamate decarboxylase activity without affecting the Km of the enzyme for glutamic acid. Taken together, these results suggest that repeated administration of estradiol benzoate increases the activity of the tubero-infundibular GABAergic system in male rats.