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Effect of novel mGluR antagonists on signal transduction in mesencephalic and striatal neurons in vitro
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18
METABOTROPIC EFFECT IONOTROPIC AGONISTS INHIBITORS.
USE OF SPECIFIC ANTIBODIES FOR STUDYING mGLUR SUBTYPES IN RAT BRAIN.
OF GLUTAMATE AND UPTAKE
Bouazzaoui M., Kannen ieser C., Procksch 0.. Gombos G., L.N.C., Centre de Neurochimie, I&NRS, F-67084 Strasbourg, France
The conditions leadin to Glu increase, however, seem to be different in that DHWJ affected only Glu transport whereas the other drugs decrease Glu transport and increase Glu release suggesting that the stimulation of IP formation is due to the accumulated extracellular Glu actin on mGluR. The absence of additivity of the e # ect of these drugs with that of the selective metabotroptc agonist (lS,3R ACPD is consistent with this inte retation. The occurence of the L effect in the presence of NBQ re suggest that KA/AMPA receptors are not involved in the metabotropic effect. The next question to be adressed is if this Glu accumulatton also occurs in the CNS in sirtr
Stefania Risso Bradley, AIlan 1. Levey, Steven M. Hersch. and P. Jeffrey Conn. Departments of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA 30322. Polyclonal antibodies that specifically react with metabotropic glutamate receptors (mGluRs) mGIuR4a or mGluR7 were produced and characterized using immunoblot and immunocytochemistry analysis. Antibodies were generated against C-terminal domains of mGluR4a and mGluR7 using synthetic peptide immunogens. Both antibodies recognized native proteins in rat brain with molecular weights similar to the molecular weights of the bands in mGluR-transfected cell lines. Immunoblot analysis revealed that the levels of mGluR4a and mGluR7 are differentially regulated in different brain regions during development. For instance, in the cerebellum, mGluR7 immunoreactivity is higher at postnatal day 7 than in adults. whereas mGluR4a immunoreactivity is higher in adults than in younger animals. In hippocampus, both mGluR4a and mGluR7 immunoreactivity is higher in young animals than in adults. mGluR4a and mGluR7 are widely but differentially distributed throughout the rat brain, and are localized in specific cellular sites. Strong mGluR4a immunoreactivity was observed throughout the brain whereas the distribution of mGluR7 immunoreactivity was more varied, with heavy staining in cortical and limbic regions and light staining in ponsimedulla and cerelbellum. Immunocytochemistry studies at the EM level are consistent with the postulated role of mGluR4a and mGluR7 as presynaptic autoreceptors and suggest that mGluR4a may also have a postsynaptic function. Supported by NIH grant NS31373.
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MOLECULAR PHARMACOLOGY OF ACID IBOTENIC ANALOGUES AT METABOTROPIC GLUTAMATE RECEPTORS.
EFFECT OF NOVEL mGLUR ANTAGONISTS ON SIGNAL TRANSDUCTION IN MESENCEPHALIC AND STRIATAL NEURONS IN VITRO.
Bmuner-Osborne, H., LB.Ebert, F. Slok. and P. Krogsgaard-Larsen. Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitet.sparken 2.2100 Copenhagen, Denmark.
L. Bresciani, N. BruneIlo*, R. Pellicciari”, G. Racagni, and A. Ambrosini. Center of Neuropharmacol., Univ. of Milan, *Dept. Pharmaceutical Sci., Univ. of Modena and “Univ. of Perugia (I).
Based on the structure of ibotenic acid (IBO) we have previously esigned AMPA, Homo-AMPA and Homo-IBO.
New selective agonists enabled us to characterise the activity of metabotropic glutamate receptors (mGluRs) in mesencephalic (Mol. Pharmacol. 47, 1057, 1995) and in striatal neurons. In both neuronal systems, the agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) was more potent on PI hydrolysis stimulation than on the modulation of CAMP levels, a difference that was more evident in striatal neurons, in which the EC50 values on the two pathways were 3 pM and 25 pM, respectively. On the contrary, (2S,3S,4S)alpha-(carboxycycIopropyl)glycine (CCGI) inhibited CAMP formation at lower concentrations than those required to activate PI turnover. In striatal neurons, CCGI EC50 values were 50 pM on the PI hydrolysis and 0.8 pM on the modulation of CAMP levels. The effects of novel antagonists proposed as selective for class I (%I-aminoindandicarboxylic acid - UPF523) and for class III (RS-alpha-methyl-4-phosphonophenylglycine - MPPG) mGluRs were tested. IJPF523 selectively antagonised the Quis and DHPGinduced inositol phosphate fonuation. MPPG (10 pM) did not affect basal CAMP values, but effectively blocked the response to aminophoposphonobutyrate, serine-O-phosphate and CCGI, confirming its pure antagonist action at mGluRs that inhibit adenylate cyclase, although a selectivity between class II and III was not evident. The pharmacological distinction of the mGluR effects may help to clarify their role in the basal ganglia.
Homo-AMPA
AMPA
When tested on ionotropic glutamate receptors Homo-IBO is a weak AMPA agonist zand AMPA is a potent and selective AMPA agonist. Homo-AMPP. is totally inactive. In this study we h.ave tested these ligands on Chinese hamster ovary cells expressirlg mGlu to, mGlu2 and mGlu6 receptorsa representing group I, II and III receptors, respectively. AMPA and Homo-AMPA were inactive on mGlu to and mGIu2 but were weak and potent mGIU6 agonists, respectivelye. Homo-IBO showed a more complex pharmacology being a antagonist on mGlut,, inactive at mGlur and a a Kindly provided by Drs.
potent
agonist at mGlu6.
N. Sekiyama
and S. Nakanishi.
Skjarbrek, B. Eben, N. Sekiyama, S. Nakanishi. and P. Krogsgaard-Larsen. J. Med. Chem. (In press).
b Br&mer-Osborne.
H., F.A. Wk,
N.
AS
18
METABOTROPIC EFFECT IONOTROPIC AGONISTS INHIBITORS.
USE OF SPECIFIC ANTIBODIES FOR STUDYING mGLUR SUBTYPES IN RAT BRAIN.
OF GLUTAMATE AND UPTAKE
Bouazzaoui M., Kannen ieser C., Procksch 0.. Gombos G., L.N.C., Centre de Neurochimie, I&NRS, F-67084 Strasbourg, France
The conditions leadin to Glu increase, however, seem to be different in that DHWJ affected only Glu transport whereas the other drugs decrease Glu transport and increase Glu release suggesting that the stimulation of IP formation is due to the accumulated extracellular Glu actin on mGluR. The absence of additivity of the e # ect of these drugs with that of the selective metabotroptc agonist (lS,3R ACPD is consistent with this inte retation. The occurence of the L effect in the presence of NBQ re suggest that KA/AMPA receptors are not involved in the metabotropic effect. The next question to be adressed is if this Glu accumulatton also occurs in the CNS in sirtr
Stefania Risso Bradley, AIlan 1. Levey, Steven M. Hersch. and P. Jeffrey Conn. Departments of Pharmacology and Neurology, Emory University School of Medicine, Atlanta, GA 30322. Polyclonal antibodies that specifically react with metabotropic glutamate receptors (mGluRs) mGIuR4a or mGluR7 were produced and characterized using immunoblot and immunocytochemistry analysis. Antibodies were generated against C-terminal domains of mGluR4a and mGluR7 using synthetic peptide immunogens. Both antibodies recognized native proteins in rat brain with molecular weights similar to the molecular weights of the bands in mGluR-transfected cell lines. Immunoblot analysis revealed that the levels of mGluR4a and mGluR7 are differentially regulated in different brain regions during development. For instance, in the cerebellum, mGluR7 immunoreactivity is higher at postnatal day 7 than in adults. whereas mGluR4a immunoreactivity is higher in adults than in younger animals. In hippocampus, both mGluR4a and mGluR7 immunoreactivity is higher in young animals than in adults. mGluR4a and mGluR7 are widely but differentially distributed throughout the rat brain, and are localized in specific cellular sites. Strong mGluR4a immunoreactivity was observed throughout the brain whereas the distribution of mGluR7 immunoreactivity was more varied, with heavy staining in cortical and limbic regions and light staining in ponsimedulla and cerelbellum. Immunocytochemistry studies at the EM level are consistent with the postulated role of mGluR4a and mGluR7 as presynaptic autoreceptors and suggest that mGluR4a may also have a postsynaptic function. Supported by NIH grant NS31373.
19
20
MOLECULAR PHARMACOLOGY OF ACID IBOTENIC ANALOGUES AT METABOTROPIC GLUTAMATE RECEPTORS.
EFFECT OF NOVEL mGLUR ANTAGONISTS ON SIGNAL TRANSDUCTION IN MESENCEPHALIC AND STRIATAL NEURONS IN VITRO.
Bmuner-Osborne, H., LB.Ebert, F. Slok. and P. Krogsgaard-Larsen. Department of Medicinal Chemistry, Royal Danish School of Pharmacy, Universitet.sparken 2.2100 Copenhagen, Denmark.
L. Bresciani, N. BruneIlo*, R. Pellicciari”, G. Racagni, and A. Ambrosini. Center of Neuropharmacol., Univ. of Milan, *Dept. Pharmaceutical Sci., Univ. of Modena and “Univ. of Perugia (I).
Based on the structure of ibotenic acid (IBO) we have previously esigned AMPA, Homo-AMPA and Homo-IBO.
New selective agonists enabled us to characterise the activity of metabotropic glutamate receptors (mGluRs) in mesencephalic (Mol. Pharmacol. 47, 1057, 1995) and in striatal neurons. In both neuronal systems, the agonist (RS)-3,5-dihydroxyphenylglycine (DHPG) was more potent on PI hydrolysis stimulation than on the modulation of CAMP levels, a difference that was more evident in striatal neurons, in which the EC50 values on the two pathways were 3 pM and 25 pM, respectively. On the contrary, (2S,3S,4S)alpha-(carboxycycIopropyl)glycine (CCGI) inhibited CAMP formation at lower concentrations than those required to activate PI turnover. In striatal neurons, CCGI EC50 values were 50 pM on the PI hydrolysis and 0.8 pM on the modulation of CAMP levels. The effects of novel antagonists proposed as selective for class I (%I-aminoindandicarboxylic acid - UPF523) and for class III (RS-alpha-methyl-4-phosphonophenylglycine - MPPG) mGluRs were tested. IJPF523 selectively antagonised the Quis and DHPGinduced inositol phosphate fonuation. MPPG (10 pM) did not affect basal CAMP values, but effectively blocked the response to aminophoposphonobutyrate, serine-O-phosphate and CCGI, confirming its pure antagonist action at mGluRs that inhibit adenylate cyclase, although a selectivity between class II and III was not evident. The pharmacological distinction of the mGluR effects may help to clarify their role in the basal ganglia.
Homo-AMPA
AMPA
When tested on ionotropic glutamate receptors Homo-IBO is a weak AMPA agonist zand AMPA is a potent and selective AMPA agonist. Homo-AMPP. is totally inactive. In this study we h.ave tested these ligands on Chinese hamster ovary cells expressirlg mGlu to, mGlu2 and mGlu6 receptorsa representing group I, II and III receptors, respectively. AMPA and Homo-AMPA were inactive on mGlu to and mGIu2 but were weak and potent mGIU6 agonists, respectivelye. Homo-IBO showed a more complex pharmacology being a antagonist on mGlut,, inactive at mGlur and a a Kindly provided by Drs.
potent
agonist at mGlu6.
N. Sekiyama
and S. Nakanishi.
Skjarbrek, B. Eben, N. Sekiyama, S. Nakanishi. and P. Krogsgaard-Larsen. J. Med. Chem. (In press).
b Br&mer-Osborne.
H., F.A. Wk,
N.
AS