RESUMEN
The 5-HT(6) receptor is predominantly expressed in the CNS and has been implicated in the regulation of cognitive function. Antagonists of the 5-HT(6) receptor improve cognitive performance in a number of preclinical models and have recently been found to be effective in Alzheimer's disease patients. Systemic administration of 5-HT(6) antagonists increases the release of acetylcholine and glutamate in the frontal cortex and dorsal hippocampus. In contrast, the selective 5-HT(6) agonist, WAY-181187, can elicit robust increases in extracellular levels of GABA. The reported behavioral and neurochemical effects of 5-HT(6) receptor ligands raise the possibility that the 5-HT(6) receptor may modulate synaptic plasticity in the hippocampus. In the present study, selective pharmacological tools were employed to determine the effect of 5-HT(6) receptor activation on long-term potentiation (LTP) in brain slices containing area CA1 of the hippocampus. While having no effect on baseline synaptic transmission, the results demonstrate that the selective 5-HT(6) agonist, WAY-181187, attenuated LTP over a narrow dose range (100-300 nM). The increase in the slope of the field excitatory post synaptic potential (fEPSP) caused by theta burst stimulation in brain slices treated with the most efficacious dose of WAY-181187 (200 nM) was 80.1+/-4.0% of that observed in controls. This effect was dose-dependently blocked by the selective 5-HT(6) antagonist, SB-399885. WAY-181187 also increased the frequency of spontaneous GABA release in area CA1. As assessed by measuring and evaluating spontaneous inhibitory postsynaptic currents (sIPSCs), 200 nM WAY-181187 increased sIPSC frequency by 3.4+/-0.9 Hz. This increase in GABA sIPSCs was prevented by the selective 5-HT(6) antagonist SB-399885 (300 nM). Taken together, these results suggest that the 5-HT(6) receptor plays a role in the modulation of synaptic plasticity in hippocampal area CA1 and that the regulation of GABAergic interneuron activity may underlie the cognition enhancing effects of 5-HT(6) antagonists.
Asunto(s)
Región CA1 Hipocampal/fisiología , Potenciación a Largo Plazo/fisiología , Células Piramidales/fisiología , Receptores de Serotonina/metabolismo , Transmisión Sináptica/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Región CA1 Hipocampal/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/fisiología , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Piperazinas/farmacología , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/administración & dosificación , Agonistas de Receptores de Serotonina/farmacología , Sulfonamidas/farmacología , Transmisión Sináptica/efectos de los fármacos , Ritmo Teta/efectos de los fármacos , Tiazoles/administración & dosificación , Tiazoles/farmacología , Triptaminas/administración & dosificación , Triptaminas/farmacologíaRESUMEN
The mGluR5 antagonist 2-methyl-6-(phenylethynyl) pyridine (MPEP) produces anxiolytic or antidepressant effects in several rodent models through incompletely described mechanisms. Anxiolytics and antidepressants share several neuroendocrine features, including acute activation of the hypothalamic-pituitary-adrenal (HPA)-axis, desensitization of neuroendocrine responses with repeated dosing, and desensitization of the HPA axis to 5-HT1A agonist stimulation. We characterized these neuroendocrine parameters in rats treated systemically with MPEP and compared them to those induced by the anxiolytic buspirone. Acutely, MPEP dose-dependently (0.1-10 mg/kg i.p.) increased plasma corticosterone concentrations. These responses were blocked by 50% with the 5-HT1A antagonist WAY100635. The corticosterone responses to both 3 mg/kg MPEP and buspirone were decreased by 80% after 5 days of twice-daily injections. Repeated injection with MPEP decreased HPA-axis sensitivity to buspirone challenge by 75%. This desensitization was not associated with changes in mGluR5 or 5-HT1A receptor binding properties, expression of G-protein subunits coupled to these receptors, or in 5-HT-stimulated binding of [(3)H]-GTPgammaS to membranes. We conclude that MPEP acutely disinhibits the HPA axis, in part through uncharacterized changes in serotonergic signaling. Desensitization of 5-HT1A responses after repeated MPEP administration may indicate that, like other anxiolytics and antidepressants, plasticity in 5-HT signal transduction pathways has occurred.
Asunto(s)
Antagonistas de Aminoácidos Excitadores/farmacología , Sistema Hipotálamo-Hipofisario/efectos de los fármacos , Sistema Hipófiso-Suprarrenal/efectos de los fármacos , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Serotonina/farmacología , Animales , Relación Dosis-Respuesta a Droga , Sistema Hipotálamo-Hipofisario/metabolismo , Masculino , Sistema Hipófiso-Suprarrenal/metabolismo , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptor del Glutamato Metabotropico 5 , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de Serotonina/metabolismo , Receptores de Serotonina 5-HT1 , Serotonina/metabolismo , Agonistas de Receptores de Serotonina/farmacologíaRESUMEN
Recent evidence suggests that the functions of presynaptic metabotropic glutamate receptors (mGluRs) are tightly regulated by protein kinases. We previously reported that cAMP-dependent protein kinase (PKA) directly phosphorylates mGluR2 at a single serine residue (Ser843) on the C-terminal tail region of the receptor, and that phosphorylation of this site inhibits coupling of mGluR2 to GTP-binding proteins. This may be the mechanism by which the adenylyl cyclase activator forskolin inhibits presynaptic mGluR2 function at the medial perforant path-dentate gyrus synapse. We now report that PKA also directly phosphorylates several group III mGluRs (mGluR4a, mGluR7a, and mGluR8a), as well as mGluR3 at single conserved serine residues on their C-terminal tails. Furthermore, activation of PKA by forskolin inhibits group III mGluR-mediated responses at glutamatergic synapses in the hippocampus. Interestingly, beta-adrenergic receptor activation was found to mimic the inhibitory effect of forskolin on both group II and III mGluRs. These data suggest that a common PKA-dependent mechanism may be involved in regulating the function of multiple presynaptic group II and group III mGluRs. Such regulation is not limited to the pharmacological activation of adenylyl cyclase but can also be elicited by the stimulation of endogenous G(s)-coupled receptors, such as beta-adrenergic receptors.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sinapsis/fisiología , Agonistas Adrenérgicos beta/farmacología , Secuencia de Aminoácidos , Animales , Fraccionamiento Celular , Cerebelo/citología , Colforsina/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Técnicas In Vitro , Isoproterenol/farmacología , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Propionatos/farmacología , Estructura Terciaria de Proteína , Ratas , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sinapsis/efectos de los fármacosRESUMEN
One of the primary physiological roles of group II and group III metabotropic glutamate receptors (mGluRs) is to presynaptically reduce synaptic transmission at glutamatergic synapses. Interestingly, previous studies suggest that presynaptic mGluRs are tightly regulated by protein kinases. cAMP analogs and the adenylyl cyclase activator forskolin inhibit the function of presynaptic group II mGluRs in area CA3 of the hippocampus. We now report that forskolin has a similar inhibitory effect on putative mGluR2-mediated responses at the medial perforant path synapse and that this effect of forskolin is blocked by a selective inhibitor of cAMP-dependent protein kinase (PKA). A series of biochemical and molecular studies was used to determine the precise mechanism by which PKA inhibits mGluR2 function. Our studies reveal that PKA directly phosphorylates mGluR2 at a single serine residue (Ser(843)) on the C-terminal tail region of the receptor. Site-directed mutagenesis combined with biochemical measures of mGluR2 function reveal that phosphorylation of this site inhibits coupling of mGluR2 from GTP-binding proteins
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Sulfonamidas , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adenina/análogos & derivados , Adenina/farmacología , Secuencia de Aminoácidos , Animales , Anticonvulsivantes/farmacología , Células CHO , Cricetinae , Ciclopropanos/farmacología , Giro Dentado/citología , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Glicina/análogos & derivados , Glicina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Isoquinolinas/farmacología , Datos de Secuencia Molecular , Mutagénesis/fisiología , Neuronas/citología , Neuronas/enzimología , Vía Perforante/citología , Fosforilación , Unión Proteica/fisiología , Ratas , Receptores de Glutamato Metabotrópico/genética , Serina/metabolismo , TransfecciónAsunto(s)
Proteínas de Unión al GTP/metabolismo , Hipocampo/enzimología , Proteína Quinasa C/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacología , Animales , Dihidropiridinas/farmacología , Activación Enzimática , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Hipocampo/metabolismo , Forbol 12,13-Dibutirato/farmacología , Forboles/farmacología , Propionatos/farmacología , Receptor de Adenosina A3 , Receptores de Glutamato Metabotrópico/clasificación , Receptores Purinérgicos P1/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
One of the most prominent roles of metabotropic glutamate receptors (mGluRs) in the CNS is to serve as presynaptic receptors that inhibit transmission at glutamatergic synapses. Previous reports suggest that the presynaptic effect of group II mGluRs at corticostriatal synapses can be inhibited by activators of protein kinase C (PKC). We now report that activation of PKC inhibits the ability of group II and group III mGluRs to regulate transmission at three major synapses in the hippocampal formation. Thus, this effect may be a widespread phenomenon that occurs at glutamatergic synapses throughout the CNS. We also report that this response is not limited to PKC-activating phorbol esters but that activation of A3 adenosine receptors induces a PKC-dependent inhibition of group III mGluR function at the Schaffer collateral-CA1 synapse. In addition to inhibiting mGluR modulation of excitatory synaptic transmission, we found that activation of PKC reduces inhibition of forskolin-stimulated cAMP accumulation by group II and group III mGluRs, suggesting that the effect of PKC on mGluR signaling is not specific to their effects on neurotransmitter release. This led us to test the hypothesis that PKC acts upstream from effector proteins regulated by mGluRs and acts at the level of the receptor or GTP-binding protein. Interestingly, we found that PKC inhibited mGluR-induced increases in [35S]-GTPgammaS binding in cortical synaptosomes. These data suggest that PKC-induced inhibition of mGluR signaling may be mediated by the inhibition of coupling of mGluRs to GTP-binding proteins.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Proteínas de Unión al GTP/metabolismo , Isoenzimas/fisiología , Terminales Presinápticos/metabolismo , Proteína Quinasa C/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Receptores Purinérgicos P1/fisiología , Animales , Colforsina/farmacología , AMP Cíclico/metabolismo , Hipocampo/fisiología , Masculino , Inhibición Neural/fisiología , Vía Perforante/fisiología , Forbol 12,13-Dibutirato/farmacología , Agonistas del Receptor Purinérgico P1 , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Sinapsis/fisiologíaRESUMEN
Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.
Asunto(s)
Cuerpo Estriado/efectos de los fármacos , AMP Cíclico/biosíntesis , Agonistas de Aminoácidos Excitadores/farmacología , Neuronas/efectos de los fármacos , Receptores de Glutamato Metabotrópico/agonistas , Adenosina-5'-(N-etilcarboxamida)/farmacología , Animales , Autorradiografía , Células Cultivadas , Cuerpo Estriado/citología , Cuerpo Estriado/metabolismo , Técnica del Anticuerpo Fluorescente , Ácido Glutámico/farmacología , Glicina/análogos & derivados , Glicina/farmacología , Hidrólisis , Hibridación in Situ , Neuronas/metabolismo , Fosfatidilinositoles/metabolismo , Ratas , Resorcinoles/farmacologíaRESUMEN
The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.
Asunto(s)
Compuestos Bicíclicos con Puentes/metabolismo , Corteza Cerebral/metabolismo , Agonistas de Aminoácidos Excitadores/metabolismo , Hipocampo/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Tálamo/metabolismo , Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Sistema de Transporte de Aminoácidos X-AG , Animales , Unión Competitiva , Membrana Celular/metabolismo , Técnicas In Vitro , Masculino , Ensayo de Unión Radioligante , RatasRESUMEN
In rat cortical primary cultures, group II- and III-metabotropic glutamate receptor-selective agonists concentration-dependently reduced KCl-induced [3H]GABA release, with IC50 values of 11 nM for LY354740, 80 nM for L(+)-2-amino-4-phosphonobutyric acid (L-AP4), 180 nM for DCG-IV, and 330 nM for L-SOP. The group II antagonists, LY341495 and EGLU, reversed the effect of LY354740, and the group III antagonist MTPG reversed the effect of L-AP4. In the presence of omega-conotoxin GVIA, LY354740 inhibited the remaining [3H]GABA release, whereas L-AP4 was inactive. In contrast, in the presence of nifedipine, L-AP4 inhibited the remaining [3H]GABA release, but LY354740 was no longer active. The PKA inhibitor, H89, blocked the effects of both L-AP4 and LY354740, whereas the PKC inhibitor Ro 31-8220 blocked only the effect of LY354740. Both Ro 31-8220 and H89 reduced the [3H]GABA release to 60% of control. In whole-cell, voltage-clamp experiments, LY354740 and L-AP4 inhibited voltage-gated calcium channel currents with IC50 values of 28 nM and 22 microM, respectively. The results suggest that, in these cells, KCl-induced [3H]GABA release is modulated by two different mechanisms, one involving group II receptors and a direct control of the Ca2+ channel activity, and the other mediated by group III receptors and possibly involving a regulation located downstream of the Ca2+ channel activation.
Asunto(s)
Corteza Cerebral/metabolismo , Cloruro de Potasio/farmacología , Receptores de Glutamato Metabotrópico/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Western Blotting , Células Cultivadas , Corteza Cerebral/citología , Corteza Cerebral/efectos de los fármacos , AMP Cíclico/metabolismo , Hibridación in Situ , Técnicas de Placa-Clamp , Ratas/embriología , Ratas Endogámicas , Receptores de Glutamato Metabotrópico/agonistas , TritioRESUMEN
1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).
Asunto(s)
Ciclopropanos/metabolismo , Glicina/análogos & derivados , Receptores de Glutamato Metabotrópico/metabolismo , Animales , Células CHO , Membrana Celular/metabolismo , Colforsina/farmacología , Cricetinae , AMP Cíclico/biosíntesis , Glicina/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Unión Proteica , Ratas , Proteínas Recombinantes/metabolismo , Radioisótopos de Azufre , Transfección , TritioRESUMEN
3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.
Asunto(s)
Corteza Cerebral/fisiología , AMP Cíclico/fisiología , Glicina/farmacología , Receptores de Glutamato Metabotrópico/fisiología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Corteza Cerebral/efectos de los fármacos , Fluorescencia , Glicina/análogos & derivados , Glicina/química , Hidrólisis , Técnicas In Vitro , Fosfatidilinositoles/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato Metabotrópico/efectos de los fármacos , Transducción de Señal/fisiología , Relación Estructura-ActividadRESUMEN
1. The effects of selective agonists at group I, II and III metabotropic glutamate receptors (mGluRs) on adenosine A2 receptor-mediated cyclic AMP formation were compared in cross-chopped slices of adult and neonatal (8 days old) rat striatum, in the presence of 1 u ml(-1) adenosine deaminase. 2. The group II selective agonist, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), elicited a potentiation of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated cyclic AMP production with similar potencies in adult (EC50 value 122 +/- 35 nM) and neonatal (EC50 value 285 +/-6 nM) brain. In contrast, the group I selective agonist (S)-dihydroxyphenylglycine ((S)-DHPG) augmented the NECA cyclic AMP response in neonatal striatum (EC50 value 9 +/- 1 microM), but at a concentration of 100 microM, (S)-DHPG failed to affect the NECA response in adult striatal slices. 3. The potentiation evoked by (S)-DHPG was specific for group I mGluRs as (2S,3S,4S,)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a group II antagonist, was ineffective on the (S)-DHPG (100 microM) response at a concentration (500 microM) which reversed a similar augmentation elicited by DCG-IV (300 nM). Furthermore, a protein kinase C inhibitor (Ro 31-8220, 10 microM) markedly reversed the effect of (S)-DHPG without affecting the response to DCG-IV. 4. The mGluR agonist (2S,3S,4S,)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited a greater potentiation of NECA-stimulated cyclic AMP production in neonatal striatum in comparison with that observed in adult rat brain. Moreover, EC50 values obtained from adult and neonatal striatum were 2 +/-1 microM and 9 +/-1 microM, respectively. These differences in potency might reflect co-activation of both group I and group II mGluRs by L-CCG-I in neonatal striatum. 5. Distinct patterns of mGluR expression in various brain areas might account for previous conflicting data on the nature of the mGluR able to evoke such potentiated responses.
Asunto(s)
Cuerpo Estriado/metabolismo , AMP Cíclico/biosíntesis , Receptores de Glutamato/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Adenosina-5'-(N-etilcarboxamida) , Animales , Animales Recién Nacidos , Femenino , Glicina/farmacología , Técnicas In Vitro , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The pharmacological profile of mGlu receptors negatively linked to adenylyl cyclase was characterized in adult rat striatal slices. Among the mGlu agonists tested, (+)-2-aminobicyclo-[3.1.0]-hexane-2,6-di carboxylate (LY354740), was the most potent inhibitor of forskolin-stimulated cAMP formation (EC50 = 11 +/- 2 nM). Inhibition of forskolin stimulation by the group III agonist L-2-amino-4-phosphono-butanoate (L-AP4) was biphasic, the two parts of the concentration curve having EC50 values of 6 +/- 1 microM and 260 +/- 4 microM, suggesting a sequential recruitment of mGlu4/8 and mGlu7. The effects of several new phenylglycine derivative antagonists were tested on the inhibition of forskolin cAMP response by (2S,1'S,2'S)-2-(carboxy-cyclopropyl)-glycine (L-CCG I) and L-AP4. At 500 microM, (RS)-alpha-methyl-3-carboxy-methyl-pheny lglycine was unable to antagonize the effect of L-CCG I or L-AP4 but (S)-alpha-methyl-3-carboxy-phenylalanine inhibited the effect of L-AP4 with a low potency. Finally, (RS)-alpha-methyl-4-tetrazolylphenylglyc ine and particularly (RS)-alpha-methyl-4-phosphonophenylglyci ne, appeared to be the most potent and selective antagonists of L-AP4 induced inhibition of forskolin-stimulated cAMP production in adult rat striatal slices.