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1.
Cell Signal ; 36: 56-66, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28445805

RESUMO

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions including subretinal neovascularization (SRN), proliferative vitreoretinopathy (PVR) and, importantly, as a consequence of retinal surgery. Therefore, the elucidation of the mechanisms underlying RPE trans-differentiation and migration is essential for devising effective treatments aimed to the prevention of these disorders. A common event in these pathologies is the alteration of the blood-retina barrier (BRB), which allows the interaction of RPE cells with thrombin, a pro-inflammatory protease contained in serum. Our previous work has demonstrated that thrombin induces RPE cell cytoskeletal remodeling and migration, hallmark processes in the development of PVR; however, the molecular mechanisms involved are still unclear. Cell migration requires the disassembly of focal adhesions induced by Focal Adhesion Kinase (FAK) phosphorylation, together with the formation of actin stress fibers. The aim of the present work was to identify thrombin-activated signaling pathways leading to FAK phosphorylation and to determine FAK participation in thrombin-induced RPE cell migration. Results demonstrate that the activation of PAR1 by thrombin induces FAK autophosphorylation at Y397 and the subsequent phosphorylation of Y576/577 within the activation loop. FAK phosphorylation was shown to be under the control of c/nPKC and PI3K/PKC-ζ, as well as by Rho/ROCK, since the inhibition of these pathways prevented thrombin-induced FAK phosphorylation and the consequent disassembly of focal adhesions, in parallel to FAK-dependent actin stress fiber formation and RPE cell migration. These findings demonstrate, for the first time, that thrombin stimulation of RPE cell transformation and migration are regulated by FAK tyrosine phosphorylation. Thus, targeting FAK phosphorylation may provide a strategical basis for PVR treatment.


Assuntos
Movimento Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/enzimologia , Trombina/farmacologia , Actinas/metabolismo , Animais , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Isoenzimas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Proteína Quinase C/metabolismo , Ratos Long-Evans , Receptor PAR-1 , Transdução de Sinais/efeitos dos fármacos , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/metabolismo
2.
Biochim Biophys Acta ; 1813(10): 1758-66, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21736902

RESUMO

The retinal pigment epithelium (RPE) plays an essential role in the survival and function of the neural retina. RPE uncontrolled proliferation leads to the development of proliferative ocular pathologies, among which proliferative vitreoretinopathy (PVR) is the main cause of retinal surgery failure. Upon the breakdown of the BRB due to trauma or metabolic imbalance the contact of RPE with serum-contained thrombin has been shown to stimulate the proliferation of otherwise quiescent RPE cells. Although the molecular mechanisms involved in this effect are still undetermined, thrombin proteolytic activation of protease-activated G protein coupled receptor-1 (PAR-1) activates PI3K and Akt, known to play an essential role in proliferation. The present study demonstrates that: 1) thrombin stimulates Ser 473 Akt phosphorylation without affecting Thr 308 basal phosphorylation in RPE cells; 2) thrombin-induced Akt stimulation promotes cyclin D1 accumulation through the phosphorylation/ inhibition of GSK-3ß, thus preventing Thr 286 cyclin D1 phosphorylation, nuclear export and degradation; 3) Akt signaling requires the upstream activation of PI3K and PLC. Since the pharmacological inhibition of these pathways or the silencing of cyclin expression prevent thrombin-induced RPE cell proliferation, these results contribute relevant evidence for establishing the mechanism involved in the development of proliferative eye diseases.


Assuntos
Proliferação de Células , Ciclina D1/metabolismo , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Trombina/farmacologia , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ciclina D1/antagonistas & inibidores , Ciclina D1/genética , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Long-Evans , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Trombina/metabolismo , Trombina/fisiologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética
3.
J Neurosci Res ; 73(3): 369-83, 2003 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-12868071

RESUMO

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. The N-methyl-D-aspartate glutamate receptor (NMDAR) is assembled as a tetramer containing NR1 and NR2, and possibly NR3 subunits, NR1 being essential for the formation of the ion channel. The NMDAR1 (NR1) gene encodes for mRNAs that generate at least eight functional variants by alternative splicing of exon 5 (cassette N1), 21 (cassette C1), or 22 (cassettes C2 or C2'). NR1 splice variants were identified in the mature chick retina, and their variation during embryonic development (ED) was analyzed. NR1 was shown to lack N1 in early ED, shifting to N1-containing variants in the mature retina, which could contribute to explaining the distinct biochemical properties of retinal NMDARs compared with the CNS. Sequence analysis of C-terminal variants containing C1 and C2 cassettes suggests a membrane-targeting mechanism for avian NMDARs distinct from that in mammals. An NR1 variant containing a novel alternative C-terminal splice exon named C3 was found, which encodes six amino acids containing a predicted casein kinase II phosphorylation site. This new variant is expressed in the retina during a restricted period of ED, coincident with the generation of spontaneous calcium activity waves, which precedes synapse formation in the retina, suggesting its participation in this process.


Assuntos
Processamento Alternativo/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Receptores de N-Metil-D-Aspartato/genética , Retina/embriologia , Retina/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Embrião de Galinha , Primers do DNA , Masculino , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
J Comp Physiol A ; 187(5): 349-57, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11529479

RESUMO

The octapeptide red pigment-concentrating hormone is capable of eliciting the aggregation of intracellular pigment granules in distal retinal pigment cells of isolated retinas of the crayfish Procambarus clarkii (Girard). The final level and the time course of pigment aggregation are dose dependent within a range of 10(-10) mol l(-1) to 10(-4) mol l(-1). The effect of red pigment-concentrating hormone is prevented by previous incubation with an anti- red pigment-concentrating hormone antibody; however, application of the antibody after the onset of the red pigment-concentrating hormone effect, does not prevent its full development. A similar effect to that elicited by red pigment-concentrating hormone is induced by the calcium ionophores ionomycin and A-23187. Red pigment-concentrating hormone evokes entry of 45Ca2+ to retinal cells. However, the red pigment-concentrating hormone-induced pigment aggregation persists in the presence of the calcium channel blocker verapamil and in Ca2+-free solutions. Caffeine and thapsigargin, known to release calcium from intracellular stores, elicit distal pigment aggregation, while ryanodine and dantrolene, blockers of intracellular calcium release, as well as the intracellular calcium chelator bapta-AM suppress the effect of red pigment-concentrating hormone. These results suggest that red pigment-concentrating hormone elicits distal retinal pigment aggregation by increasing intracellular calcium concentration, acting via a dual mechanism: (1) promoting calcium entry, and (2) releasing intracellular calcium.


Assuntos
Cálcio/farmacocinética , Oligopeptídeos/metabolismo , Células Fotorreceptoras de Invertebrados/fisiologia , Pigmentos da Retina/metabolismo , Animais , Anticorpos/farmacologia , Astacoidea , Quelantes/farmacologia , Relação Dose-Resposta a Droga , Ácido Egtázico/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Oligopeptídeos/imunologia , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/análogos & derivados
5.
J Neurosci Res ; 64(3): 218-22, 2001 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-11319765

RESUMO

Glial cells possess transport systems for the three major amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA) and glycine, involved in the arrest of neurotransmission mediated by these compounds. Two glycine transporters have been cloned: GLYT1, mainly expressed by glial cells and shown to colocalize with NMDA receptors, and GLYT2, exclusively expressed by neurons and colocalized with the inhibitory glycine receptors. The way in which the regulation of extracellular glycine concentration by glial glycine transporters affects physiological and pathological conditions is discussed. The presence, differential pharmacology and specific regulation of glycine transporters in glial cells strongly support an important role for glia in the modulation of both, excitatory and inhibitory neurotransmission.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Transporte/metabolismo , Proteínas de Membrana Transportadoras , Neuroglia/metabolismo , Sistema X-AG de Transporte de Aminoácidos , Esclerose Lateral Amiotrófica/metabolismo , Animais , Encéfalo/metabolismo , Proteínas da Membrana Plasmática de Transporte de GABA , Proteínas da Membrana Plasmática de Transporte de Glicina , Humanos , Neurônios/metabolismo , Esquizofrenia/metabolismo , Medula Espinal/metabolismo
6.
J Neurosci Res ; 63(6): 453-60, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11241580

RESUMO

The termination of chemical neurotransmission in the CNS involves the rapid removal of neurotransmitter from synapses by specific transport systems. Such mechanism operates for the three major amino acid neurotransmitters glutamate, gamma-aminobutyric acid (GABA) and glycine. To date, five different high-affinity Na(+)-dependent glutamate (Glu) transporters have been cloned: GLT1, GLAST, EAAC1, EAAT4 and EAAT5. The first two are expressed mainly by glial cells, and seem to be the predominant Glu transporters in the brain. A major function of Glu uptake in the nervous system is to prevent extracellular Glu concentrations from raising to neurotoxic levels in which glial transporters seem to play a critical role in protecting neurons from glutamate-induced excitotoxicity. Under particular conditions, glial GluTs have been shown to release Glu by reversal of activity, in a Ca(2+)--and energy-independent fashion. Furthermore, an activity of these transporters as ion channels or transducing units coupled to G-proteins has recently been reported. The localization, stoichiometry, and regulation of glial GluTs are outlined, as well as their possible contributions to nervous system diseases as ALS, AD and ischemic damage.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Neuroglia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sistema X-AG de Transporte de Aminoácidos , Animais , Humanos
7.
J Neurosci Res ; 63(6): 461-8, 2001 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-11241581

RESUMO

The termination of chemical neurotransmission in the central nervous system (CNS) involves the rapid removal of neurotransmitter from synapses. This is fulfilled by specific transport systems in neurons and glia, including those for gamma-aminobutyric acid (GABA), the main inhibitory neurotransmitter in the brain. Glial cells express the cloned Na(+)/Cl(-)-dependent, high-affinity GABA transporters (GATs) GAT1, GAT2, and GAT3, as well as the low-affinity transporter BGT1. In situ hybridization and immunocytochemistry have revealed that each transporter shows distinct regional distribution in the brain and the retina. The neuronal vs. glial localization of the different transporters is not clear-cut, and variations according to species, neighboring excitatory synapses, and developmental stage have been reported. The localization, stoichiometry, and regulation of glial GATs are outlined, and the participation of these structures in development, osmoregulation, and neuroprotection are discussed. A decrease in GABAergic neurotransmission has been implicated in the pathophysiology of several CNS disorders, particularly in epilepsy. Since drugs which selectively inhibit glial but not neuronal GABA uptake exert anticonvulsant activity, clearly the establishment of the molecular mechanisms controlling GATs in glial cells will be an aid in the chemical treatment of several CNS-related diseases.


Assuntos
Proteínas de Transporte/metabolismo , Ácido Glutâmico/metabolismo , Glicina/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Neuroglia/metabolismo , Transportadores de Ânions Orgânicos , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas da Membrana Plasmática de Transporte de GABA , Humanos
8.
Brain Res ; 854(1-2): 1-5, 2000 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-10784099

RESUMO

Spermine has been shown to influence NMDA receptor function through an interaction at the coagonist site for glycine in the central nervous system (CNS) and the retina. In order to support a role for spermine as neurotransmitter or neuromodulator in the chick retina, specific stimulated-release of spermine should be demonstrated. Isolated chick retinas, preloaded with [3H]spermine, were stimulated with 1 mM NMDA and other glutamate agonists at ionotropic receptors, in a continuous superfusion system. [3H]spermine was released from the retina by depolarization with 50 mM KCl, in a Ca2+-independent manner. Inhibition of Na+/K+-ATPase by ouabain or digitoxigenin also induced spermine release following 36 min in the presence of the drugs; such effect seems unrelated to changes in Na+ electrochemical gradients, since nigericin and veratrine did not induce release in Na+ containing medium. The lack of effect of glutamate, NMDA and kainate at 1 mM concentration, suggests that release of spermine in the retina is mediated by the reversal of uptake and not necessarily linked to EAA-receptor activation.


Assuntos
Cálcio/fisiologia , Retina/metabolismo , Espermina/metabolismo , Animais , Animais Recém-Nascidos , Galinhas , Digitoxigenina/farmacologia , Inibidores Enzimáticos/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , N-Metilaspartato/farmacologia , Ouabaína/farmacologia , Cloreto de Potássio/farmacologia , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , Trítio
9.
Epilepsy Res ; 39(1): 13-26, 2000 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-10690749

RESUMO

The sharp interruption of the intracortical instillation of exogenous gamma-aminobutyric acid (GABA), generates an epileptic focus in mammals. Seizures elicited by GABA withdrawal last several days or weeks. The present work reports that GABA withdrawal-induced hyperexcitability can be produced in vitro: a sudden withdrawal of GABA (5 mM; 120 min) or benzodiazepine (60 microM flunitrazepam) from the superfusion, induced a gradual increase in the amplitude of the evoked population spike (PS) recorded on neocortical slices. PS enhancement reached 150% above the control value 2.5 h after GABA withdrawal. GABA withdrawal-induced hyperexcitability was facilitated by progesterone. PS enhancement induced by GABA withdrawal was associated with an impairment of GABA transmission occurring before epileptiform discharges were fully established. Paired pulse inhibition and evoked [3H]-GABA release appear decreased; suggesting that cortical hyperexcitability as a result of GABA withdrawal involves pre-synaptic changes. Specific muscimol binding decreased during GABA superfusion but recovered after GABA withdrawal. However, the sensitivity of the post-synaptic response to 3alpha-OH-5alpha-pregnan-20-one or allopregnanolone (alloP) was enhanced after GABA withdrawal, suggesting a functional change in the GABA(A) receptors. The changes described may be the cellular correlates of the withdrawal syndromes appearing after interruption of the administration of GABA(A) receptor agonists.


Assuntos
Neocórtex/efeitos dos fármacos , Convulsões/induzido quimicamente , Síndrome de Abstinência a Substâncias/metabolismo , Transmissão Sináptica/efeitos dos fármacos , Ácido gama-Aminobutírico/efeitos adversos , Animais , Flunitrazepam/efeitos adversos , Agonistas GABAérgicos/metabolismo , Moduladores GABAérgicos/efeitos adversos , Muscimol/metabolismo , Neocórtex/metabolismo , Pregnanolona/efeitos adversos , Progesterona/efeitos adversos , Ratos , Ratos Wistar , Convulsões/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/farmacocinética
10.
Brain Res ; 844(1-2): 150-6, 1999 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-10536271

RESUMO

The binding of [3H]spermine to synaptosomal membranes from chick retina was examined. Saturable specific binding of [3H]spermine to synaptosomal membranes from plexiform layers of retina (P1 and P2) has been characterized, and found to concentrate in the inner plexiform layer compared to the outer plexiform layer (Bmax=9.3 and 37 pmol/mg protein for P1 and P2, respectively). Kinetics of specific [3H]spermine binding yield a sigmoidal saturation curve, indicating positive cooperativity (nH: 2.4 and 3.2 for P1 and P2, respectively) with high affinity: Kapp=61 and 67 nM for P1 and P2. The time required to attain equilibrium at room temperature was less than 5 min in both fractions. Dose-response curves for spermine, spermidine, and diethylene-triamine (DET) show different potencies for inhibiting [3HDET. Our results support a role for polyamines (PA) as neurotransmitters or neuromodulators in the vertebrate retina.


Assuntos
Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Espermina/metabolismo , Espermina/farmacologia , Sinaptossomos/metabolismo , Animais , Galinhas , Maleato de Dizocilpina/farmacologia , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/farmacologia , Cinética , Poliaminas/metabolismo , Poliaminas/farmacologia , Ensaio Radioligante , Retina/química , Espermidina/metabolismo , Espermidina/farmacologia , Trítio
11.
Brain Res ; 838(1-2): 200-4, 1999 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-10446333

RESUMO

Müller glial cells express two transport systems for glycine (Gly): one with low affinity and another identified as GLYT1 with high affinity. The latter colocalizes with NMDA receptors in the CNS. Gly is considered as an obligatory coagonist at NMDA receptors, and, hence, the Gly transport system could contribute to the modulation of glutamate (Glu) excitatory transmission in the vertical pathways of the retina. For this reason, the regulation of Gly transport by cAMP was studied. We report here a non-specific effect of MDL-12330A, a compound reported to inhibit adenylate cyclase (AC), on Gly transport in Müller glia. This effect might be due to a toxic action on the cells, decreasing cell viability, and not to a specific inhibition of the adenylate cyclase. Non-specific effects of this drug should be considered when the participation of cAMP in any biological process is studied. We have clearly demonstrated that cAMP does not participate in the regulation of Gly transport in Müller glia.


Assuntos
Inibidores de Adenilil Ciclases , Inibidores Enzimáticos/farmacologia , Glicina/metabolismo , Iminas/farmacologia , Neuroglia/efeitos dos fármacos , Retina/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Galinhas , AMP Cíclico/fisiologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Neuroglia/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Retina/citologia , Retina/metabolismo
13.
Glia ; 26(4): 273-9, 1999 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10383046

RESUMO

Rapid termination of the synaptic action of glutamate (Glu) and glycine (Gly) is achieved by uptake into the presynaptic terminal and glial cells. In the vertebrate CNS, Gly acts both as an inhibitory neurotransmitter and as a Glu modulator or coagonist at postsynaptic N-methyl-D-aspartate (NMDA) receptors. We have previously described NMDA receptors in Müller cells of chick retina coupled to the phosphoinositide cascade, the entry of calcium, and the activation of protein kinase C (PKC; López-Colomé et al. Glia 9:127-135, 1993). A colocalization of Gly transporters and NMDA receptors has been reported in brain tissue (Smith et al. Neuron 8:927-936, 1992); since the concentration of Gly could participate in the modulation of Glu excitatory transmission in the vertical pathways of the retina, transport of Gly in monolayer cultures of Müller cells was studied. Gly transport was found pH-sensitive with an optimum at pH 7.4. Kinetic analysis of the saturation curve for Gly within a concentration range of 0.01-2 mM, revealed two components of transport: a low-affinity system with Km = 1.7 mM, Vmax = 30 nmol/10 min/mg protein, and a high-affinity one with a Km = 27 microM, Vmax = 3 nmol/10 min/mg protein. Both systems were Na+ -dependent; the high-affinity system proved also dependent on external Cl- and was inhibited by sarcosine, characteristic of GLYT1 transporters. The inhibition of low-affinity uptake by 2-(methylamino)isobutyric acid (MeAIB) and 2-aminoisobutyric acid (AIB) suggests the presence of transport system A in Müller cells. The process is energy-requiring, since Gly transport was decreased by metabolic inhibitors. Data obtained are in keeping with a modulatory role for Müller glia on excitatory transmission in the retina.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros , Proteínas de Transporte/metabolismo , Glicina/metabolismo , Neuroglia/metabolismo , Retina/metabolismo , 2,4-Dinitrofenol/farmacologia , Animais , Transporte Biológico , Células Cultivadas , Embrião de Galinha , Cloretos , Inibidores Enzimáticos/farmacologia , Glicina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Glicina , Concentração de Íons de Hidrogênio , Iodoacetatos/farmacologia , Íons , Cinética , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Ouabaína/farmacologia , Cianeto de Potássio/farmacologia , Retina/citologia , Retina/embriologia , Sódio , Fatores de Tempo
14.
Vis Neurosci ; 16(2): 263-9, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10367961

RESUMO

Excitatory amino acid (EAA)-induced production of inositolphosphates (IPs) was studied in primary cultures of chick retinal pigment epithelium (RPE) following in vitro incorporation of [3H] myo-inositol. Glutamic acid (L-glu) significantly increased [3H]-IPs accumulation (215%). L-glu agonists stimulated [3H]IPs accumulation in the following order of efficiency: N-methyl-D-aspartate (NMDA) > or = L-glu > quisqualate > or = kainate > (IS,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (ACPD). Stimulation was dependent on external Ca2+. The NMDA-induced response was blocked by (+)-5-methyl-10,11-dihydro-5H-dibenzo-cyclohepten-5,10-imine maleate (MK-801) and 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP) and was decreased by the L-Ca2+-channel blockers verapamil and nifedipine as well as by dantrolene. The metabotropic glutamate receptor (mGluR) antagonist (+)-alpha-methyl-4-carboxyphenylglycine (+)MCPG inhibited 3,5-dihydroxyphenylglycine (DHPG) and ACPD-induced stimulation, which demonstrates the presence in RPE of mGluRs 1 and/or 5, as well as NMDA receptors coupled directly, or through the influx of external Ca2+, to phospholipase C activation. L-glu agonists showed no effect either on basal level of intracellular cyclic adenosine monophosphate, nor on forskolin- or carbachol-induced stimulation of adenylyl cyclase. Since L-glu is released from the retina upon illumination, and receptors for this compound are present in RPE, the activation of the inositide pathway could be involved in the regulation of retina-RPE interaction, which is essential for the visual process.


Assuntos
Aminoácidos Excitatórios/farmacologia , Fosfatos de Inositol/biossíntese , Epitélio Pigmentado Ocular/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Embrião de Galinha , AMP Cíclico/metabolismo , Antagonistas de Aminoácidos Excitatórios/farmacologia , Epitélio Pigmentado Ocular/embriologia , Epitélio Pigmentado Ocular/metabolismo
15.
Neurochem Res ; 23(11): 1363-9, 1998 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-9814546

RESUMO

Saturable specific binding of glycine to synaptosomal membranes from plexiform layers of the retina has been described, which seems to correspond to the modulatory site on NMDA-receptors (26). Spermine inhibited specific [3H]glycine binding to membranes from synaptosomal fractions from the outer (P1) and the inner (P2) plexiform layers of 1-3 day-old chick retinas in a dose-dependent manner with an IC50 = 35 microM for the P1 fraction and 32 microM for the P2 fraction. Kinetic experiments and non-linear regression analysis of [3H]glycine-specific binding showed a Kd approximately 100-150 nM in both fractions, and a higher Bmax (4.11 +/- 0.47 pmol/mg protein) for the inner plexiform layer compared to the outer plexiform layer (Bmax = 2.76 +/- 0.25 pmol/mg protein). Strychnine-insensitive [3H]glycine binding was inhibited by 100 microM spermine, due to a reduction in Bmax (P1 = 0.84 +/- 0.16 pmol/mg protein; P2 = 0.81 +/- 0.16 pmol/mg protein) without affecting the Kd. Association and dissociation constants in the absence and presence of 50 microM spermine remained unchanged. Results demonstrate the presence of a single modulatory site for spermine on NMDA receptors, in both synaptic layers of the chick retina.


Assuntos
Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/efeitos dos fármacos , Espermina/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Galinhas , Relação Dose-Resposta a Droga , Retina/metabolismo , Retina/ultraestrutura , Sinaptossomos/metabolismo , Trítio
16.
Int J Dev Neurosci ; 16(5): 413-21, 1998 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-9829177

RESUMO

The pharmacology and kinetics of strychnine-insensitive [3H] glycine binding to synaptic membranes from the outer (P1) and the inner (P2) plexiform layers of chick retina was studied. Inhibition curves for glycine, D-serine, 1-amincyclopropanecarboxylic acid (ACPC) and strychnine were analyzed by non-linear regression. Hill slopes for glycine and D-serine were not different from unity, whereas those for ACPC were < 1 in both fractions, revealing heterogeneity of binding sites in these membranes. Non-linear regression analysis of time course and saturation experiments strengthen the idea that [3H] glycine binds to more than one class of sites, with similar affinities at equilibrium. Antagonists of strychnine-insensitive glycine receptors in the CNS did not inhibit [3H] glycine binding to these membranes, which demonstrates that NMDA receptors in the retina have different structural requirements for ligand interaction at these sites. pH affected the specific binding, in agreement with the participation of specific amino acid residues at glycine binding sites on NMDA receptors, and also with functional studies in which the modulation of affinity at this site by protons has been observed. These results support previous studies regarding CPP and MK-801 binding, and provide evidence which indicates that the pharmacophore for glycine and other NMDA-related ligands is distinct for the retina, compared to the CNS, mainly regarding the effects of glycine-site antagonists.


Assuntos
Glicina/metabolismo , Retina/efeitos dos fármacos , Estricnina/farmacologia , Sinaptossomos/efeitos dos fármacos , Animais , Galinhas , Concentração de Íons de Hidrogênio , Hibridização In Situ , Cinética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Ensaio Radioligante , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/metabolismo , Retina/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Sinaptossomos/metabolismo , Trítio
17.
Brain Res Mol Brain Res ; 58(1-2): 40-6, 1998 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-9685580

RESUMO

The expression of neurotransmitter receptors in glial cells has suggested a regulatory role of these cells in synaptic function. In radial glia, glutamate receptors elicit a cascade from the membrane to the nucleus and a consequent change in gene expression. In order to gain insight into this process, we address the question of whether receptor activation leads to changes in the repertoire of AMPA/KA glutamate receptor subunits in Bergmann and Müller glial cells. Of the subunits investigated, only GluR4 was up-regulated in Bergmann glial cells both at mRNA and protein levels. In contrast, in Müller glial cells Glu treatment leads to a reduction in GluR4 mRNA and protein expression. Both effects are receptor-mediated and must probably involve group I of metabotropic glutamate receptors. Accordingly, using Northern blot analysis and RT-PCR we detected the expression of both mGluR1 and mGluR5 transcripts in the cultured cells. Our results confirm that glutamate receptors in Bergmann and Müller cells modulate gene expression and further strengthen a plausible role of glial cells in long-lasting changes in the central nervous system.


Assuntos
Ácido Glutâmico/farmacologia , Neuroglia/metabolismo , Receptores de AMPA/biossíntese , Receptores de Glutamato Metabotrópico/biossíntese , Animais , Células Cultivadas , Cerebelo/embriologia , Cerebelo/metabolismo , Embrião de Galinha , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Ácido Caínico/farmacologia , Cinética , N-Metilaspartato/farmacologia , Neuroglia/citologia , Neuroglia/efeitos dos fármacos , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , Receptor de Glutamato Metabotrópico 5 , Retina/citologia , Retina/metabolismo , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologia
18.
Neurochem Res ; 22(6): 679-85, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9178950

RESUMO

The effect of L-glutamate (Glu) and its structural analogs N-methyl-D-aspartate (NMDA), kainate (KA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), on the activation of p42 mitogen activated protein kinase (MAPK) was examined in cultured chick radial glia cells, namely retinal Muller cells and cerebellar Bergmann cells. Glu, NMDA, AMPA and KA evoked a dose and time dependent increase in MAPK activity. AMPA and KA responses were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) whereas NMDA responses were sensitive to 3-[(RS)-2-carboxypiperazin-4-yl)]-propyl-1-phosphonate (CPP) indicating that the increase in MAPK activity is mediated by AMPA/low affinity KA and NMDA subtypes of Glu receptors. The present findings open the possibility of a MAPK cascade involvement in the regulation of Glu-induced gene expression in radial glia.


Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/farmacologia , Ácido Glutâmico/farmacologia , Neuroglia/efeitos dos fármacos , Proteínas Tirosina Quinases/farmacologia , Animais , Células Cultivadas , Embrião de Galinha , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática , Agonistas de Aminoácidos Excitatórios/farmacologia , Proteína Quinase 1 Ativada por Mitógeno , Neuroglia/enzimologia , Proteínas Recombinantes/farmacologia
19.
Brain Res ; 759(1): 141-8, 1997 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-9219872

RESUMO

Rats showing disrupted taste aversion due to insular cortex lesions, received either homotopic or heterotopic (occipital) cortical fetal brain grafts. Behavioral results showed that the recovery of the ability to acquire conditioned taste aversions induced by fetal grafts depended on post-graft time (45 but not at 15 days) and tissue specificity (homotopic but not heterotopic). In vivo analysis of acetylcholine (ACh) release revealed that only the group receiving homotopic grafts and tested 45 days post graft had a release of ACh after KCl stimulation similar to that in the control group. Furthermore, homotopic grafts and lesioned groups showed significantly weaker specific receptor binding of [3H]L-glutamate compared with controls. These results suggest that ACh is specifically involved in the process of behavioral recovery induced by homotopic cortical transplants.


Assuntos
Aprendizagem da Esquiva/fisiologia , Córtex Cerebral/embriologia , Córtex Cerebral/fisiologia , Transplante de Tecido Fetal , Paladar/fisiologia , Acetilcolina/metabolismo , Animais , Córtex Cerebral/metabolismo , Condicionamento Psicológico/fisiologia , Espaço Extracelular/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Microdiálise , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Transplante Heterotópico
20.
Neurochem Res ; 22(3): 305-12, 1997 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9051666

RESUMO

Glutamate (L-glu) receptors coupled to phosphoinositide hydrolysis in primary cultures of Bergmann cells from chick cerebellum were characterized biochemically and pharmacologically. Both ionotropic and metabotropic receptor agonists stimulated [3H] inositol phosphates accumulation in the following order of potency: QA > NMDA > L-glu > KA approximately QA > AMPA > > t-ACPD. QA showed a biphasic dose-response curve (EC50 = 0.07 and 53 microM), suggesting interaction with two populations of receptors; L-glu was the most efficient agonist. Stimulation by NMDA was blocked by CPP, AP5 and MK-801; that by AMPA and KA was inhibited 100% by CNQX and DNQX, whereas the effect of QA was decreased both by CNQX and the metabotropic antagonist 4-CPG. Stimulation of PIP2 hydrolysis induced by metabotropic L-glu receptor agonist t-ACPD was blocked by 4-CPG but was only moderately inhibited by MCPG. EAA-induced [3H]IPs accumulation was dependent on external Ca2+ and was not affected by nifedipine verapamil, or dantrolene; thapsigargin increased the effect. Results suggest that EAA activate the PI pathway in Bergmann glia through ionotropic (NMDA and AMPA/KA) as well as metabotropic receptor subtypes (t-ACPD) which could act jointly influencing neurotransmission at the parallel fiber-Purkinje cell synapses in the cerebellum.


Assuntos
Cerebelo/efeitos dos fármacos , Ácido Glutâmico/farmacologia , Neuroglia/efeitos dos fármacos , Fosfatidilinositóis/metabolismo , Receptores de Glutamato/efeitos dos fármacos , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Cerebelo/embriologia , Cerebelo/metabolismo , Embrião de Galinha , Cicloleucina/análogos & derivados , Cicloleucina/farmacologia , Hidrólise , Neuroglia/metabolismo , Ácido Quisquálico/farmacologia , Receptores de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Estimulação Química
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