RESUMO
Radioligand binding studies with [(3)H](2E)-(5-hydroxy-5,7,8,9-tetrahydro-6H-benzo[a][7]annulen-6-ylidene) ethanoic acid ([(3)H]NCS-382), an antagonist of gamma-hydroxybutyric acid (GHB) receptor, revealed specific binding sites in the rat cerebral cortex and hippocampus. However, there was very little binding in the rat cerebellum, heart, kidney, liver, and lung membranes. Binding was rapid and reached equilibrium in about 5 min. Scatchard analysis of saturation isotherms revealed two different populations of binding sites in the rat cerebral cortex (K(d1), 795 nM, B(max1), 25.4 pmol/mg of protein; K(d2), 21 microM; B(max2), 178 pmol/mg of protein) as well as in the rat hippocampus (K(d1), 441 nM; B(max1), 16.2 pmol/mg of protein; K(d2), 9.8 microM; B(max2), 255 pmol/mg of protein). (+/-)Baclofen (500 microM) and gamma-aminobutyric acid (100 microM) inhibited the binding only partially, whereas (+)bicuculline, muscimol, picrotoxinin, and phaclofen did not modify the binding. Interestingly, potassium chloride (100-300 mM) inhibited [(3)H]NCS-382 binding (34-56%), and this inhibitory effect was not affected by picrotoxinin. GHB and NCS-382 completely inhibited the [(3)H]NCS-382 (16 nM) binding in the rat cerebrocortical and hippocampal membranes, and NCS-382 was found to be about 10 times more potent than GHB in this regard. A variety of ligands for other receptors did not modify the [(3)H]NCS-382 binding, thereby suggesting selectivity of this radioligand for the GHB receptor sites in the brain. Based on these observations, [(3)H]NCS-382 seems to be a better radioligand than [(3)H]GHB for investigating the role of the GHB receptors in various pharmacological actions.
Assuntos
Benzocicloeptenos/farmacologia , Encéfalo/efeitos dos fármacos , Receptores de Superfície Celular/antagonistas & inibidores , Animais , Anticonvulsivantes/farmacologia , Bicuculina/farmacologia , Ligação Competitiva , Encéfalo/metabolismo , Interações Medicamentosas , Masculino , Muscimol/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/metabolismo , TrítioRESUMO
This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Larry P. Gonzalez. The presentations were (1) EEG indices of sensitization in a murine model of repeated ethanol withdrawals, by Lynn M. Veatch; (2) Long-term changes in central nervous system function after repeated alcohol withdrawals: Recommendations for the treatment of acute withdrawal, by Larry P. Gonzalez; (3) Differential regulation of GABAA and NMDA receptors by repeated ethanol treatment in cultured mammalian neurons, by Maharaj K. Ticku; and (4) Involvement of GABAA and NMDA receptors in alcohol withdrawal kindling: Implications for treatment, by Howard C. Becker.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Eletroencefalografia/efeitos dos fármacos , Etanol/farmacologia , Excitação Neurológica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Células Cultivadas , Depressores do Sistema Nervoso Central/efeitos adversos , Etanol/efeitos adversos , Humanos , Excitação Neurológica/fisiologia , Camundongos , Neurônios/fisiologia , Receptores de GABA-A/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Síndrome de Abstinência a Substâncias/terapiaRESUMO
Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) inhibited the binding of [(3)H]flunitrazepam (2 nM), [(3)H]muscimol (5 nM) and 4 nM [(35)S]t-butylbicyclophosphorothionate [(35)S]TBPS in the rat cerebellum as well as cerebral cortex. DHEAS-induced inhibition of binding of these radioligands (62% to 100%) was more pronounced as compared to that in the case of DHEA (5% to 31%). DHEAS, unlike DHEA, inhibited [(3)H]flunitrazepam binding significantly to a lesser extent in the cerebellum of ethanol-dependent rats as compared to the control group (I(max):82+/-1vs.92+/-2%, p<0.005). However, DHEA, unlike DHEAS, inhibited [(35)S]TBPS binding to a greater extent in the ethanol-dependent rat cerebellum as compared to the control group (I(max):31+/-2vs.19+/-2%, p<0.005). Furthermore, DHEA was more potent in inhibiting [(35)S]TBPS binding in the cerebellum (IC(50):55+/-5 vs. 74+/-7 microM, p<0.05) and cerebral cortex (IC(50):26+/-4vs.64+/-9 microM, p<0.05) of ethanol-dependent rats as compared to the control group. These observations indicate that unsulfated and sulfated androstane-steroids modulate the GABA(A) receptors in the control as well as the ethanol-dependent rats differentially, and also suggest that the androstane-steroid binding sites associated with the GABA(A) receptors play an important role during ethanol dependence.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Esteroides/farmacologia , Animais , Ansiolíticos/farmacocinética , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Depressores do Sistema Nervoso Central/efeitos adversos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Desidroepiandrosterona/farmacologia , Sulfato de Desidroepiandrosterona/farmacologia , Etanol/efeitos adversos , Flunitrazepam/farmacocinética , Agonistas GABAérgicos/metabolismo , Muscimol/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
This protocol describes an in vitro assay for characterization of the picrotoxin site of GABA(A) receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics and competition assays (IC50, Ki determination) are included in the protocol.
Assuntos
Química Encefálica , Picrotoxina/análise , Ensaio Radioligante/métodos , Receptores de GABA-A/análise , Ácido gama-Aminobutírico/análise , Animais , Sítios de Ligação , Ligação Competitiva , Compostos Bicíclicos com Pontes , Compostos Bicíclicos Heterocíclicos com Pontes , Cinética , Membranas/química , Membranas/metabolismo , Nitrilas , Picrotoxina/metabolismo , Ratos , Receptores de GABA-A/metabolismo , Manejo de Espécimes , Estatística como Assunto/métodos , Radioisótopos de Enxofre , Ácido gama-Aminobutírico/metabolismoRESUMO
NMDA receptors are glutamate-gated ion channels, mediating excitatory neurotransmission in the brain. These widely distributed receptors are known to play a role in neuronal development and synaptic plasticity, but over stimulation of these receptors can lead to neurotoxicity. In recent years, NMDA receptors have emerged as an important site of action of ethanol. It is believed that at least some of the deleterious effects of ethanol like alcohol dependence, development of tolerance to alcohol and alcohol withdrawal syndrome are mediated via NMDA receptors. The sensitivity of NMDA receptors to ethanol, however, varies regionally. This diversity of NMDA receptor sensitivity is believed to result, at least in part, from heterogeneity of receptor subunit composition. Ethanol's effects on NMDA receptors, including alteration in receptor function and number, probably result from interplay of multiple mechanisms some of which are discussed here.
Assuntos
Etanol/toxicidade , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Encéfalo/efeitos dos fármacos , Humanos , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Chronic administration of Nomega-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, diminished the ability of 5alpha-pregnan-3alpha-ol-20-one, a neurosteroid, to potentiate the [3H]muscimol (5 nM) binding in the rat hippocampus but not in the cerebellum or cerebral cortex. This effect of NAME was stereospecific and susceptible to reversal by the pre-treatment of rats with L-arginine. However, chronic administration of L-NAME did not affect the modulation of the [3H]flunitrazepam (2 nM) or [35S]TBPS (4 nM) binding by the neurosteroid in any of the brain regions investigated in this study. These results suggest that nitric oxide may be involved in some of the effects of neurosteroids in hippocampus.
Assuntos
Encéfalo/efeitos dos fármacos , Moduladores GABAérgicos/farmacologia , Óxido Nítrico/fisiologia , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Animais , Arginina/farmacologia , Encéfalo/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , NG-Nitroarginina Metil Éster/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-DawleyRESUMO
There is evidence that phosphorylation plays a crucial role in the regulation of the NMDA receptors in the brain. In this study we examined the effect of acute and chronic ethanol treatment on the phosphorylation of the R2B subunit of the NMDA receptors in fetal cortical neurons. Additionally, the effect of acute ethanol treatment on the phosphorylation of the R2B subunit in adult cerebral cortex and hippocampus was also examined. The results show that acute or chronic ethanol treatments did not affect the total phosphorylation of the R2B subunit in cortical neurons. In adult mice, we observed that acute ethanol treatment increased the tyrosine phosphorylation of the R2B subunit in hippocampus but not in cerebral cortex. We also observed that acute or chronic ethanol treatments did not alter the Fyn or Csk levels in cortical neurons. Although Fyn, but not Csk, was present in adult cerebral cortex, ethanol did not phosphorylate the R2B subunit in this region. Like ethanol, MK-801 (NMDA antagonist) did not affect the phosphorylation of the R2B subunit in cortical neurons. Taken together, these results suggest that acute and chronic ethanol and MK-801 treatments do not affect the phosphorylation of the R2B subunit in fetal cortical neurons and adult cerebral cortex. Based on these observations, we speculate that the R2B subunit of the NMDA receptors is regulated by multiple cascades and in a brain region specific manner.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Recent advances in molecular biology and complementary information derived from neuropharmacology, biochemistry and behavior have dramatically increased our understanding of various aspects of GABAA receptors. These studies have revealed that the GABAA receptor is derived from various subunits such as alpha1-alpha6, beta1-beta3, gamma1-gamma3, delta, epsilon, pi, and rho1-3. Furthermore, two additional subunits (beta4, gamma4) of GABAA receptors in chick brain, and five isoforms of the rho-subunit in the retina of white perch (Roccus americana) have been identified. Various techniques such as mutation, gene knockout and inhibition of GABAA receptor subunits by antisense oligodeoxynucleotides have been used to establish the physiological/pharmacological significance of the GABAA receptor subunits and their native receptor assemblies in vivo. Radioligand binding to the immunoprecipitated receptors, co-localization studies using immunoaffinity chromatography and immunocytochemistry techniques have been utilized to establish the composition and pharmacology of native GABAA receptor assemblies. Partial agonists of GABAA receptors are being developed as anxiolytics which have fewer and less severe side effects as compared to conventional benzodiazepines because of their lower efficacy and better selectivity for the GABAA receptor subtypes. The subunit requirement of various drugs such as anxiolytics, anticonvulsants, general anesthetics, barbiturates, ethanol and neurosteroids, which are known to elicit at least some of their pharmacological effects via the GABAA receptors, have been investigated during the last few years so as to understand their exact mechanism of action. Furthermore, the molecular determinants of clinically important drug-targets have been investigated. These aspects of GABAA receptors have been discussed in detail in this review article.
Assuntos
Receptores de GABA-A/fisiologia , Animais , Agonistas de Receptores de GABA-A , Antagonistas de Receptores de GABA-A , Humanos , Receptores de GABA-A/efeitos dos fármacosRESUMO
The anti-alpha1 antibody elicited higher immunoprecipitation (%) values of the [3H]flunitrazepam and [3H]muscimol binding activity in the rat cerebellum vs. cerebral cortex, whereas immunoprecipitation values for [3H]Ro 15-4513 and [3H]zolpidem were comparable in these brain regions. Chronic ethanol administration neither changed the radioligand binding to the immunoprecipitated pellet nor the percentage immunoprecip-itation values, thereby indicating that chronic ethanol did not result in down-regulation of the GABAA receptor assemblies containing alpha1-subunit.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Cerebelo/química , Córtex Cerebral/química , Etanol/farmacologia , Receptores de GABA-A/análise , Marcadores de Afinidade/farmacologia , Alcoolismo/metabolismo , Animais , Azidas/farmacologia , Benzodiazepinas/farmacologia , Química Encefálica/efeitos dos fármacos , Doença Crônica , Flunitrazepam/farmacologia , Agonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacologia , Hipnóticos e Sedativos/farmacologia , Masculino , Muscimol/farmacologia , Testes de Precipitina , Piridinas/farmacologia , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Trítio , ZolpidemRESUMO
The aim of this study was to study the potential mechanism(s) involved in the antagonist induced upregulation of the N-methyl-d-aspartate receptor (NMDA) NR2B subunit. The results show that chronic treatment of cortical neurons with tyrosine kinase inhibitor (genistein) resulted in downregulation of the NR2B subunit polypeptide levels, while daidzein, an inactive analog of genistein, did not alter the levels of NR2B subunit, implying that tyrosine kinases may be involved in the regulation of the NMDA NR2B subunit content. Chronic treatment of cortical neurons with the NMDA receptor antagonist, (+)-5-methyl-10,11-dihydro-5H-dibenzo[a, d]cycloheptane-5,10-iminemaleate (MK-801) enhanced the membrane associated tyrosine kinase activity and upregulated the NR2B receptor subunit. These results suggest that MK-801 induced upregulation of NMDA (NR2B) receptor subunit might be mediated by tyrosine kinases.
Assuntos
Neurônios/química , Neurônios/enzimologia , Proteínas Tirosina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Células Cultivadas , Córtex Cerebral/citologia , Maleato de Dizocilpina/farmacologia , Inibidores Enzimáticos/farmacologia , Estrogênios não Esteroides/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Genisteína/farmacologia , Isoflavonas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Gravidez , Frações Subcelulares/enzimologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
In this study, we investigated the modulatory effect of 5alpha-pregnan-3alpha-ol-20-one, a neurosteroid, on the binding characteristics of [3H]flunitrazepam (2 nM), [3H]muscimol (5 nM), and 4 nM [35S]t-butylbicyclophosphorothionate (TBPS) in cerebral cortex, cerebellum, and hippocampus of control, ethanol-dependent, and ethanol-withdrawn rats. 5alpha-Pregnan-3alpha-ol-20-one potentiated the binding of [3H]flunitrazepam and [3H]muscimol in all the rat brain regions investigated in this study. There was a significant increase in the maximal potentiation of [3H]flunitrazepam as well as [3H]muscimol binding (Emax) in the ethanol-dependent rat cerebellum as compared to control group (p<0. 025). Furthermore, 5alpha-pregnan-3alpha-ol-20-one elicited a biphasic response, i.e., it potentiated the binding of [35S]TBPS at lower concentrations (<=100 nM) and inhibited the binding at higher concentrations (>100 nM). There was a significant higher inhibition of [35S]TBPS binding (-Emax) by 5alpha-pregnan-3alpha-ol-20-one in the hippocampus of ethanol-dependent as well as ethanol-withdrawn rats (p<0.025). These observations suggest that the neurosteroid binding site associated with the gamma-aminobutyric acidA (GABAA) receptors in cerebellum and hippocampus plays an important role during ethanol-dependence and ethanol-withdrawal, and some of the changes following ethanol dependence and its withdrawal may be mediated through the neurosteroid binding site.
Assuntos
Encéfalo/metabolismo , Etanol/administração & dosagem , Moduladores GABAérgicos/farmacologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Cerebelo/metabolismo , Córtex Cerebral/metabolismo , Etanol/farmacologia , Flunitrazepam/metabolismo , Agonistas GABAérgicos/metabolismo , Moduladores GABAérgicos/metabolismo , Hipocampo/metabolismo , Masculino , Muscimol/metabolismo , Pregnanolona/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We investigated the effect of chronic ethanol administration and its withdrawal on the polypeptide levels of NMDA receptor subunits such as NR1, NR2A, and NR2B in the rat cerebral cortex and hippocampus using Western blot analysis technique. Our results indicate that chronic ethanol treatment upregulates NMDA receptor subunits NR1, NR2A, and NR2B ( approximately 35%). At 48 h of last dose of ethanol administration, the protein content returned to almost control level, thereby demonstrating the reversibility of the changes.
Assuntos
Alcoolismo/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Regulação para Cima , Alcoolismo/genética , Animais , Substâncias Macromoleculares , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biossíntese , Receptores de N-Metil-D-Aspartato/química , Síndrome de Abstinência a SubstânciasRESUMO
It is widely accepted that calorie restriction is an effective way of delaying the aging process. Also, there is an indication that the beneficial effects exerted by dietary manipulation may be due to a direct effect at the molecular level like gene expression. The studies were conducted to determine whether calorie restriction prevents any age-related changes in the structural and molecular aspects of the GABAA-BZ receptor. In aged (24-month old diet ad libitum) rats, the binding of [35S]t-butyl-bicyclophosphorothionate (TBPS) was significantly reduced in the cerebellum. In contrast, [35S]TBPS binding remained unchanged in the cerebellum of calorie restricted old rats. In order to evaluate the molecular basis of these changes, the alpha sub-unit mRNA levels were measured. The GABAA receptor alpha1 sub-unit mRNA level remained unchanged in both the old groups of rats. The alpha2 subunit mRNA level was significantly decreased in the cerebellum of aged rats (24-month old ad libitum), whereas it remained unchanged in the cerebellum of calorie restricted old animals. These findings indicate a selective age and diet related modulation in the stoichiometry of the GABAA receptor in aging.
Assuntos
Envelhecimento/metabolismo , Cerebelo/metabolismo , Ingestão de Energia , Regulação da Expressão Gênica/fisiologia , Receptores de GABA-A/genética , Animais , Córtex Cerebral/metabolismo , Masculino , RNA Mensageiro/biossíntese , Ratos , Ratos Endogâmicos F344 , Receptores de GABA-A/metabolismoRESUMO
Previous studies have shown that chronic ethanol treatment up-regulates the expression of the N-methyl-D-aspartate (NMDA) receptor number and function both in vitro and in vivo. In vitro chronic ethanol treatment specifically augments mRNA levels of the R2B subunit without altering R1 subunit mRNA levels, although similar treatment results in increased levels of both R1 and R2B polypeptides. To further delineate the molecular mechanisms involved in differential regulation of NMDA receptor subunits by chronic ethanol treatment (50 mM, 5 days), we have determined the mRNA stability of the NMDA R1 and R2B subunits and the transcription rate of the respective genes using mouse fetal cortical neurons. Our observations demonstrated that ethanol stabilized the NMDA R1 mRNA over the time period examined (24 h) without altering the stability of the R2B mRNA. Chronic exposure of fetal cortical neurons to ethanol enhanced the rate of R2B gene transcription approximately twofold. Taken together, these results suggest that up-regulation of the NMDA receptor number seen in cultured cortical neurons after chronic ethanol treatment is due to the regulation of the NMDA R2B receptor subunit at the transcriptional level and that of the NMDA R1 subunit mainly at the posttranscriptional level.
Assuntos
Córtex Cerebral/fisiologia , Etanol/farmacologia , Feto/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/genética , Animais , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Estabilidade de Medicamentos , Feto/citologia , Feto/metabolismo , Camundongos/embriologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/química , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The purpose of this study of GT1-7 neurons was to partially characterize basal Cl- transport and GABAA mediated Cl- efflux and to test the effect of ethanol on a GABAA receptor that lacks a gamma subunit. We measured GABAA function and Cl- transport with 36Cl-. Our results show that basal 36Cl- efflux varied with temperature at 4 degrees C, 23 degrees C, and 37 degrees C. At 23 degrees C, DIDS, an inhibitor of anion exchange, reduced basal 36Cl- efflux maximally by 79.6% with an IC50 of 42.1 microM, whereas bumetanide, an inhibitor of (Na-K-Cl) cotransport, had no effect on basal 36Cl- efflux at concentrations up to 150 microM. At 4 degrees C, muscimol, a GABAA receptor agonist, stimulated 36Cl- efflux with an EC50 of 1.47 microM. Bicuculline, a GABAA receptor antagonist, completely reversed the effect of 20 microM muscimol with an IC50 of 6.08 microM. Ethanol, at concentrations up to 87 mM (0.4% (w/v)), had no effect on muscimol-induced 36Cl- efflux at 4 degrees C or at 32 degrees C. Our results indicate that stimulation of GABAA receptors causes an efflux of Cl- from GT1-7 neurons. This finding is consistent with the concept that stimulation of GABAA receptors produces depolarization of the plasma membrane, increase in cytosolic [Ca2+], and GnRH release. Our results represent the first description of chloride transport in GT1-7 neurons and suggest the presence of a Cl- exchange, but not (Na-K-Cl), transporter mechanism. Furthermore, the lack of an effect of ethanol observed in this study is consistent with the idea that a gamma 2L subunit may be necessary for the effects of low concentrations of ethanol at GABAA receptors.
Assuntos
Estimulantes do Sistema Nervoso Central/farmacologia , Cloretos/metabolismo , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Animais , Bicuculina/farmacologia , Bumetanida/farmacologia , Células Cultivadas , Diuréticos/farmacologia , Agonistas GABAérgicos/farmacologia , Antagonistas GABAérgicos/farmacologia , Moduladores GABAérgicos/farmacologia , Camundongos , Muscimol/farmacologia , Neurônios/química , Neurônios/citologia , Pentobarbital/farmacologia , Receptores de GABA-A/química , TemperaturaRESUMO
Chronic ethanol exposure has been reported to alter NMDA and GABA(A) receptor function and gene expression in brain regions of animals and mammalian cultured cortical neurons. In the present study, we investigated the effects of another model of chronic, but intermittent, ethanol treatment (CIE) on GABA(A) and NMDA receptor systems in cortical neurons. CIE (50 mM ethanol, 12 h exposure/12 h withdrawal, 5 cycles) exposure produced increased [3H]MK-801 binding and diazepam insensitive binding sites as measured by [3H]Ro15-4513 binding to cortical cultured neuronal membranes, at 0 h following the last treatment cycle relative to control neurons. The NMDA mediated increase in intracellular calcium [Ca2+]i was also increased following similar CIE treatment. CIE treatment also increased the ability of pentylenetetrazol (PTZ) to inhibit GABA mediated 36Cl- influx relative to control neurons. These effects were not reversible following 1 week ethanol withdrawal, implying enhanced sensitivity of PTZ to inhibit GABA(A) receptor mediated inhibition, and an increased NMDA receptor function in CIE treated cortical neurons. These alterations are consistent with the behavioral studies in animals, and suggest that both GABA(A) and NMDA receptors play an important role in ethanol withdrawal following either chronic or CIE exposure. Furthermore, this provides a feasible in vitro model for further biochemical and molecular studies of the mechanism underlying the CIE induced kindling-like phenomenon observed in humans.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Etanol/efeitos adversos , Excitação Neurológica , Neurônios/efeitos dos fármacos , Síndrome de Abstinência a Substâncias , Animais , Células Cultivadas , Córtex Cerebral/citologia , Diazepam/farmacologia , Esquema de Medicação , Etanol/administração & dosagem , Feminino , Moduladores GABAérgicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Pentilenotetrazol/farmacologiaRESUMO
The interactions of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) were investigated with various binding sites of the gamma-aminobutyric acid (GABA(A)) receptor complex to rat brain membranes, and on GABA-induced [36Cl-] influx in mammalian cortical cultured neurons. DHEAS and DHEA did not affect the binding of [3H]flunitrazepam to the benzodiazepine binding sites. In contrast, DHEAS, but not DHEA, inhibited the binding of [3H]GABA and [35S]TBPS to rat brain cerebral cortical and cerebellar membranes in a concentration-dependent manner. DHEAS decreased the Bmax values of both the high and low affinity GABA receptor binding sites without affecting their affinity constants. In contrast, DHEAS inhibited [35S]TBPS binding competitively, as analyzed by Scatchard analysis. In dissociation kinetic studies, DHEAS dissociated [35S]TBPS from rat cerebral cortical membranes in a monophasic pattern that was similar to that observed with inhibitors of GABA(A) receptors such as TBPS and picrotoxin but different from pentobarbital and GABA. Taken together, these results suggest that DHEAS binds to the TBPS/picrotoxin site of the GABA(A) receptor complex, and this interaction may be responsible for the noncompetitive inhibition of GABA responses observed with DHEAS. Furthermore, we confirmed that DHEAS inhibits GABA responses, as measured by GABA-induced [36Cl-] influx in cultured cortical neurons. Studies with DHEA indicate that this neurosteroid does not interact with the GABA(A) receptor complex.
Assuntos
Sulfato de Desidroepiandrosterona/metabolismo , Picrotoxina/metabolismo , Receptores de GABA-A/metabolismo , Animais , Ligação Competitiva , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cloretos/metabolismo , Desidroepiandrosterona/farmacologia , Sulfato de Desidroepiandrosterona/farmacologia , Técnicas In Vitro , Transporte de Íons , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Ácido gama-Aminobutírico/metabolismoRESUMO
We have recently shown that chronic neurosteroid, 5 alpha 3 alpha, treatment produced down-regulation of the GABA receptor binding and function, and heterologous uncoupling on the GABAA receptor complex in cultured mammalian cortical neurons. In order to explore the underlying mechanism of these observed down-regulation and heterologous uncoupling phenomenon, we investigated the effect of chronic 5 alpha 3 alpha (1 microM; 5 days) treatment on the GABAA receptor subunits mRNA levels, using RNase protection assay. We found that chronic neurosteroid, 5 alpha 3 alpha, treatment decreased the beta- and alpha-subunits mRNA levels while not altering the gamma 2S-subunit mRNA levels in the cortical neurons. The decrease in the beta-subunits mRNA levels suggests a decrease in the presence of the beta-subunits in the composition of GABAA receptors. This phenomenon may explain the down-regulation of the GABAA receptor binding and function. A decrease in the alpha 3-subunit mRNA level suggests a corresponding decrease in the alpha 3-subunit in the composition of GABAA receptor isoforms, relative to other isoforms. This observation may be responsible for the chronic neurosteroid-induced uncoupling and decreased efficacy. In summary, chronic 5 alpha 3 alpha treatment produced down-regulation of the GABAA receptor beta- and alpha-subunit mRNA levels, and these changes may be associated with the down-regulation, heterologous uncoupling, and decreased efficacy of GABAA receptor complex in the cultured mammalian cortical neurons.
Assuntos
Córtex Cerebral/citologia , Regulação para Baixo/efeitos dos fármacos , Moduladores GABAérgicos/farmacologia , Proteínas do Tecido Nervoso/biossíntese , Neurônios/efeitos dos fármacos , Pregnanolona/farmacologia , RNA Mensageiro/biossíntese , Receptores de GABA-A/biossíntese , Isomerases de Aminoácido/biossíntese , Isomerases de Aminoácido/genética , Animais , Proteínas de Transporte/biossíntese , Proteínas de Transporte/genética , Células Cultivadas , Feminino , Moduladores GABAérgicos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Neurônios/metabolismo , Peptidilprolil Isomerase , Pregnanolona/administração & dosagem , Conformação Proteica , RNA Mensageiro/genética , Receptores de GABA-A/química , Receptores de GABA-A/genéticaRESUMO
The present study was designed to determine the effects of chronic neonatal exposure to the NMDA receptor antagonist phencyclidine (PCP) on [3H]MK-801 binding and on gene expression of NMDA receptor subunits in juvenile male rats. Rat pups were injected daily with PCP from day 5 to 15 and killed on day 21. [3H]MK-801 binding was measured by quantitative autoradiography. A sensitive RNase protection assay was employed to determine simultaneously the mRNA levels of NR1 subunit (comprising all different splice variants) and three NR2 subunits (NR2A-NR2C). The relative distribution profile of NMDA receptor subunits in the cerebral cortex was NR2B > NR1 > NR2A > NR2C and in the cerebellum NR2C = NR1 > NR2A = NR2B. Chronic PCP administration in postnatal rats produced significant reduction in both [3H]MK-801 binding and mRNA level of the NR2B subunit in the cerebral cortex. Expression of the other NMDA receptor subunits in the cerebral cortex did not change following the drug treatment. In the cerebellum, neither [3H]MK-801 binding nor any of the NMDA receptor subunit expression levels showed any alteration. Together, these data provide a molecular correlate for chronic postnatal PCP-induced down-regulation of [3H]MK-801 binding in rat cerebral cortex and suggest that the NR2B subunit plays an important role in developmental plasticity.