RESUMO
Activation of NMDA receptors (NMDARs) within the CNS represents a major signal for persistent alterations in glutamatergic signaling, such as long-term potentiation (LTP) and long-term depression. NMDARs are composed of a combination of NR1 and NR2 subunits, with distinct NR2 subunits imparting distinct characteristics on the receptor. One particular NR2 subunit, NR2A (NRepsilon1), has been proposed to play an integral role in LTP induction in the hippocampus and cortex. Here, we report studies investigating the role of NR2A in LTP induction in the dorsolateral bed nucleus of the stria terminalis (dlBNST). The putative NR2A-specific inhibitor NVP-AAM077 (AAM077) has been used previously to demonstrate the dependence of cortical and hippocampal LTP on NMDARs containing NR2A subunits. We report here the same sensitivity of LTP to pretreatment with AAM077 (0.4 microm) in the dlBNST. However, inconsistent with the conclusion that LTP in the dlBNST is NR2A dependent, we see intact LTP in the dlBNST of NR2A knock-out mice. Because we also see blockade of this dlBNST LTP in NR2A knock-out mice after pretreatment with AAM077, we conclude that the antagonist is targeting non-NR2A subunit-containing receptors. Using a variety of cultured cell types, we find that AAM077 (0.4 microm) can attenuate transmission of NR2B subunit-containing NMDARs when preapplied rather than coapplied with an agonist. Therefore, we conclude that NR2A is not obligatory for the induction of LTP in the dlBNST. Furthermore, our data demonstrate that care must be exercised in the interpretation of data generated with AAM077 when the compound is applied before an agonist.
Assuntos
Potenciação de Longa Duração/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Linhagem Celular , Humanos , Rim , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Técnicas de Patch-Clamp , Receptores de N-Metil-D-Aspartato/deficiência , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/metabolismo , Núcleos Septais/fisiologia , TransfecçãoRESUMO
Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.
Assuntos
Terapia de Alvo Molecular , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Compostos de Fenilureia/química , Compostos de Fenilureia/farmacologia , Quinolinas/química , Quinolinas/farmacologia , Receptores de N-Metil-D-Aspartato/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Anti-Inflamatórios/sangue , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células CHO , Doença Crônica , Cricetinae , Cricetulus , Células HEK293 , Humanos , Inflamação/tratamento farmacológico , Masculino , Compostos de Fenilureia/sangue , Compostos de Fenilureia/uso terapêutico , Isoformas de Proteínas/metabolismo , Quinolinas/sangue , Quinolinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/sangue , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/farmacologia , Bloqueadores dos Canais de Sódio/uso terapêuticoRESUMO
N-methyl-D-aspartate (NMDA) receptors are important target sites of alcohol action in the central nervous system. Alcohol inhibits NMDA receptor current by an action on ion channel gating, apparently through a direct action on a region of the NMDA receptor accessible from the extracellular environment. Our previous studies have revealed an important role for a methionine residue (Met823) in membrane-associated domain 4 (M4) of the NR2A subunit in channel gating as well as alcohol sensitivity of the NMDA receptor. The role of sites in M4 of the NMDA receptor NR2A subunit adjacent to Met823 was investigated using tryptophan-scanning mutagenesis and electrophysiological recording. Receptors containing NR1 and NR2A(V820W) or NR2A(M817W) mutant subunits expressed in HEK 293 cells were not functional. The mutation Ala826Trp modified apparent desensitization, and the mutations Ala825Trp and Ala826Trp changed the mean open time of the channel as determined by fluctuation analysis. In addition, the mutations Tyr822Trp and Ala825Trp significantly altered the concentration-response curves for ethanol inhibition. The changes in mean open time did not appear to be able to account for the observed differences in ethanol sensitivity. These results indicate that this region in M4 of the NR2A subunit may be involved in the action of alcohol.
Assuntos
Membrana Celular/fisiologia , Etanol/farmacologia , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Linhagem Celular , Membrana Celular/genética , Relação Dose-Resposta a Droga , Humanos , Dados de Sequência Molecular , Estrutura Terciária de Proteína/genética , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores de N-Metil-D-Aspartato/genéticaRESUMO
Previously, we have used CsCl gradient-purified recombinant adenovirus (AdV) to successfully transfer genes into hippocampal neurons cultured on microisland substrate. Here, we report that purification of AdV particles is not required and efficient gene expression can be achieved using either crude AdV lysates or HEK 293 cells infected with AdV. The advantages of the simplified procedure are greatly reduced preparation time and reduced requirements for equipment and expertise.
Assuntos
Adenoviridae/genética , Expressão Gênica/fisiologia , Hipocampo/citologia , Neurônios/metabolismo , Adenoviridae/metabolismo , Animais , Animais Recém-Nascidos , Baclofeno/farmacologia , Células Cultivadas , Interações Medicamentosas , Embrião de Mamíferos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Agonistas GABAérgicos/farmacologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/farmacologia , Técnicas de Transferência de Genes , Vetores Genéticos , Hipocampo/virologia , Humanos , Potenciais da Membrana/genética , Neurônios/virologia , Técnicas de Patch-Clamp , Toxina Pertussis/farmacologia , Ratos , Ratos Sprague-DawleyRESUMO
Long-term potentiation or depression of synaptic function often requires Ca2+ influx via NMDA-type glutamate receptors (NMDARs) and changes in the autophosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII) at Thr286. Autophosphorylated CaMKII binds directly to NMDAR subunits, co-localizes with NMDARs in the postsynaptic density, and phosphorylates NR2B subunits at Ser1303. Here, we demonstrate that CaMKIIalpha enhances the extent and/or rate of desensitization of NMDA-induced macroscopic currents in HEK293 cells co-expressing NR2B with either the NR1(011) or NR1(101) splice variants, without significantly changing other current parameters. In contrast, the extent of desensitization of NMDARs containing NR2A in place of NR2B is significantly decreased by co-expression of CaMKIIalpha. Kinases harboring K42R (inactive kinase) or T286A (autophosphorylation-deficient) mutations are defective in enhancing the desensitization of NR1/NR2B channels. In addition, the CaMKII-dependent enhancement of NR1/NR2B channel desensitization is abrogated by intracellular loading with BAPTA. These data suggest a novel mechanism for Ca2+-dependent negative-feedback regulation of NR2B-containing NMDARs in a CaMKII activity- and autophosphorylation-dependent manner that may modulate NMDAR-mediated synaptic plasticity.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Plasticidade Neuronal/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Linhagem Celular , Eletrofisiologia , Humanos , Rim/citologia , Mutagênese , Fosforilação , Splicing de RNA , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
The N-methyl-d-aspartate (NMDA) subtype of ionotropic glutamate receptor is an important mediator of the behavioral effects of ethanol in the central nervous system. Although ethanol is known to inhibit NMDA receptors by influencing ion-channel gating, its molecular site of action and the mechanism underlying this effect have not been established. We have previously identified a conserved methionine residue in the fourth membrane-associated domain of the NMDA receptor NR2A subunit (Met823) that influences desensitization and gating of the ion channel. Here we report that this residue plays an important role in mediating the effect of ethanol on the NMDA receptor. Ethanol IC50 values among functional substitution mutants at this position varied over the range approximately 130-225 mm. There was a weak correlation between ethanol IC50 and mean open time of NR2A(Met823) mutants that was dependent on inclusion of the value for the tryptophan mutant. In the absence of this value, there was no trend toward a correlation among the remaining mutants. Desensitization appeared to influence the action of ethanol, because ethanol IC50 of the mutants was correlated with the steadystate to peak current ratio. With the exception of tryptophan, ethanol sensitivity was significantly related to the molecular volume and hydrophobicity of the substituent. The relation between ethanol sensitivity and the molecular volume and hydrophobicity at this position suggests that this residue interacts with or forms part of a site of ethanol action and that the presence of a tryptophan residue in this site disrupts its ability to interact with ethanol.
Assuntos
Etanol/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Humanos , Mutação , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
BACKGROUND: It has been suggested that abnormalities seen in fetal alcohol syndrome are linked with NMDA receptor malfunction. Our laboratory has previously shown that prenatal ethanol treatment decreases [3H]MK-801 binding density at postnatal day 21, when NMDA receptor subunit protein levels were unaltered. Thus, the focus of the present study was to examine whether prenatal ethanol modifies native NMDA receptor levels. METHODS: Cerebral cortices were taken from offspring born to three treatment groups of pregnant Sprague Dawley(R) rats: an ethanol group given an ethanol liquid diet during the gestational period, a pair-fed control group that received a liquid diet without ethanol, and an ad libitum group fed rat chow and tap water. Western blot studies were carried out at postnatal days 1, 7, 14, and 21 to examine total protein expression of NR1 and NR1b splice variants. NR2 subunit levels were examined by [3H]MK-801 binding studies using spermidine, an endogenous polyamine, and ifenprodil, a selective NR2B antagonist. RESULTS: [3H]MK-801 binding density was significantly reduced in prenatal ethanol-treated groups compared with ad libitum and pair-fed control groups. Spermidine increased [3H]MK-801 binding, although potentiation by spermidine was not significantly different among all three experimental groups. Furthermore, no significant differences in total protein expression of NR1 or NR1b splice variants were observed in cortical membrane homogenates at postnatal days 1 through 21. [3H]MK-801 binding in the presence of ifenprodil showed that prenatal ethanol treatment significantly decreased low-affinity ifenprodil binding. High-affinity ifenprodil binding was reduced in both pair-fed and ethanol-treated groups. CONCLUSIONS: These results suggest that prenatal ethanol treatment reduces [3H]MK-801 binding and that this reduction may be due to a decrease in NR2A subunits.
Assuntos
Maleato de Dizocilpina/metabolismo , Etanol/farmacologia , Efeitos Tardios da Exposição Pré-Natal , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Processamento Alternativo/efeitos dos fármacos , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Relação Dose-Resposta a Droga , Feminino , Piperidinas/farmacologia , Gravidez , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/biossíntese , Receptores de N-Metil-D-Aspartato/genética , Espermidina/farmacologia , Trítio/metabolismoRESUMO
The N-methyl-d-aspartate (NMDA) receptor has four membrane-associated domains, three of which are membrane-spanning (M1, M3, and M4) and one of which is a re-entrant pore loop (M2). The M1-M3 domains have been demonstrated to influence the function of the ion channel, but a similar role for the M4 domain has not been reported. We have identified a methionine residue (Met(823)) in the M4 domain of the NR2A subunit that regulates desensitization and ion channel gating. A tryptophan substitution at this site did not alter the EC(50) for glycine or the peak NMDA EC(50) but decreased the steady-state NMDA EC(50) and markedly increased apparent desensitization, mean open time, and peak current density. Results of rapid solution exchange experiments revealed that changes in microscopic desensitization rates and closing rates could account for the changes in macroscopic desensitization, steady-state NMDA EC(50), and current density. Other amino acid substitutions at this site could increase or decrease the rate of desensitization and mean open time of the ion channel. Both mean open time and desensitization were dependent primarily upon the hydrophobic character of the amino acid at the position. These results demonstrate an important role for hydrophobic interactions at Met(823) in regulation of NMDA receptor function.