RESUMO
The cellular mechanisms underlying picrotoxin-induced convulsive activity were studied by using mouse spinal neurons growing in tissue culture. Picrotoxin-induced convulsive activity in most but not all of the cells studied. The activity could be inverted by polarizing to positive potentials and eliminated either by decreasing the ratio of calcium to magnesium or by applying tetrodotoxin. When applied locally to individual cells, picrotoxin lowered spike threshold and induced spontaneous firing in some but not all cells tested. The results suggest that picrotoxin-induced convulsive activity involves rapidly summating synaptic activity which may be evoked by high-frequency repetitive firing.
Assuntos
Picrotoxina/farmacologia , Convulsões/induzido quimicamente , Medula Espinal/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/farmacologia , Células Cultivadas , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Medula Espinal/fisiologiaRESUMO
In the mammalian central nervous system, receptors for excitatory amino acid neurotransmitters such as the alpha-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA)-kainate receptor mediate a large fraction of excitatory transmission. Currents induced by activation of the AMPA-kainate receptor were potentiated by agents that specifically stimulate adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A (PKA) activity or were supported by intracellular application of the catalytic subunit of PKA by itself or in combination with cAMP. Furthermore, depression of these currents by a competitive inhibitor of PKA indicates that AMPA-kainate receptors are regulated by endogenous PKA. Endogenous protein phosphatases also regulate these receptors because an inhibitor of cellular phosphates enhanced kainate currents. Modulation of PKA and phosphatases may regulate the function of these receptors and thus contribute to synaptic plasticity in hippocampal neurons.
Assuntos
Éteres Cíclicos/farmacologia , Hipocampo/fisiologia , Ácido Caínico/metabolismo , Neurônios/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismo , Receptores de Neurotransmissores/fisiologia , Animais , Células Cultivadas , AMP Cíclico/farmacologia , AMP Cíclico/fisiologia , Feto , Homeostase , Cinética , Substâncias Macromoleculares , Potenciais da Membrana/efeitos dos fármacos , Camundongos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Ácido Okadáico , Inibidores de Proteínas Quinases , Receptores de Ácido Caínico , Receptores de Neurotransmissores/efeitos dos fármacosRESUMO
Mouse spinal neurons grown in tissue culture were used to study the membrane effects of the benzodiazepine flurazepam and the naturally occurring purine nucleoside inosine, which competes for benzodiazepine receptor sites in the central nervous system. Application of inosine elicited two types of transmitter-like membrane effects: a rapidly desensitizing excitatory response and a nondesensitizing inhibitory response. Flurazepam produced a similar excitatory response which showed cross-desensitization with the purine excitation. Flurazepam also blocked the inhibitory inosine response. The results provide electrophysiological evidence that an endogenous purine can activate two different conductances on spinal neurons and that flurazepam can activate one of the conductances and antagonize the other.
Assuntos
Benzodiazepinas/metabolismo , Inosina/metabolismo , Receptores de Droga/metabolismo , Medula Espinal/metabolismo , Animais , Células Cultivadas , Condutividade Elétrica , Flurazepam/antagonistas & inibidores , Inosina/farmacologia , Ligantes , Camundongos , Neurotransmissores/metabolismo , Receptores de Neurotransmissores/metabolismoRESUMO
The efficiency with which N-methyl-D-aspartate receptors (NMDARs) trigger intracellular signaling pathways governs neuronal plasticity, development, senescence, and disease. In cultured cortical neurons, suppressing the expression of the NMDAR scaffolding protein PSD-95 (postsynaptic density-95) selectively attenuated excitotoxicity triggered via NMDARs, but not by other glutamate or calcium ion (Ca2+) channels. NMDAR function was unaffected, because receptor expression, NMDA currents, and 45Ca2+ loading were unchanged. Suppressing PSD-95 blocked Ca2+-activated nitric oxide production by NMDARs selectively, without affecting neuronal nitric oxide synthase expression or function. Thus, PSD-95 is required for efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to excitotoxic Ca2+ signaling.
Assuntos
Cálcio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Óxido Nítrico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Canais de Cálcio/metabolismo , Sobrevivência Celular , Células Cultivadas , Proteína 4 Homóloga a Disks-Large , Ativação Enzimática , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , N-Metilaspartato/toxicidade , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico Sintase Tipo I , Núcleosídeo-Fosfato Quinase/metabolismo , Oligodesoxirribonucleotídeos Antissenso , Técnicas de Patch-Clamp , Sistemas do Segundo Mensageiro , Transdução de SinaisRESUMO
Ligand-gated ion channels gated by glutamate constitute the major excitatory neurotransmitter system in the mammalian brain. The functional modulation of GluR6, a kainate-activated glutamate receptor, by adenosine 3',5'-monophosphate-dependent protein kinase A (PKA) was examined with receptors expressed in human embryonic kidney cells. Kainate-evoked currents underwent a rapid desensitization that was blocked by lectins. Kainate currents were potentiated by intracellular perfusion of PKA, and this potentiation was blocked by co-application of an inhibitory peptide. Site-directed mutagenesis was used to identify the site or sites of phosphorylation on GluR6. Although mutagenesis of two serine residues, Ser684 and Ser666, was required for complete abolition of the PKA-induced potentiation, Ser684 may be the preferred site of phosphorylation in native GluR6 receptor complexes. These results indicate that glutamate receptor function can be directly modulated by protein phosphorylation and suggest that a dynamic regulation of excitatory receptors could be associated with some forms of learning and memory in the mammalian brain.
Assuntos
Encéfalo/fisiologia , Ácido Caínico/farmacologia , Proteínas Quinases/metabolismo , Receptores de Glutamato/fisiologia , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Concanavalina A/farmacologia , Potenciais Evocados/efeitos dos fármacos , Humanos , Rim , Cinética , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Receptores de Glutamato/efeitos dos fármacos , Receptores de Glutamato/genética , Receptores de Ácido Caínico , Serina , Aglutininas do Germe de Trigo/farmacologiaRESUMO
Long-term potentiation (LTP) of excitatory transmission in the hippocampus likely contributes to learning and memory. The mechanisms underlying LTP at these synapses are not well understood, although phosphorylation and redistribution of AMPA receptors may be responsible for this form of synaptic plasticity. We show here that miniature excitatory postsynaptic currents (mEPSCs) in cultured hippocampal neurons reliably demonstrate LTP when postsynaptic NMDA receptors are briefly stimulated with glycine. LTP of these synapses is accompanied by a rapid insertion of native AMPA receptors and by increased clustering of AMPA receptors at the surface of dendritic membranes. Both LTP and glycine-facilitated AMPA receptor insertion are blocked by intracellular tetanus toxin (TeTx), providing evidence that AMPA receptors are inserted into excitatory synapses via a SNARE-dependent exocytosis during LTP.
Assuntos
Potenciação de Longa Duração/fisiologia , Neurônios/metabolismo , Receptores de AMPA/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Animais , Cálcio/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Glicina/metabolismo , Glicina/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Fusão de Membrana/efeitos dos fármacos , Camundongos , Neurônios/citologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidoresRESUMO
Long-term potentiation (LTP) is an activity-dependent enhancement of synaptic efficacy, considered a model of learning and memory. The biochemical cascade producing LTP requires activation of Src, which upregulates the function of NMDA receptors (NMDARs), but how Src becomes activated is unknown. Here, we show that the focal adhesion kinase CAKbeta/Pyk2 upregulated NMDAR function by activating Src in CA1 hippocampal neurons. Induction of LTP was prevented by blocking CAKbeta/Pyk2, and administering CAKbeta/Pyk2 intracellularly mimicked and occluded LTP. Tyrosine phosphorylation of CAKbeta/Pyk2 and its association with Src was increased by stimulation that produced LTP. Finally, CAKbeta/Pyk2-stimulated enhancement of synaptic AMPA responses was prevented by blocking NMDARS, chelating intracellular Ca(2+), or blocking Src. Thus, activating CAKbeta/Pyk2 is required for inducing LTP and may depend upon downstream activation of Src to upregulate NMDA receptors.
Assuntos
Potenciação de Longa Duração/fisiologia , Proteínas Tirosina Quinases/metabolismo , Células Piramidais/fisiologia , Receptores de AMPA/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Quinases da Família src/fisiologia , Animais , Quinase 2 de Adesão Focal , Hipocampo/fisiologia , Masculino , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologia , Regulação para Cima/fisiologia , Quinases da Família src/metabolismoRESUMO
Small-scale clinical trials show that treatment of cystic fibrosis (CF) patients with ibuprofen, a nonsteroidal anti-inflammatory drug, improves the symptoms of CF and slows down the decline of lung function. Paradoxically, ibuprofen inhibits ligand-stimulated CF transmembrance conductance regulator (CFTR) activity. The aim of the present study was to investigate the effects of ibuprofen on CFTR function under different conditions. Patch-clamp recordings were performed in two lines of human airway epithelial cells: IB3-8-3-7 cells, which express wild-type CFTR; and IB3-1 cells, which express the variant CFTR with deletion of phenylalanine 580 (DeltaF580CFTR). Addition of ibuprofen to the extracellular solution caused a rapid inhibition of CFTR activity in IB3-8-3-7 cells in the presence of a high intracellular concentration of cAMP, whereas ibuprofen enhanced the CFTR conductance at low levels of cAMP. Introducing ibuprofen into the interior of cells occluded the enhancing effect of ibuprofen. Notably, the variant CFTR-mediated conductance was detected in IB3-1 cells treated with myoinositol and was enhanced by ibuprofen at endogenous levels of cAMP. In summary, nonsteroidal anti-inflammatory drugs increase the function of both wild-type cystic fibrosis transmembrane conductance regulator and the phenylalanine 580 deletion in cultured human airway epithelial cells at endogenous levels of cAMP.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Epitélio/microbiologia , Pulmão/microbiologia , Mutação , Regulação para Cima , Linhagem Celular , AMP Cíclico/metabolismo , Epitélio/metabolismo , Humanos , Ibuprofeno/farmacologia , Pulmão/efeitos dos fármacos , Pulmão/patologia , Modelos Biológicos , Técnicas de Patch-Clamp , Receptores de GABA/metabolismoRESUMO
In central neurons, the second messenger cGMP is believed to induce long-term changes in efficacy at glutamatergic synapses through activation of protein kinase G (PKG). Stimulating nitric oxide synthase, activating soluble guanylyl cyclase or elevating concentrations of intracellular cGMP depressed excitatory synaptic transmission in CA1 hippocampal neurons. Unexpectedly, intracellular cGMP depressed responses of AMPA receptors and inhibited excitatory postsynaptic currents in hippocampal neurons independently of phosphorylation. Our findings demonstrate that cGMP's modulation of excitatory transmission may involve a coupling of AMPA channel activity to levels of cGMP.
Assuntos
GMP Cíclico/metabolismo , Inibição Neural/fisiologia , Proteínas Quinases/metabolismo , Receptores de AMPA/metabolismo , Animais , Proteína Quinase Tipo 1 Dependente de Cálcio-Calmodulina , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico , Dibutiril GMP Cíclico/farmacologia , Relação Dose-Resposta a Droga , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Retroalimentação/fisiologia , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Hipocampo/metabolismo , Técnicas In Vitro , N-Metilaspartato/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Técnicas de Patch-Clamp , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de AMPA/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Proteína Quinase C/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Células Piramidais/efeitos dos fármacos , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Receptores Muscarínicos/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Transdução de Sinais/fisiologia , Alcaloides , Sequência de Aminoácidos , Animais , Benzofenantridinas , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genisteína/farmacologia , Isoflavonas/farmacologia , Lisofosfolipídeos/farmacologia , Camundongos , Camundongos Knockout , Microinjeções , Dados de Sequência Molecular , Muscarina/farmacologia , Plasticidade Neuronal , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenantridinas/farmacologia , Fenóis/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Células Piramidais/fisiologia , Ratos , Ratos Wistar , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Ácidos Lisofosfatídicos , Receptores Muscarínicos/efeitos dos fármacos , Salicilatos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/química , Acetato de Tetradecanoilforbol/farmacologia , Xenopus laevis , meta-AminobenzoatosRESUMO
At CA1 synapses, activation of NMDA receptors (NMDARs) is required for the induction of both long-term potentiation and depression. The basal level of activity of these receptors is controlled by converging cell signals from G-protein-coupled receptors and receptor tyrosine kinases. Pituitary adenylate cyclase activating peptide (PACAP) is implicated in the regulation of synaptic plasticity because it enhances NMDAR responses by stimulating Galphas-coupled receptors and protein kinase A (Yaka et al., 2003). However, the major hippocampal PACAP1 receptor (PAC1R) also signals via Galphaq subunits and protein kinase C (PKC). In CA1 neurons, we showed that PACAP38 (1 nM) enhanced synaptic NMDA, and evoked NMDAR, currents in isolated CA1 neurons via activation of the PAC1R, Galphaq, and PKC. The signaling was blocked by intracellular applications of the Src inhibitory peptide Src(40-58). Immunoblots confirmed that PACAP38 biochemically activates Src. A Galphaq pathway is responsible for this Src-dependent PACAP enhancement because it was attenuated in mice lacking expression of phospholipase C beta1, it was blocked by preventing elevations in intracellular Ca2+, and it was eliminated by inhibiting either PKC or cell adhesion kinase beta [CAKbeta or Pyk2 (proline rich tyrosine kinase 2)]. Peptides that mimic the binding sites for either Fyn or Src on receptor for activated C kinase-1 (RACK1) also enhanced NMDAR in CA1 neurons, but their effects were blocked by Src(40-58), implying that Src is the ultimate regulator of NMDARs. RACK1 serves as a hub for PKC, Fyn, and Src and facilitates the regulation of basal NMDAR activity in CA1 hippocampal neurons.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Hipocampo/fisiologia , Neurônios/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/fisiologia , Proteína Quinase C/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Ativação Enzimática/fisiologia , Hipocampo/enzimologia , Hipocampo/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neurônios/enzimologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Ratos , Ratos WistarRESUMO
Excitatory synaptic activity governs excitotoxicity and modulates the distribution of NMDA receptors (NMDARs) among synaptic and extrasynaptic sites of central neurons. We investigated whether NMDAR localization was functionally linked to excitotoxicity by perturbing F-actin, a cytoskeletal protein that participates in targeting synaptic NMDARs in dendritic spines. Depolymerizing F-actin did not affect NMDA-evoked whole-cell currents. However, the number of dendritic NMDAR clusters and the NMDAR-mediated component of miniature spontaneous EPSCs were reduced, whereas the number of AMPA receptor clusters and AMPA receptor-mediated component of EPSCs was unchanged. This selective perturbation of synaptically activated NMDARs had no effect on neuronal death or the accumulation of (45)Ca(2+) evoked by applying exogenous NMDA or L-glutamate, which reach both synaptic and extrasynaptic receptors. However, it increased survival and decreased (45)Ca(2+) accumulation in neurons exposed to oxygen glucose deprivation, which causes excitotoxicity by glutamate release at synapses. Thus, synaptically and extrasynaptically activated NMDARs are equally capable of excitotoxicity. However, their relative contributions vary with the location of extracellular excitotoxin accumulation, a factor governed by the mechanism of extracellular neurotransmitter accumulation, not the synaptic activation of NMDARs.
Assuntos
Neurônios/fisiologia , Neurotoxinas/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Sinapses/fisiologia , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Actinas/efeitos dos fármacos , Actinas/fisiologia , Animais , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Citocalasina D/farmacologia , Dendritos/química , Dendritos/metabolismo , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Glucose/farmacologia , Ácido Glutâmico/farmacologia , Hipocampo/citologia , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Camundongos , Neurônios/química , Neurônios/ultraestrutura , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oxigênio/farmacologia , Receptores de AMPA/fisiologia , Sinapses/química , Sinapses/efeitos dos fármacos , Tiazóis/farmacologia , TiazolidinasRESUMO
The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.
Assuntos
Actinas/metabolismo , Quinase de Cadeia Leve de Miosina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/metabolismo , Carbocianinas , Separação Celular , Células Cultivadas , Dendritos/metabolismo , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Corantes Fluorescentes , Hipocampo , Camundongos , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/farmacologia , N-Metilaspartato/farmacologia , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Propofol (2,6-di-isopropylphenol) has multiple actions on GABA(A) receptor function that act in concert to potentiate GABA-evoked currents. To understand how propofol influences inhibitory IPSCs, we examined the effects of propofol on responses to brief applications of saturating concentrations of GABA (1-30 mM). GABA was applied using a fast perfusion system to nucleated patches excised from hippocampal neurons. In this preparation, propofol (10 microM) had no detectable agonist effect but slowed the decay, increased the charge transfer (62%), and enhanced the peak amplitude (8%) of currents induced by brief pulses (3 msec) of GABA. Longer pulses (500 msec) of GABA induced responses that desensitized with fast (tau(f) = 1.5-4.5 msec) and slow (tau(s) = 1-3 sec) components and, after the removal of GABA, deactivated exponentially (tau(d) = 151 msec). Propofol prolonged this deactivation (tau(d) = 255 msec) and reduced the development of both fast and slow desensitization. Recovery from fast desensitization, assessed using pairs of brief pulses of GABA, paralleled the time course of deactivation, indicating that fast desensitization traps GABA on the receptor. With repetitive applications of pulses of GABA (0.33 Hz), the charge transfer per pulse declined exponentially (tau approximately 15 sec) to a steady-state value equal to approximately 40% of the initial response. Despite the increased charge transfer per pulse with propofol, the time course of the decline was unchanged. These experimental data were interpreted using computer simulations and a kinetic model that assumed fast and slow desensitization, as well as channel opening developed in parallel from a pre-open state. Our results suggest that propofol stabilizes the doubly liganded pre-open state without affecting the isomerization rate constants to and from the open state. Also, the rate constants for agonist dissociation and entry into the fast and slow desensitization states were reduced by propofol. The recovery rate constant from fast desensitization was slowed, whereas that from slow desensitization appeared to be unchanged. Taken together, the effects of propofol on GABA(A) receptors enhance channel opening, particularly under conditions that promote desensitization.
Assuntos
Anestésicos Gerais/farmacologia , Propofol/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Animais , Células Cultivadas , Simulação por Computador , Condutividade Elétrica , Cinética , Camundongos , Modelos Neurológicos , Técnicas de Patch-Clamp , Receptores de GABA-A/fisiologia , Fatores de Tempo , Ácido gama-Aminobutírico/administração & dosagem , Ácido gama-Aminobutírico/farmacologiaRESUMO
The protein-tyrosine kinase Src is known to potentiate the function of NMDA receptors, which is necessary for the induction of long-term potentiation in the hippocampus. With recombinant receptors composed of NR1-1a/NR2A or NR1-1a/2B subunits, Src reduces voltage-independent inhibition by the divalent cation Zn2+. Thereby the function of recombinant NMDA receptors is potentiated by Src only when the Zn2+ level is sufficient to cause tonic inhibition. Here we investigated whether the Src-induced potentiation of NMDA receptor function in neurons is caused by reducing voltage-independent Zn2+ inhibition. Whereas chelating extracellular Zn2+ blocked the Src-induced potentiation of NR1-1a/2A receptors, we found that Zn2+ chelation did not affect the potentiation of NMDA receptor (NMDAR) currents by Src applied into hippocampal CA1 or CA3 neurons. Moreover, Src did not alter the Zn2+ concentration-inhibition relationship for NMDAR currents in CA1 or CA3 neurons. Also, chelating extracellular Zn2+ did not prevent the upregulation of NMDA single-channel activity by endogenous Src in membrane patches from spinal dorsal horn neurons. Taking these results together we conclude that Src-induced potentiation of NMDAR currents is not mediated by reducing Zn2+ inhibition in hippocampal and dorsal horn neurons.
Assuntos
Hipocampo/fisiologia , Potenciais da Membrana/fisiologia , Receptores de N-Metil-D-Aspartato/fisiologia , Medula Espinal/citologia , Medula Espinal/fisiologia , Zinco/farmacologia , Quinases da Família src/fisiologia , Animais , Eletrofisiologia , Hipocampo/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Medula Espinal/efeitos dos fármacosRESUMO
Massive activation of glutamate receptors can result in excessive rises in cytoplasmic Ca2+ that are thought to underlie the fundamental processes ultimately leading to neuronal death. Preventing such cellular Ca2+ rises in the brain may reduce considerably the neuronal damage produced by stroke, head trauma, or epilepsy. Activation of NMDA receptors is instrumental in this type of neurotoxicity. Recent findings, discussed here by Istvan Mody and John MacDonald, indicate that a large proportion of the neurotoxic Ca2+ that enters nerve cells following NMDA receptor activation originates from an intracellular Ca2+ pool. The release of Ca2+ from this pool is sensitive to the skeletal muscle relaxant dantrolene, and this may constitute a novel and alternative therapeutic approach against NMDA receptor-mediated excitotoxicity.
Assuntos
Cálcio/metabolismo , Aminoácidos Excitatórios/toxicidade , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Humanos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacosRESUMO
Ion channels coupled to NMDA, kainate and AMPA receptors are the target of pharmacological regulation by a variety of drugs and ions. While these channels are all nonselectively permeated by Na+ and K+ ions, the NMDA receptor-channel complex contains a number of pharmacological sites distinct from those found on the others. For example, Mg2+ ions rapidly and reversibly block open NMDA channels in a highly voltage-dependent manner. Its extreme voltage dependence suggests that the Mg2+ binding site lies deep within the ion channel pore. By contrast the voltage-dependent block of activated channels by the dissociative anesthetic 'slow channel blockers' has unusual characteristics. In the fourth article in our series on excitatory amino acids, John MacDonald and Linda Nowak analyse the characteristics of these two types of block and describe the hypotheses that have been put forward to explain the mechanisms involved.
Assuntos
Receptores de Superfície Celular/metabolismo , Animais , Eletrofisiologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Receptores de AminoácidoRESUMO
The hypothalamic-pituitary-adrenal (HPA) axis is susceptible to programming during fetal life. Such programming occurs, at least partially, at the level of the hippocampus. The hippocampus plays a central role in regulation of the HPA axis and release of endogenous glucocorticoids, via mediation of glucocorticoid negative feedback. Fetal exposure to synthetic glucocorticoids can permanently alter glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) levels within the hippocampus, and serotonin is thought to be involved in this process. In the present study, we hypothesised that dexamethasone, cortisol and serotonin exposure would modify GR mRNA expression within fetal guinea-pig hippocampal cultures. Cultures were derived from 40-day-old guinea-pig fetuses, and were exposed to 0, 1, 10 and 100 nM dexamethasone, cortisol or serotonin for 4 days. Expression of GR and MR mRNA was examined by in situ hybridisation followed by high-resolution silver emulsion autoradiography. Four-day exposure to dexamethasone (P < 0.05; 100 nM) or cortisol (P = 0.08; 100 nM) downregulated the expression of GR mRNA within neurons. There was no change in the expression of MR mRNA levels following cortisol treatment. Exposure to serotonin (100 nM) significantly increased GR mRNA levels in hippocampal neurons. We conclude that synthetic and endogenous glucocorticoids, as well as serotonin, can influence GR expression during hippocampal development and in this way may act to permanently programme HPA function.
Assuntos
Retroalimentação Fisiológica/efeitos dos fármacos , Feto/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Animais , Autorradiografia , Dexametasona/farmacologia , Cobaias , Hipocampo/citologia , Hidrocortisona/farmacologia , Hibridização In Situ , Técnicas In Vitro , RNA Mensageiro/genética , Receptores de Glucocorticoides/genética , Serotonina/farmacologiaRESUMO
In human pregnancy, cortisol and PGs are involved in the onset of labor and play an important role in the mechanisms leading to parturition. Recent studies have shown that at term, cortisol increases PG synthesis and decreases PG metabolism in chorion trophoblast (CT) cells. In CT, 11 beta-hydroxysteroid oxidase type 1 (11 beta-HSD1) converts biologically inactive cortisone to cortisol to regulate cortisol availability. In the present study, we have investigated whether 11 beta-HSD1 activity could be influenced by PGs. We have shown that in CT, PGF2alpha rapidly increased 11 beta-HSD1 reductase activity in a dose-dependent manner via the PGF2alpha receptor, localized in the fetal membranes. PGF2alpha stimulated 11 beta-HSD1 activity through increased intracellular calcium mobilization, activation of PKC, and the phosphorylation of the 11 beta-HSD enzyme. We propose that within CT there is a novel feed forward loop by which PGF2alpha acts to promote cortisol production from cortisone through increases in 11beta-HSD1, and this in turn leads to further net PG output for the onset of labor and birth.
Assuntos
Dinoprosta/farmacologia , Hidrocortisona/biossíntese , Hidroxiesteroide Desidrogenases/biossíntese , Trabalho de Parto/fisiologia , 11-beta-Hidroxiesteroide Desidrogenases , Adulto , Cálcio/metabolismo , Células Cultivadas , Retroalimentação/fisiologia , Feminino , Feto/metabolismo , Corantes Fluorescentes , Fura-2 , Humanos , Imuno-Histoquímica , Luteolíticos/farmacologia , Membranas/metabolismo , Testes de Precipitina , Gravidez , Prostaglandinas F Sintéticas/farmacologia , Receptores de Prostaglandina/metabolismo , Estimulação Química , Trofoblastos/metabolismoRESUMO
Synaptic plasticity, or long-term potentiation (LTP), of excitatory synapses in the hippocampus contributes to learning and the establishment of spatial memories. In the CA1 region, induction of LTP enhances the function of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors (AMPARs) because of the Ca2+-calmodulin kinase II (CaMKII)-dependent phosphorylation of this subtype of glutamate receptor. Entry of Ca2+, via N-methyl-D-aspartate receptors (NMDARs), during strong synaptic stimulation provides the stimulus to trigger phosphorylation of AMPARs. However, this induction also requires activation of a protein kinase C (PKC)-dependent tyrosine kinase signal cascade and a concomitant upregulation of NMDARs. This review focuses upon NMDARs as potential targets of PKC and/or of the PKC-dependent tyrosine kinase cascade. PKC, acting via the CAKbeta/Src tyrosine kinase cascade, enhances NMDAR activation and may increase the number of receptors expressed in synapses. In contrast, direct phosphorylation of NMDARs by PKC increases the sensitivity of NMDA channel inactivation to intracellular Ca2+. In CAI neurons, PKC provides a point of convergence of control of NMDARs and synaptic plasticity for a wide variety of G-protein coupled and growth factor receptors.