RESUMO
The RSH/Smith--Lemli--Opitz syndrome (RSH/SLOS) is a human autosomal recessive syndrome characterized by multiple malformations, a distinct behavioral phenotype with autistic features and mental retardation. RSH/SLOS is due to an inborn error of cholesterol biosynthesis caused by mutation of the 3 beta-hydroxysterol Delta(7)-reductase gene. To further our understanding of the developmental and neurological processes that underlie the pathophysiology of this disorder, we have developed a mouse model of RSH/SLOS by disruption of the 3 beta-hydroxysterol Delta(7)-reductase gene. Here we provide the biochemical, phenotypic and neurophysiological characterization of this genetic mouse model. As in human patients, the RSH/SLOS mouse has a marked reduction of serum and tissue cholesterol levels and a marked increase of serum and tissue 7-dehydrocholesterol levels. Phenotypic similarities between this mouse model and the human syndrome include intra-uterine growth retardation, variable craniofacial anomalies including cleft palate, poor feeding with an uncoordinated suck, hypotonia and decreased movement. Neurophysiological studies showed that although the response of frontal cortex neurons to the neurotransmitter gamma-amino-n-butyric acid was normal, the response of these same neurons to glutamate was significantly impaired. This finding provides insight into potential mechanisms underlying the neurological dysfunction seen in this human mental retardation syndrome and suggests that this mouse model will allow the testing of potential therapeutic interventions.
Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Oxirredutases/genética , Síndrome de Smith-Lemli-Opitz/genética , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Oxirredutases/deficiência , Oxirredutases/metabolismo , Fenótipo , Síndrome de Smith-Lemli-Opitz/metabolismo , Síndrome de Smith-Lemli-Opitz/patologiaRESUMO
The effect of ethanol on current activated by extracellular adenosine 5'-triphosphate (ATP) was studied in freshly isolated adult rat hippocampal CA1 neurons using whole-cell patch-clamp recording. ATP activated an inward current with an EC(50) value of 18 microM. The inward current was also activated by 2-methylthio ATP (2-MeSATP) and alpha,beta-methylene ATP (alpha,beta-MeATP), inhibited by pyridoxal-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and potentiated by Zn(2+). Ethanol inhibited current activated by 10 microM ATP with an IC(50) value of 83 mM in a voltage-independent manner. Ethanol, 100 mM, shifted the ATP concentration-response curve to the right, increasing the EC(50) value for ATP from 18 to 33 microM, but did not reduce the maximal response to ATP. The results suggest that ethanol can inhibit the function of P2X receptors in adult rat hippocampal neurons by decreasing the apparent affinity of the binding site for ATP.
Assuntos
Trifosfato de Adenosina/farmacologia , Etanol/farmacologia , Hipocampo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Neurônios/citologia , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2/metabolismoRESUMO
1. The effect of ethanol on the function of P2X(4) receptors expressed in Xenopus oocytes was studied using two-electrode voltage-clamp recording. 2. The amplitude of current activated by 1 microM ATP was decreased by ethanol in a concentration-dependent manner over the concentration range 1 - 500 mM. The concentration of ethanol that produced 50% inhibition (IC(50)) of current activated by 1 microM ATP was 58 mM. 3. Ethanol inhibition of ATP-activated current was not dependent on membrane potential from -60 to +20 mV, and ethanol did not change the reversal potential of ATP-activated current. 4. Ethanol, 50 mM, shifted the ATP concentration-response curve to the right, increasing the EC(50) for ATP from 9.1 to 16.0 microM, but did not reduce the maximal response to ATP. 5. The results suggest that ethanol may inhibit P2X(4) receptors by decreasing the apparent affinity of the binding site for ATP. 6. Since the P2X(4) receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system.
Assuntos
Etanol/farmacologia , Antagonistas do Receptor Purinérgico P2 , Trifosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Oócitos , Fosfato de Piridoxal/análogos & derivados , Fosfato de Piridoxal/farmacologia , RNA Complementar/administração & dosagem , RNA Complementar/genética , Ratos , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X4 , Suramina/farmacologia , Triazinas/farmacologia , Xenopus laevisRESUMO
Extracellular adenosine 5'-triphosphate (ATP) has been reported to produce excitatory actions in the nervous system, such as excitatory postsynaptic potentials or currents in both central and peripheral neurons, via activation of a class of ATP-gated membrane ion channels designated P2X receptors. This article reviews studies of alcohol effects on these receptor-channels. Ethanol has been found to inhibit ATP-gated ion channel function by shifting the agonist concentration-response curve to the right in a parallel manner, increasing the EC50 without affecting Emax of this curve. To distinguish whether this inhibition involves competitive antagonism of agonist action or a decrease in the affinity of the agonist binding site, the kinetics of activation and deactivation of agonist-activated current were studied. Ethanol was found to decrease the time-constant of deactivation of ATP-gated ion channels without affecting the time-constant of activation, indicating that ethanol inhibits the function of these receptors by an allosteric decrease in the affinity of the agonist binding site. The inhibition of ATP-gated ion channel function by a number of alcohols was found to exhibit a distinct cutoff effect that appeared to be related to the molecular volume of the alcohols. For alcohols with a molecular volume of < or = 42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of > or = 46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. This cutoff effect has been interpreted as evidence that alcohols inhibit the function of ATP-gated ion channels by interacting with a hydrophobic pocket of circumscribed dimensions on the receptor protein. To evaluate the localization of this presumed alcohol binding site, the effect of the intracellular application of ethanol was studied on the inhibition of ATP-activated current by extracellularly applied ethanol. The intracellular application of 100 mM ethanol did not affect the inhibition of current by 100 mM extracellular ethanol, suggesting that the alcohol inhibition of ATP-gated ion channel function involves the extracellular domain of the receptor. Finally, recent studies suggest that the alcohol sensitivity of ATP-gated channels may be regulated by physiological mechanisms.
Assuntos
Trifosfato de Adenosina/fisiologia , Etanol/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Animais , Membrana Celular/efeitos dos fármacos , Receptores Purinérgicos P2/fisiologiaRESUMO
Differential Modulation by Copper and Zinc of P2X2 and P2X4 Receptor Function. The modulation by Cu2+ and Zn2+ of P2X2 and P2X4 receptors expressed in Xenopus oocytes was studied with the two-electrode, voltage-clamp technique. In oocytes expressing P2X2 receptors, both Cu2+ and Zn2+, in the concentration range 1-130 microM, reversibly potentiated current activated by submaximal concentrations of ATP. The Cu2+ and Zn2+ concentrations that produced 50% of maximal potentiation (EC50) of current activated by 50 microM ATP were 16.3 +/- 0.9 (SE) microM and 19.6 +/- 1.5 microM, respectively. Cu2+ and Zn2+ potentiation of ATP-activated current was independent of membrane potential between -80 and +20 mV and did not involve a shift in the reversal potential of the current. Like Zn2+, Cu2+ increased the apparent affinity of the receptor for ATP, as evidenced by a parallel shift of the ATP concentration-response curve to the left. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally effective concentration of Zn2+, suggesting a common site or mechanism of action of Cu2+ and Zn2+ on P2X2 receptors. For the P2X4 receptor, Zn2+, from 0.5 to 20 microM enhanced current activated by 5 microM ATP with an EC50 value of 2.4 +/- 0.2 microM. Zn2+ shifted the ATP concentration-response curve to the left in a parallel manner, and potentiation by Zn2+ was voltage independent. By contrast, Cu2+ in a similar concentration range did not affect ATP-activated current in oocytes expressing P2X4 receptors, and Cu2+ did not alter the potentiation of ATP-activated current produced by Zn2+. The results suggest that Cu2+ and Zn2+ differentially modulate the function of P2X2 and P2X4 receptors, perhaps because of differences in a shared site of action on both subunits or the absence of a site for Cu2+ action on the P2X4 receptor.
Assuntos
Cobre/farmacologia , Receptores Purinérgicos P2/efeitos dos fármacos , Zinco/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Relação Dose-Resposta a Droga , Condutividade Elétrica , Feminino , Oócitos , Concentração Osmolar , Técnicas de Patch-Clamp , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2X2 , Receptores Purinérgicos P2X4 , Xenopus laevisRESUMO
Rat dorsal root ganglion neurons can be classified into at least three distinct groups based on cell size, afferent fiber diameter, electrophysiological properties, sensitivity to vanilloid agonists such as capsaicin, and function. In the present study, ATP-activated current in these neurons was characterized using whole-cell patch-clamp recording. Small diameter (<30 microm) cells had high capsaicin sensitivity, high affinity for ATP, and rapidly desensitizing ATP-activated current. Medium diameter (30-50 microm) cells had no capsaicin sensitivity, lower affinity for ATP and slowly desensitizing ATP-activated current. Large diameter (>50 microm) cells were insensitive to both capsaicin and ATP. These findings suggest that distinct types of ATP receptor-ion channels are expressed in different types of dorsal root ganglion neurons, and may contribute to the functional differences among these types of neurons.
Assuntos
Trifosfato de Adenosina/farmacologia , Gânglios Espinais/fisiologia , Neurônios/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/fisiologia , Animais , Capsaicina/farmacologia , Tamanho Celular , Gânglios Espinais/citologia , Técnicas In Vitro , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Fibras Nervosas/ultraestrutura , Neurônios/classificação , Neurônios/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Tionucleotídeos/farmacologiaRESUMO
NMDA and GABA(A) receptors are believed to be important CNS targets of alcohol action. In mouse hippocampal neurons, n-alcohols from ethanol to dodecanol enhanced GABA-activated ion current, whereas higher alcohols had no effect. Alcohols below pentanol affected NMDA receptors more potently than GABA(A) receptors. Increasing alcohol carbon chain length produced a greater average change in apparent binding energy and potency for modulation of GABA(A) than of NMDA receptor-channels, with the result that alcohols above pentanol affected GABA(A) receptors more potently than NMDA receptors. The anesthetic potency of n-alcohols in rats more closely reflected NMDA receptor modulatory potency for lower alcohols and GABA(A) receptor modulatory potency for higher alcohols. The results suggest that there may be fundamental differences in the sites through which alcohols affect NMDA and GABA(A) receptor function.
Assuntos
Etanol/farmacologia , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de GABA-A/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Animais , Hipocampo/citologia , Hipocampo/metabolismo , Modelos Lineares , Camundongos , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos , Solubilidade , Água/químicaRESUMO
1. The effects of the active metabolite of chloral derivative sedative-hypnotic agents, 2,2,2-trichloroethanol (trichloroethanol), and its analog 2,2,2-trifluoroethanol (trifluoroethanol), were studied on ion current activated by the excitatory amino acids N-methyl-D-aspartate (NMDA) and kainate in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents in a concentration-dependent manner. Trichloroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 6.4 and 12 mM, respectively, while trifluoroethanol inhibited NMDA- and kainate-activated currents with IC50 values of 28 and 35 mM, respectively. 3. Both trichloroethanol and trifluoroethanol appeared to be able to inhibit excitatory amino acid-activated currents by 100 per cent. 4.Concentration-response analysis of NMDA- and kainate-activated current revealed that trichloroethanol decreased the maximal response to both agonists without significantly affecting their EC50 values. 5. Both trichloroethanol and trifluoroethanol inhibited excitatory amino acid-activated currents more potently than did ethanol. The inhibitory potency of trichloroethanol and trifluoroethanol appears to be associated with their increased hydrophobicity. 6. The observation that trichloroethanol inhibits excitatory amino acid-activated currents at anaesthetic concentrations suggests that inhibition of excitatory amino acid receptors may contribute to the CNS depressant effects of chloral derivative sedative-hypnotic agents.
Assuntos
Etilenocloroidrina/análogos & derivados , Hipocampo/efeitos dos fármacos , N-Metilaspartato/farmacologia , Trifluoretanol/farmacologia , Animais , Células Cultivadas , Etilenocloroidrina/farmacologia , Hipocampo/citologia , Hipocampo/fisiologia , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologiaRESUMO
Ethanol inhibits a neuronal P2X purinoceptor by shifting the ATP concentration-response curve to the right in an apparently competitive manner. However, the underlying mechanism has not been determined. We investigated the effects of ethanol on the activation and deactivation time constants for ATP-activated current in bullfrog dorsal root ganglion neurones. Ethanol decreased the time constant of deactivation of ATP-gated ion channels without affecting the time constant of activation. The observations are not consistent with a competitive mechanism of inhibition by ethanol, but may be explained by an allosteric action of ethanol to decrease apparent agonist affinity. This represents a novel mechanism of action of ethanol on a neurotransmitter-gated ion channel.
Assuntos
Depressores do Sistema Nervoso Central/farmacologia , Etanol/farmacologia , Neurônios/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Trifosfato de Adenosina/fisiologia , Animais , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Neurônios/química , Técnicas de Patch-Clamp , Rana catesbeiana , Receptores Purinérgicos P2/biossíntese , Receptores Purinérgicos P2/químicaRESUMO
1. The action of ethanol on N-methyl-D-aspartate (NMDA)-activated ion current was studied in mouse hippocampal neurones in culture using whole-cell patch-clamp recording. 2. Ethanol inhibited NMDA-activated current in a voltage-independent manner, and did not alter the reversal potential of NMDA-activated current. 3. Concentration-response analysis of NMDA- and glycine-activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4. The polyamine spermine (1 microM) increased amplitude of NMDA-activated current but did not alter the percentage inhibition of ethanol. 5. Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA-activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6. The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 7. Mg2+ (10, 100, 500 microM), (5, 20 microM) or ketamine (2, 10 microM) decreased the amplitude of NMDA-activated current, but did not affect the percentage inhibition by ethanol. 8. The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non-competitive mechanism that does not involve several modulatory sites on the NMDA receptor-ionophore complex.
Assuntos
Etanol/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Hipocampo/citologia , Hipocampo/metabolismo , Concentração de Íons de Hidrogênio , Ketamina/farmacologia , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , N-Metilaspartato/metabolismo , Neurônios/metabolismo , Oxirredução , Técnicas de Patch-Clamp , Espermina/farmacologia , Zinco/farmacologiaRESUMO
The effect of dynorphin A(1-13) on N-methyl-D-aspartate (NMDA)-activated currents was investigated in the presence of low extracellular glycine concentrations in Xenopus oocytes expressing recombinant heteromeric NMDA receptors and in cultured hippocampal neurons with the use of voltage-clamp techniques. At an extracellular added glycine concentration of 100 nM, dynorphin A(1-13) (10 microM) greatly increased the amplitude of NMDA-activated currents for all heteromeric subunit combinations tested; on average, the potentiation was: epsilon1/zeta1, 3,377 +/- 1,416% (mean +/- SE); epsilon2/zeta1, 1,897 +/- 893%; epsilon3/zeta1, 4,356 +/- 846%; and epsilon4/zeta1, 1,783 +/- 503%. Potentiation of NMDA-activated current by dynorphin A(1-13) was concentration dependent between 0.1 and 10 microM dynorphin A(1-13), with a half-maximal concentration value of 2.77 microM and an apparent Hill coefficient of 2.53, for epsilon2/zeta1 subunits at 100 nM added extracellular glycine. Percentage potentiation by dynorphin A(1-13) was maximal at the lowest glycine concentrations tested (0.01 and 0.1 microM), and decreased with increasing glycine concentration. No significant potentiation was observed at glycine concentrations > 0.1 microM for epsilon1/zeta1, epsilon2/zeta1, and epsilon4/zeta1 subunits, or at > 1 microM for epsilon3/zeta1 subunits. Potentiation of NMDA-activated currents by dynorphin A(1-13) was not inhibited by 1 microM of the kappa-opioid receptor antagonist nor-binaltorphimine, and potentiation was not observed with 10 microM of the kappa-opioid receptor agonist trans-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl] benzene-acetamide. Potentiation of NMDA-activated current by dynorphin A(1-13) was inhibited by the glycine antagonist kynurenic acid (50 microM). NMDA-activated current was also potentiated at low glycine concentrations by 10 microM dynorphin A(2-13) or (3-13), both of which have a glycine as the first amino acid, but not by 10 microM dynorphin A(4-13), which does not have glycine as an amino acid. In hippocampal neurons, 10 microM dynorphin A(1-13) or (2-13) potentiated steady-state NMDA-activated current in the absence of added extracellular glycine. The extracellular free glycine concentration, determined by high-performance liquid chromatography, was between 26 and 36 nM for the bathing solution in presence or absence of 10 microM dynorphin A(1-13), (2-13), (3-13), or (4-13), and did not differ significantly among these solutions. The observations are consistent with the potentiation of NMDA-activated current at low extracellular glycine concentrations resulting from an interaction of the glycine amino acids in dynorphin A(1-13) with the glycine coagonist site on the NMDA receptor. Because dynorphin A is an endogenous peptide that can be coreleased with glutamate at glutamatergic synapses, the potentiation of NMDA receptor-mediated responses could be an important physiological regulator of NMDA receptor function at these synapses.
Assuntos
Analgésicos Opioides/farmacologia , Dinorfinas/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Glicina/farmacologia , Fragmentos de Peptídeos/farmacologia , Receptores de N-Metil-D-Aspartato/fisiologia , Animais , Células Cultivadas , Sinergismo Farmacológico , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Camundongos , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/agonistas , Receptores Opioides kappa/efeitos dos fármacos , Xenopus laevisRESUMO
The effect of Mg2+ on ATP-activated current in rat nodose ganglion neurons was investigated with the use of the whole cell patch-clamp technique. Mg2+ decreased the amplitude of ATP-activated current in a concentration-dependent manner over the concentration range of 0.25-8 mM, with a 50% inhibitory concentration value of 1.5 mM for current activated by 10 microM ATP. Mg2+ shifted the ATP concentration-response curve to the right in a parallel manner, increasing the 50% effective concentration value for ATP from 9.2 microM in the absence of added Mg2+ to 25 microM in the presence of 1 mM Mg2+. Mg2+ increased the deactivation rate of ATP-activated current without changing its activation rate. The observations are consistent with an action of Mg2+ to inhibit ATP-gated ion channel function by decreasing the affinity of the agonist binding site on these receptors.
Assuntos
Trifosfato de Adenosina/fisiologia , Magnésio/farmacologia , Inibição Neural/efeitos dos fármacos , Gânglio Nodoso/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Inibição Neural/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Gânglio Nodoso/fisiologia , Técnicas de Patch-Clamp , Ratos , Transmissão Sináptica/fisiologiaRESUMO
The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptor-mediated responses, potentiated 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N-terminal domain from the nACh alpha 7 receptor and the transmembrane and C-terminal domains from the 5-HT3 receptor) in a manner similar to that of the nACh alpha 7 receptor. Since the N-terminal domain of the chimeric receptor was from the nACh alpha 7 receptor, the observations suggest that the inhibition involves the N-terminal domain of the receptor.
Assuntos
Anestésicos Inalatórios/farmacologia , Antagonistas Colinérgicos/farmacologia , Oócitos/metabolismo , Receptores Colinérgicos/efeitos dos fármacos , Receptores de Serotonina/efeitos dos fármacos , Agonistas do Receptor de Serotonina/farmacologia , Animais , Halotano/farmacologia , Isoflurano/farmacologia , Oócitos/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , XenopusRESUMO
The effect of pH on ATP-activated current in bullfrog dorsal root ganglion neurons was studied using the whole-cell patch-clamp technique. ATP-activated current amplitude was highly dependent upon extracellular pH. An acid pH increased, whereas alkaline pH decreased, ATP-activated current amplitude. The half-maximal pH (EC50) for potentiation of 2.5 micro;M ATP-activated current was 7.2. Acidification alone did not activate detectable current and, at an acid pH, ATP-activated current was abolished by suramin. Proton-induced enhancement of ATP-activated current was not sensitive to membrane potential between -80 and +40 mV, and did not involve a shift in reversal potential. Lowering pH from 7.2 to 6.5 or elevating pH from 7.2 to 8.0 shifted the ATP concentration/response curve to the left or right, respectively, without changing the maximal response to ATP. Protons increased the time constant of deactivation without affecting the time constant of activation or desensitization of ATP-activated current. Alteration of patch-pipette (intracellular) pH did not affect the enhancement of ATP-activated current by extracellular protons. Diethylpyrocarbonate (DEP), dithiothreitol (DTT), 5, 5'-dithio-bis-(2-nitro-benzoic acid) (DTNB), or N-ethylmaleimide (NEM) did not affect enhancement of ATP-activated current by protons. The results suggest that extracellular protons, at physiological concentrations, can regulate the function of P2X purinoceptors by modulating the affinity of the ATP-binding site.
Assuntos
Trifosfato de Adenosina/farmacologia , Gânglios Espinais/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Masculino , Técnicas de Patch-Clamp , Prótons , Rana catesbeianaRESUMO
1. The effect of Zn2+ on ATP-activated current was studied in bullfrog dorsal root ganglion (DRG) neurones using the whole-cell patch-clamp technique. 2. Zn2+ (2-800 microM) inhibited current activated by submaximal concentrations of ATP. The Zn2+ concentration that produced 50% inhibition (IC50) of current activated by 2.5 microM ATP was 61 +/- 9.8 microM. When ATP concentrations were adjusted to account for chelation of Zn2+, the IC50 of Zn2+ was 86 +/- 18 microM. 3. The inhibitory action of Zn2+ on ATP-gated channels did not appear to be due to a decrease in the concentration of one or more species of ATP. 4. Zn2+ inhibition of ATP-activated current was independent of membrane potential between -80 and +40 mV, and did not involve a shift in the reversal potential of the current. 5. Zn2+ (100 microM) shifted the ATP concentration-response curve to the right in a parallel manner, increasing the EC50 for ATP from 2.5 +/- 0.5 microM to 5.5 +/- 0.4 microM. 6. Zn2+ decreased the time constant of deactivation of ATP-gated ion channels without affecting the time constant of activation or desensitization. 7. Dithiothreitol (DTT) reversed Zn2+ inhibition of ATP-activated current. 8. 2-Methylthio ATP, alpha,beta-methylene ATP and ADP activated current with EC50 values of 2.4 +/- 0.3. 50.1 +/- 5.8 and 303.1 +/- 53.9 microM, respectively. Adenosine, AMP or beta,gamma-methylene ATP did not evoke detectable current. 9. Reactive Blue 2 and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid inhibited ATP-activated current. 10. The results suggest that Zn2+ can inhibit P2X purinoceptor function by decreasing the affinity of the binding site for ATP. These observations provide the first evidence for this action of Zn2+ on a neurotransmitter-gated ion channel. Furthermore, the receptor-channel in these neurones appears to be a novel member of the P2X purinoceptor class.
Assuntos
Trifosfato de Adenosina/fisiologia , Neurônios/fisiologia , Receptores Purinérgicos P2/fisiologia , Raízes Nervosas Espinhais/fisiologia , Zinco/fisiologia , Trifosfato de Adenosina/análogos & derivados , Animais , Técnicas In Vitro , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/antagonistas & inibidores , Canais Iônicos/fisiologia , Técnicas de Patch-Clamp , Rana catesbeiana , Receptores Purinérgicos P2/efeitos dos fármacos , Raízes Nervosas Espinhais/citologia , Zinco/farmacologiaRESUMO
Dynorphin is an endogenous ligand for kappa-opioid receptors. We investigated the effect of dynorphin 1-13 on different heteromeric subunits of recombinant mouse N-methyl-D-aspartate (NMDA) receptors expressed in Xenopus oocytes by using voltage-clamp recording methods. Dynorphin inhibited the NMDA-activated currents of all heteromeric NMDA receptor subunits tested. The different NMDA receptor subunits, however, exhibited a differential sensitivity to dynorphin. For the epsilon-1/zeta-1 subunit combination the EC50 was 19 microM; the other NMDA receptor subunit combinations were less sensitive to dynorphin and had the following order of sensitivity: epsilon-2/zeta-1 > epsilon-4/zeta-1 > epsilon-3/zeta-1. Inhibition of NMDA-activated currents by dynorphin was not competitive with NMDA, and was voltage-independent. NMDA-activated currents were not affected by the synthetic kappa-opioid receptor agonist U50488 ¿trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzene-acetamide¿, the specific kappa-opioid receptor antagonist nor-binaltorphimine1 or the nonspecific opioid receptor antagonist naloxone. In addition, nor-binaltorphimine1 or naloxone did not attenuate dynorphin inhibition of NMDA-activated current. The observations suggest that dynorphin inhibition of NMDA receptor function is mediated by an interaction of dynorphin with NMDA receptors, rather than an action involving kappa-opioid receptors. The data also show that different heteromeric NMDA receptor subunits exhibit a differential sensitivity to dynorphin.
Assuntos
Dinorfinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Relação Dose-Resposta a Droga , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Receptores Opioides kappa/agonistas , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inibidores , XenopusRESUMO
The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC50S for inhibition of NMDA-activated currents by ethanol were 129 mM +/- 20 mM in hippocampal neurons and 126 +/- 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC50 of 174 +/- 23 mM, which was not significantly different from the IC50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC50 of 340 +/- 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC50 of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.
Assuntos
Córtex Cerebral/efeitos dos fármacos , Etanol/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Hipocampo/efeitos dos fármacos , N-Metilaspartato/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Análise de Variância , Animais , Células Cultivadas , Córtex Cerebral/citologia , Hipocampo/citologia , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Explosão Respiratória/efeitos dos fármacosRESUMO
1. The modulation by protons of ATP-gated ion channel responses to ATP and Zn2+ was studied in freshly isolated rat nodose ganglion neurons using the whole cell patch-clamp technique. 2. Reduced external pH enhanced, whereas elevated external pH suppressed, current activated by 10 microM ATP. The pH producing the half-maximal effect (EC50) at this ATP concentration was 7.1. 3. Acidification shifted the ATP concentration-response curve to the left, decreasing the EC50 for ATP, and alkalinization shifted the ATP concentration-response curve to the right, increasing the EC50 for ATP. Fitting the data to a single-site pH model yielded an apparent pKa of the site on the ATP-gated ion channel of 7.6. Between pH 6.8 and 7.8, a change of 0.1 pH unit was calculated to change the ATP EC50 by 4.03 microM. Changing pH did not alter the maximal response to ATP. 4. The potentiating effect of protons appeared to be due to a direct action on the ATP-gated channel, as it could not be explained by an increase in the concentration of one or more species of ATP. 5. Lowering pH also increased the potency of Zn2+ for enhancement of ATP-activated current without altering its maximal response. Changing the pH from 7.3 to 6.8 changed the Zn2+ EC50 from 12 to 1.7 microM. 6. The potentiation of ATP-activated current by protons could not be attributed solely to an increase in the affinity of the receptor for Zn2+, as the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine did not alter the effect of protons. 7. Protons and Zn2+ do not appear to act at the same site on ATP-gated channels, as responses to maximally effective concentrations of Zn2+ were enhanced further by protons and vice versa. 8. These results suggest that protons regulate the function of P2X purinoceptors in rat nodose ganglion neurons by modulating the affinity of the binding sites for ATP and Zn2+ on these receptor channels.
Assuntos
Trifosfato de Adenosina/farmacologia , Canais Iônicos/efeitos dos fármacos , Gânglio Nodoso/efeitos dos fármacos , Prótons , Zinco/farmacologia , Animais , Relação Dose-Resposta a Droga , Concentração de Íons de Hidrogênio , Masculino , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
Several lines of evidence suggest a physiological role for Cu2+ in regulating nervous system function. In the present study using whole-cell patch-clamp recording, Cu2+ greatly enhanced current activated by 10 microM ATP in the majority of rat nodose ganglion neurons. The enhancement was concentration-dependent between 1 and 50 microM Cu2+, and had an EC50 of 6.1 microM. Cu2+ shifted the ATP concentration-response curve to the left in a parallel manner. However, Cu2+ did not enhance ATP-activated current in the presence of a maximally-effective concentration of Zn2+. The observations suggest that Cu2+ increases the affinity of the receptor for ATP by acting at the Zn2+ modulatory site. In addition, a subset of neurons in the nodose ganglion express ATP-gated receptor-channels that are insensitive to modulation by physiological concentrations of Cu2+, Zn2+ and protons.
Assuntos
Trifosfato de Adenosina/farmacologia , Cobre/farmacologia , Canais Iônicos/efeitos dos fármacos , Gânglio Nodoso/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Recent studies have suggested that alcohols can affect the function of neurotransmitter-gated ion channels by a direct interaction with the receptor protein. However, the molecular region of the receptor protein that mediates the alcohol action is not known. To address this question, we studied the effect of ethanol on the function of recombinant nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptors, 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and a chimeric receptor constructed from these two receptors. The receptors were expressed in Xenopus oocytes and their function was studied using the two-electrode voltage-clamp technique. Ethanol inhibited the response of nACh alpha 7 receptors in a concentration-dependent manner over the concentration range of 5-100 mM; the EC50 for this inhibition was 33 mM ethanol. Ethanol decreased the maximal amplitude (Emax) of the nACh alpha 7 receptor agonist concentration-response curve, without significantly affecting the EC50. In contrast, ethanol potentiated 5-HT3 receptor-mediated responses at low agonist concentrations. The potentiation was concentration-dependent over the concentration range of 10-100 mM; the EC50 for this potentiation was 57 mM ethanol. The magnitude of the ethanol potentiation of 5-HT3 receptor-mediated responses decreased with increasing agonist concentration. The chimeric receptor had the amino-terminal domain from the nACh alpha 7 receptor and the transmembrane and carboxyl-terminal domains from the 5-HT3 receptor. Ethanol was found to inhibit the function of this chimeric receptor in a manner similar to that of nACh alpha 7 receptors. Because the inhibition transfers with the amino-terminal domain of the receptor, the observations suggest that the amino-terminal domain of the receptor is involved in the inhibition.