RESUMO
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
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 recombinant N-methyl-D-aspartate (NMDA) receptor subunit zeta 1 and the heteromeric subunit combinations epsilon 1/zeta 1, epsilon 2/zeta 1, and epsilon 3/zeta 1 were expressed in Xenopus oocytes and their sensitivities to ethanol were investigated using the two-electrode voltage-clamp technique. NMDA-activated currents in oocytes expressing subunit combinations epsilon 1/zeta 1 or epsilon 2/zeta 1 were significantly inhibited by 50 mM ethanol, whereas NMDA-activated currents associated with the homomeric expression of zeta 1 or the heteromeric epsilon 3/zeta 1 combination were not significantly affected by 50 mM ethanol. Ethanol decreased the maximal amplitude (Emax) of the concentration-response curve for NMDA-activated current, without significantly affecting the EC50. The values of percentage inhibition by ethanol were not significantly different, regardless of the amplitude of current activated by NMDA concentrations from 10 to 250 microM. Different NMDA receptor subunits and subunit combinations exhibited differences in the concentration-response curves for ethanol. NMDA-activated current associated with the epsilon 1/zeta 1 subunit combination was increasingly inhibited by increasing concentrations of ethanol from 25 to 100 mM, whereas 25 mM ethanol elicited nearly maximal inhibition of NMDA-activated current associated with the epsilon 2/zeta 1 subunits, i.e., the inhibition by 50 or 100 nM ethanol was not significantly different. NMDA-activated current associated with the epsilon 3/zeta 1 subunit combination, on the other hand, was significantly inhibited only by 100 mM ethanol, and NMDA-activated current associated with the homomeric zeta 1 subunit was not significantly affected by ethanol concentrations of < or = 100 mM. Because NMDA receptor subunits are differentially distributed throughout the brain, the observations suggest that the differential sensitivity of NMDA receptor subunits to ethanol may contribute to the differences in ethanol sensitivity observed in different types of neurons.
Assuntos
Etanol/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Análise de Variância , Animais , Clonagem Molecular , DNA Complementar/química , DNA Complementar/metabolismo , Eletrofisiologia , Feminino , Oócitos/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/fisiologia , Proteínas Recombinantes/efeitos dos fármacos , Xenopus laevisRESUMO
To investigate the mechanism of intracellular Ca2+ ([Cai]) increase in human burst-forming unit-erythroid-derived erythroblasts by erythropoietin, we measured [Cai] with digital video imaging, cellular phosphoinositides with high performance liquid chromatography, and plasma membrane potential and currents with whole cell patch clamp. Chelation of extracellular free Ca2+ abolished [Cai] increase induced by erythropoietin. In addition, the levels of inositol-1,4,5-trisphosphate did not increase in erythropoietin-treated erythroblasts. These results indicate that in erythropoietin-stimulated cells, Ca2+ influx rather than intracellular Ca2+ mobilization was responsible for [Cai] rise. Both Ni2+ and moderately high doses of nifedipine blocked [Cai] increase, suggesting involvement of ion channels. Resting membrane potential in human erythroblasts was -10.9 +/- 1.0 mV and was not affected by erythropoietin, suggesting erythropoietin modulated a voltage-independent ion channel permeable to Ca2+. No voltage-dependent ion channel but a Ca(2+)-activated K+ channel was detected in human erythroblasts. The magnitude of erythropoietin-induced [Cai] increase, however, was insufficient to open Ca(2+)-activated K+ channels. Our data suggest erythropoietin modulated a voltage-independent ion channel permeable to Ca2+, resulting in sustained increases in [Cai].
Assuntos
Eritroblastos/efeitos dos fármacos , Eritroblastos/metabolismo , Eritropoetina/farmacologia , Canais Iônicos/efeitos dos fármacos , Adulto , Cálcio/metabolismo , Humanos , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/análise , Nifedipino/farmacologia , Canais de Potássio/efeitos dos fármacosRESUMO
The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.
Assuntos
Cálcio/metabolismo , Endotélio Vascular/fisiologia , Canais Iônicos/fisiologia , Veia Porta/fisiologia , Serotonina/farmacologia , Animais , Cátions , Condutividade Elétrica , Canais Iônicos/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Toxina Pertussis , Ratos , Ratos Endogâmicos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
Rat and bovine adrenal zona glomerulosa (ZG) cells possess a low-threshold, voltage-dependent Ca2+ current that was characterized using whole cell voltage clamp techniques. Activation of this current is observed at membrane potentials above -80 mV with maximal peak Ca2+ current elicited near -30 mV. Inactivation of the Ca2+ current was half-maximal between -74 and -58 mV, depending on the external Ca2+ concentration and was nearly complete at -40 mV. The voltage dependency of the current indicates that a calcium current could be sustained at membrane potentials between -80 and -40 mV and thereby elevates cytosolic calcium (Cai) levels. Under basal conditions, Cai is stable in single rat ZG cells, whereas more than half of the bovine ZG cells produce repeated Cai transients. These Cai transients, which are blocked by removal of external Ca2+ or addition of Ni2+, are likely due to repetitive electrical activity in bovine ZG cells. Cai responses can be elicited by small increases in external K+ concentration (5-10 mM) in both rat and bovine ZG cells, indicating the opening of low-threshold Ca2+ channels. However, these Cai changes remain robust at high external K+ concentrations (20-40 mM). In experiments combining Cai measurements and whole cell voltage clamp, a steep dependence of Cai on membrane potential was revealed beginning at depolarizing voltages near a holding membrane potential of -80 mV. A maximal increase in Cai occurred near -30 mV (equivalent to an external K+ concentration of 40 mM), a membrane voltage at which sustained current through low-threshold Ca2+ channels should be negligible. These data raise the possibility of additional voltage-dependent pathways for Ca2+ influx.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Zona Glomerulosa/metabolismo , 4-Aminopiridina/farmacologia , Animais , Bário/farmacologia , Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Bovinos , Células Cultivadas , Césio/farmacologia , Citosol/metabolismo , Potenciais Evocados/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potássio/farmacologia , Ratos , Ratos Endogâmicos , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
Bile-canaliculus contraction in rat hepatocyte doublets is postulated to involve activation of an actin-myosin system. We examined this hypothesis by determining the relationship between canalicular contraction and cystolic free Ca2+ ([Ca2+]i) concentration after extracellular addition of ATP or microdialysis of myosin light chain kinase or its Ca(2+)-independent fragment, which retains catalytic activity. After incubation of doublets with 200 mumol/L ATP in the absence of extracellular Ca2+, [Ca2+]i peaked at 40 sec and 71% of canaliculi contracted within 4 min. Decreasing effects were observed with equimolar ADP, AMP and nonhydrolyzable ATP, but no effect was observed with adenosine. The effect of extracellular ATP on [Ca2+]i and canalicular contraction was dose dependent. Addition of extracellular Ca2+ and ATP resulted in a plateau level of [Ca2+]i. Cytochalasin D, which depolymerizes actin filaments, inhibited ATP-induced canalicular contraction, but not the increase in [Ca2+]i. Microdialysis of myosin light chain kinase and its Ca(2+)-independent fragment (but not the heat-denatured fragment, albumin, trypsin plus soybean inhibitor or buffer) into one hepatocyte of a doublet resulted in canalicular contraction in 86% of doublets. Injection of myosin light chain kinase or its Ca(2+)-independent fragment did not increase [Ca2+]i within 5 min. These results indicate that (a) the basolateral plasma membrane of hepatocytes has a P2Y-class purinoceptor, (b) increased [Ca2+]i after incubation with ATP is initially due to mobilization from internal sites and (c) canalicular contraction is directly related to [Ca2+]i and activation of an actin-myosin system. The physiological role of extracellular ATP in canalicular contraction is uncertain.
Assuntos
Trifosfato de Adenosina/metabolismo , Canalículos Biliares/fisiologia , Cálcio/metabolismo , Espaço Extracelular/metabolismo , Membranas Intracelulares/metabolismo , Fígado/metabolismo , Animais , Canalículos Biliares/efeitos dos fármacos , Citocalasina D/farmacologia , Diálise , Fígado/citologia , Quinase de Cadeia Leve de Miosina/metabolismo , Nucleotídeos/metabolismo , Concentração Osmolar , RatosRESUMO
Angiotensin II (ANG II) is a principal secretagogue of adrenal zona glomerulosa (ZG) cells. The transduction process includes a depolarization of the plasma membrane and the activation of calcium influx. The ANG II-induced depolarization is associated with an increase in total membrane resistance. To directly address the mechanism underlying these observations, we examined the effect of ANG II on K+ currents of rat, bovine, and human ZG cells, using whole cell patch clamp. Although some differences were seen in the characteristics of K+ currents between species, ANG II consistently blocked outward currents in ZG cells [rat: 47.1 +/- 4.5% (SE), n = 17; bovine: 38.6 +/- 3.3%, n = 21; and human: 13-63%, n = 3]. With the use of the cell-attached mode, single-channel recordings in bovine ZG cells demonstrated K+ channels that were reversibly blocked when ANG II was added to the bath solution. This indicates that the block of K+ channels by ANG II involves a diffusible intracellular messenger rather than a direct receptor-channel interaction. The decreased conductance of K+ can account for the ANG II-induced membrane depolarization.