RESUMO
Patch-clamp recording combined with indo-l measurement of free intracellular calcium concentration ([Ca2+]i) was used to determine the homeostatic systems involved in the maintenance of resting [Ca2+]I and in the clearance of Ca2+ transients following activation of voltage-gated Ca2+ channels in neurones cultured from rat superior cervical ganglion (SCG). The Ca2+ binding ratio was estimated to be approximately 500 at 100 nM, decreasing to approximately 250 at [Ca2+]i approximately 1 pM, and to involve at least two buffering systems with different affinities for Ca2+. Removal of extracellular Ca2+ led to a decrease in[Ca2+]i that was mimicked by the addition of La3+, and was more pronounced after inhibition of the endoplasmic reticulum Ca2+ uptake system (SERCA). Inhibition of the plasma membrane Ca2+ pump (PMCA) by extracellular allkalinisation (pH 9) or intracellular carboxyeosin both increased resting [Ca2+]i and prolonged the recovery of Ca2+ transients at peak [Ca2+]i C 500 nM. For [Ca2+]i loads >500 nM, recovery showed an additional plateau phase that was abolished i nm-chlorophenylhydrazone (CCCP) or on omitting intracellular Na+. Inhibition of the plasma membrane Na+ -Ca2+ exchanger (NCX) and of SERCA had a small but significant additional effect on the rate of decay of these larger Ca2+ transients. In conclusion, resting [Ca2+]i is maintained by passive Ca2+ influx and regulated by a large Ca2+ buffering system, Ca2+ extrusion via a PMCA and Ca2+ transport from the intracellular stores. PMCA is also the principal Ca2+ extrusion system at low Ca2+ loads, with additional participation of the NCX and intracellular organelles at high [Ca2+]i.
Assuntos
ATPases Transportadoras de Cálcio/fisiologia , Cálcio/metabolismo , Membranas Intracelulares/metabolismo , Neurônios/metabolismo , Gânglio Cervical Superior/metabolismo , Animais , Soluções Tampão , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions , Células Cultivadas , Eletrofisiologia , Homeostase , Imuno-Histoquímica , Mitocôndrias/fisiologia , Modelos Neurológicos , Concentração Osmolar , Técnicas de Patch-Clamp , ATPases Transportadoras de Cálcio da Membrana Plasmática , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Trocador de Sódio e Cálcio/fisiologia , Gânglio Cervical Superior/citologia , Fatores de TempoRESUMO
M-type K(+) currents (I(K(M))) play a key role in regulating neuronal excitability. In sympathetic neurons, M-channels are thought to be composed of a heteromeric assembly of KCNQ2 and KCNQ3 K(+) channel subunits. Here, we have tried to identify the KCNQ subunits that are involved in the generation of I(K(M)) in hippocampal pyramidal neurons cultured from 5- to 7-day-old rats. RT-PCR of either CA1 or CA3 regions revealed the presence of KCNQ2, KCNQ3, KCNQ4 and KCNQ5 subunits. Single-cell PCR of dissociated hippocampal pyramidal neurons gave detectable signals for only KCNQ2, KCNQ3 and KCNQ5; where tested, most also expressed mRNA for the vesicular glutamate transporter VGLUT1. Staining for KCNQ2 and KCNQ5 protein showed punctate fluorescence on both the somata and dendrites of hippocampal neurons. Staining for KCNQ3 was diffusely distributed whereas KCNQ4 was undetectable. In perforated patch recordings, linopirdine, a specific M-channel blocker, fully inhibited I(K(M)) with an IC(50) of 3.6 +/- 1.5 microM. In 70 % of these cells, TEA fully suppressed I(K(M)) with an IC(50) of 0.7 +/- 0.1 mM. In the remaining cells, TEA maximally reduced I(K(M)) by only 59.7 +/- 5.2 % with an IC(50) of 1.4 +/- 0.3 mM; residual I(K(M)) was abolished by linopirdine. Our data suggest that KCNQ2, KCNQ3 and KCNQ5 subunits contribute to I(K(M)) in these neurons and that the variations in TEA sensitivity may reflect differential expression of KCNQ2, KCNQ3 and KCNQ5 subunits.
Assuntos
Hipocampo/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , Animais , Células Cultivadas , Condutividade Elétrica , Imunofluorescência , Indóis/farmacologia , Canais de Potássio KCNQ , Canal de Potássio KCNQ2 , Canal de Potássio KCNQ3 , Reação em Cadeia da Polimerase , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Piridinas/farmacologia , Ratos , Tetraetilamônio/farmacologia , Distribuição TecidualRESUMO
Antisense approaches are increasingly used to dissect signaling pathways linking cell surface receptors to intracellular effectors. Here we used a recombinant adenovirus to deliver G-protein alpha(q) antisense into rat superior cervical ganglion (SCG) neurons and neuronal cell lines to dissect G(alpha)(q)-mediated signaling pathways in these cells. This approach was compared with other G(alpha)(q) gene knockdown strategies, namely, antisense plasmid and knockout mice. Infection with adenovirus expressing G(alpha)(q) antisense (G(alpha)(q)AS AdV) selectively decreased immunoreactivity for the G(alpha)(q) protein. Expression of other G(alpha) protein subunits, such as G(alpha)(oA/B,) was unaltered. Consistent with this, modulation of Ca(2+) currents by the G(alpha)(q)-coupled M(1) muscarinic receptor was severely impaired in neurons infected with G(alpha)(q)AS AdV whereas modulation via the G(alpha)(oA)-coupled M(4) muscarinic receptor was unchanged. In agreement, activation of phospholipase C and consequent mobilization of intracellular Ca(2+) by UTP receptors was lost in NG108-15 cells infected with G(alpha)(q)AS AdV but not in cells infected with the control GFP-expressing adenovirus. Results obtained with this recombinant AdV strategy qualitatively and quantitatively replicated results obtained using SCG neurons microinjected with G(alpha)(q) antisense plasmids or SCG neurons from G(alpha)(q) knockout mice. This combined antisense/recombinant adenoviral approach can therefore be useful for dissecting signal transduction mechanisms in SCG and other neurons.
Assuntos
Sinalização do Cálcio/fisiologia , Neurônios/fisiologia , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Gânglio Cervical Superior/fisiologia , Adenoviridae/genética , Animais , Sequência de Bases , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Vetores Genéticos , Modelos Neurológicos , Neurônios/efeitos dos fármacos , Ratos , Ratos Mutantes , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/fisiologiaRESUMO
1. The relationship between muscarinic receptor activation, phosphoinositide turnover, calcium mobilisation and M-current inhibition has been studied in rat superior cervical ganglion (SCG) neurones in primary culture. 2. Phosphoinositide-specific phospholipase C (PLC) stimulation was measured by the accumulation of [3H]-cytidine monophosphate phosphatidate (CMP-PA) after incubation with [3H]-cytidine in the presence of Li+. The muscarinic agonist oxotremorine methiodide (oxo-M) stimulated PLC in a dose-dependent manner with an EC50 of approximately 3.5 microM. 3. The concentration-response curve for oxo-M was shifted to the right by a factor of about 10 by pirenzepine (100 nM), suggesting a pKB (-log of the apparent dissociation constant) of 7.9 +/- 0.4, while himbacine (1 microM) shifted the curve by a factor of about 13 (pKB approximately 7.1 +/- 0.6). This indicates involvement of the M1 muscarinic receptor in this response. 4. The accumulation of CMP-PA was localised by in situ autoradiography to SCG principal neurones, with no detectable signal in glial cells present in the primary cultures. 5. The ability of oxo-M to release Ca2+ from inositol(1,4, 5)trisphosphate (InsP3)-sensitive stores was determined by fura-2 microfluorimetry of SCG neurones voltage clamped in perforated patch mode. Oxo-M failed to evoke intracellular Ca2+ (Ca2+i) mobilisation in SCG neurones voltage clamped at -60 mV, but produced a significant Ca2+i rise (67 +/- 15 nM, n = 9) in cells voltage clamped at -25 mV. 6. Thapsigargin (0.5-1 microM) caused a 70 % inhibition of the oxo-M-induced Ca2+i increase, indicating its intracellular origin, while oxo-M-induced inhibition of M-current in the same cells was unaffected by thapsigargin. 7. Our results do not support the involvement of InsP3-sensitive calcium mobilisation in M-current inhibition.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Neurônios/fisiologia , Fosfatidilinositóis/metabolismo , Canais de Potássio/fisiologia , Receptores Muscarínicos/fisiologia , Sistema Nervoso Simpático/fisiologia , Algoritmos , Animais , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Monofosfato de Citidina/metabolismo , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Agonistas Muscarínicos/farmacologia , Neurônios/efeitos dos fármacos , Oxotremorina/farmacologia , Fosfatidilinositóis/fisiologia , Canais de Potássio/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M1 , Receptores Muscarínicos/efeitos dos fármacos , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/efeitos dos fármacos , Fosfolipases Tipo C/metabolismoRESUMO
Using the ratiometric Ca2+ indicator, indo-1, the antigen-induced increase in intracellular Ca2+ concentration ([Ca2+]i) was measured in individual RBL-2H3 cells which had been passively sensitized with monoclonal antibody to the dintrophenyl (DNP) haptenic group. Antigenic stimulation using DNP-human serum albumin conjugate (DNP-HSA) induced concentration-dependent asynchronous Ca2+ oscillations, or irregular spikes. To achieve a quantitative comparison of the effects of different concentrations of antigen on changes in Ca2+[i, the area under the curve (AUC) of Ca2+ oscillations in each cell was calculated. The dose-response curve of the calculated AUC is consistent with the bell-shaped dose-response curve for antigen-induced mediator release, depolarization and 86Rb(+)-efflux. Ca2+ oscillations induced by antigenic stimulation were abolished by removal of external Ca2+ and the subsequent reintroduction of external Ca2+ caused their resumption. To investigate the role of Ca2+ oscillations in the secretory response, changes in [Ca2+]i induced by concanavalin A (Con-A), A23187, thapsigargin and NECA were also monitored. Con-A mimicked the response induced by antigen, whilst A23187 and thapsigargin induced a large transient non-oscillatory response. NECA, an adenosine receptor agonist, induced only a small transient rise in Ca2+[i without oscillatory behaviour. Since all these stimuli accept NECA-induced degranulation in these cells, it is suggested that, although Ca2+ oscillations are not essential for the initiation of secretion, they probably underlie the in-vivo physiological response of mast cells and basophils to an antigenic challenge. They also seem to enhance the efficacy of the Ca2+ signal.
Assuntos
Antígenos/metabolismo , Sinalização do Cálcio , Leucemia/metabolismo , Adenosina-5'-(N-etilcarboxamida)/farmacologia , Animais , Antígenos/farmacologia , Calcimicina/farmacologia , Polaridade Celular/efeitos dos fármacos , Concanavalina A/farmacologia , Dinitrofenóis/farmacologia , Relação Dose-Resposta a Droga , Ionóforos/farmacologia , Leucemia/tratamento farmacológico , Potenciais da Membrana/efeitos dos fármacos , Radioisótopos/metabolismo , Ratos , Receptores de IgE/efeitos dos fármacos , Receptores de IgE/metabolismo , Rubídio/metabolismo , Albumina Sérica/farmacologia , Células Tumorais Cultivadas , beta-N-Acetil-Hexosaminidases/efeitos dos fármacos , beta-N-Acetil-Hexosaminidases/metabolismoRESUMO
1. We have tested whether neurones show a swelling-induced Cl- current following hypotonic shock, by recording membrane current responses and cell volume changes in voltage clamped isolated rat sympathetic neurones during application of hypotonic solutions. 2. Using both whole-cell and perforated patch recording methods, hypotonic solution caused cell swelling and the activation of an inward Cl- current at -60 mV. This current showed weak outward rectification with no obvious time dependence. It was inhibited by SITS (0.3-1 mM), NPPB (30-300 microM) and niflumic acid (50-200 microM), but not by tamoxifen (10 microM). 3. Hypotonic solution did not cause a rise in intracellular Ca2+ concentration as measured by simultaneous indo-1 fluorescence. Also, neither the volume change nor Cl- current were affected by the removal of external Ca2+ or internal Ca2+ buffering to < or = 1 nM with EGTA. 4. The Cl- current was unaffected by an inhibitor of protein kinase C (PKC; GF109203X, 3 microM) or by omission of ATP from the pipette solution. 5. Cells exhibited a regulatory volume decrease during sustained exposure to hypotonic solution. This was completely inhibited by 0.5 mM niflumic acid. 6. It is concluded that osmotic swelling induces an outwardly rectifying, Ca2(+)- and PKC-independent Cl- current in these nerve cells. It is suggested that this current may be involved in volume regulatory mechanisms.
Assuntos
Cloretos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/metabolismo , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , Animais , Cálcio/metabolismo , Tamanho Celular , Canais de Cloreto/antagonistas & inibidores , Soluções Hipotônicas , Técnicas In Vitro , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana , Neurônios/efeitos dos fármacos , Ácido Niflúmico/farmacologia , Pressão Osmótica , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Gânglio Cervical Superior/efeitos dos fármacos , Tamoxifeno/farmacologiaRESUMO
We have studied the properties of r-eag voltage-activated potassium channels in a stably transfected human embryonic kidney cell line. It was found that r-eag channels are rapidly and reversibly inhibited by a rise in intracellular calcium from 30 to 300 nM. The inhibition does not appear to depend on the activity of calcium-dependent kinases and phosphatases. The effect of calcium on r-eag channel activity was studied in inside-out membrane patches. Calcium inhibited r-eag channel activity with a mean IC50 of 67 nM. Activation of muscarinic receptors, generating calcium oscillations in the transfected cells, induced a synchronous inhibition of r-eag mediated outward currents. This shows that calcium can mediate r-eag current inhibition following muscarinic receptor activation. The data indicate that r-eag channels are calcium-inhibitable voltage-activated potassium channels.
Assuntos
Cálcio/metabolismo , Receptores Muscarínicos/metabolismo , Animais , Linhagem Celular , Eletrofisiologia , Canais de Potássio Éter-A-Go-Go , Humanos , Ionomicina/farmacologia , Rim/citologia , Rim/metabolismo , Canais de Potássio/metabolismo , Ratos , Espectrometria de Fluorescência , TransfecçãoRESUMO
Using perforated patch recordings in combination with intracellular Ca2+ ([Ca2+]i) fluorescence measurements, we have identified a delayed Ca(2+)-dependent Cl- current in a mammalian sympathetic ganglion cell. This Cl- current is induced by the synergistic action of Ca2+ and diacylglycerol (DAG) and is blocked by inhibitors of protein kinase C. As a result, the current can be induced by acetylcholine through the conjoint activation of nicotinic receptors (to produce a rise in [Ca2+]i) and muscarinic receptors (to generate DAG). This demonstrates an unusual form of synergism between the two effects of a single transmitter mediated via separate receptors operating within a time scale that could be of physiological significance.
Assuntos
Cálcio/metabolismo , Canais de Cloreto/fisiologia , Neurônios/fisiologia , Proteína Quinase C/metabolismo , Receptores Muscarínicos/fisiologia , Acetilcolina/farmacologia , Animais , Cálcio/farmacologia , Diglicerídeos/farmacologia , Sinergismo Farmacológico , Condutividade Elétrica , Gânglios Simpáticos/citologia , Proteína Quinase C/antagonistas & inibidores , Ratos , Receptores Muscarínicos/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/fisiologiaRESUMO
Severe adverse reactions to neuroleptic medications are not uncommon and include the neuroleptic malignant syndrome, rhabdomyolysis, and acute renal failure. The neuroleptic malignant syndrome consists of hyperthermia, diaphoresis, tachycardia, tachypnea, abnormal blood pressure, alteration of consciousness, and extrapyramidal rigidity. Rhabdomyolysis--which might be due to hyperthermia, muscle rigidity, and/or metabolic changes in skeletal muscle function--results in acute renal failure. We report a patient with rhabdomyolysis and acute renal failure that developed after large doses of haloperidol were given, but without muscle rigidity or hyperthermia. This patient's presentation illustrates that high-dose haloperidol therapy might cause rhabdomyolysis and acute renal failure without significant rigidity or hyperthermia.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Antipsicóticos/efeitos adversos , Haloperidol/efeitos adversos , Síndrome Maligna Neuroléptica/diagnóstico , Rabdomiólise/induzido quimicamente , Adulto , Antipsicóticos/uso terapêutico , Haloperidol/uso terapêutico , Humanos , Masculino , Esquizofrenia/tratamento farmacológicoRESUMO
1. Acetylcholine (ACh) produces two membrane current changes when applied to NG108-15 mouse neuroblastoma x rat glioma hybrid cells transformed (by DNA transfection) to express m1 muscarinic receptors: it activates a Ca(2+)-dependent K+ conductance, producing an outward current, and it inhibits a voltage-dependent K+ conductance (the M conductance), thus diminishing the M-type voltage-dependent K+ current (IK(M)) and producing an inward current. The present experiments were undertaken to find out how far inhibition of IK(M) might be secondary to stimulation of phospholipase C, by recording membrane currents and intracellular Ca2+ changes with indo-1 using whole-cell patch-clamp methods. 2. Bath application of 100 microM ACh reversibly inhibited IK(M) by 47.3 +/- 3.2% (n = 23). Following pressure-application of 1 mM ACh, the mean latency to inhibition was 420 ms at 35 degrees C and 1.79 s at 23 degrees C. Latencies to inhibition by Ba2+ ions were 148 ms at 35 degrees C and 92 ms at 23 degrees C. 3. The involvement of a G-protein was tested by adding 0.5 mM GTP-gamma-S or 10 mM potassium fluoride to the pipette solution. These slowly reduced IK(M), with half-times of about 30 and 20 min respectively, and rendered the effect of superimposed ACh irreversible. Effects of ACh were not significantly changed after pretreatment for 24 h with 500 ng ml-1 pertussis toxin or on adding up to 10 mM GDP-beta-S to the pipette solution. 4. The role of phospholipase C and its products was tested using neomycin (to inhibit phospholipase C), inositol 1,4,5-trisphosphate (InsP3) and inositol 1,3,4,5-tetrakisphosphate (InsP4), heparin, and phorbol dibutyrate (PDBu) and staurosporin (to activate and inhibit protein kinase C respectively). Both neomycin (1 mM external) and InsP3 (100 microM intrapipette) inhibited the ACh-induced outward current and/or intracellular Ca2+ transient but did not block ACh-induced inhibition of IK(M). Intrapipette heparin (1 mM) blocked activation of IK(Ca) and reduced Ach-induced inhibitions of IK(M), but also reduced inhibition of ICa via endogeneous m4 receptors. PDBu (with or without intrapipette ATP) and staurosporin had no significant effects.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Glioma/metabolismo , Células Híbridas/metabolismo , Neuroblastoma/metabolismo , Canais de Potássio/efeitos dos fármacos , Receptores Muscarínicos/efeitos dos fármacos , Acetilcolina/farmacologia , Animais , Bradicinina/farmacologia , Cálcio/metabolismo , DNA de Neoplasias/biossíntese , DNA de Neoplasias/fisiologia , Eletrofisiologia , Proteínas de Ligação ao GTP/metabolismo , Glioma/enzimologia , Células Híbridas/efeitos dos fármacos , Células Híbridas/enzimologia , Inosina Trifosfato/metabolismo , Camundongos , Neuroblastoma/enzimologia , Óxido Nítrico/metabolismo , Fosfolipases A/antagonistas & inibidores , Fosfolipases A/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Suínos , Transfecção , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismoRESUMO
1. Patch-clamp techniques in conjunction with indo-1 fluorescent measurements were used to measure increases in intracellular free calcium concentration and membrane conductance induced by the activation of nicotinic and calcium channels in cultured rat sympathetic neurons. 2. Under voltage-clamp conditions, pressure application of the nicotinic agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide, 100 microM, 100 ms) increased [Ca2+]i by 193 +/- 26 nM at a clamp potential of -60 mV. This was accompanied by an inward current of -4.53 +/- 0.89 nA, giving a mean ratio of the delta (Ca2+]i to the total inward charge transfer of 42.7 nmoles per litre of free calcium per nanocoulomb of charge (M/q ratio). 3. The DMPP-induced current and associated delta [Ca2+]i were reduced by mecamylamine (100 nM-10 microM) but were unaffected by alpha-bungarotoxin (100 nM) or cadmium (100 microM). 4. The M/q ratio was not affected by the holding potential (from -80 to -40 mV) but was a function of the external calcium concentration. 5. The M/q ratio was reduced by increasing the intracellular calcium buffering capacity and increased by heparin but not affected by ryanodine or by depletion of the caffeine-sensitive calcium store. 6. Under the same recording conditions, we quantified the increase in [Ca2+]i associated with activation of the voltage-dependent calcium current. On average at -60 mV, the M/q ratio of this highly calcium-selective permeability was 1961 mM nC-1, which is 46 times that obtained for the nicotinic channel. 7. Assuming constant-field theory, ion-substitution experiments suggest that in 2.5 mM external calcium, the permeability sequence for the nicotinic conductance was Cs+ < Li+ < Na+ < K+ < Ca2+. 8. We conclude that the nicotinic channels in rat sympathetic neurones are significantly permeant to Ca2+ and that the influx of Ca2+ through these channels is the principal cause of the rise in [Ca2+]i seen under voltage clamp.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Receptores Nicotínicos/metabolismo , Gânglio Cervical Superior/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Iodeto de Dimetilfenilpiperazina/farmacologia , Homeostase , Líquido Intracelular/metabolismo , Potenciais da Membrana , Permeabilidade , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/efeitos dos fármacosRESUMO
We have identified the species of K+ channel that underlies the neuronal M-current in rat sympathetic ganglion cells. The channels were kinetically and pharmacologically defined using outside-out and cell-attached patches. They exhibited multiple conductance levels, predominantly 3-9 pS. Their slow gating in response to voltage change in outside-out patches was exhibited only in the presence of AIF-4 or GTP gamma S on the inner membrane surface and when the lower conductance states were dominant. In the absence of AIF-4 or GTP gamma S, the channels exhibited rapid activation and deactivation. We conclude that M-channel gating may be controlled by an associated GTP-binding protein.
Assuntos
Compostos de Alumínio , Fluoretos , Gânglios Simpáticos/metabolismo , Canais Iônicos/fisiologia , Alumínio/farmacologia , Animais , Bário/farmacologia , Condutividade Elétrica , Eletrofisiologia , Flúor/farmacologia , Gânglios Simpáticos/citologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Cinética , Tempo de ReaçãoRESUMO
Muscarinic acetylcholine receptor (mAChR) subtype (m1-m4)-specific cDNAs were transfected into NL308 neuroblastoma-fibroblast hybrid cells and clones expressing each of the individual mAChR subtypes m1, m2, m3 and m4 obtained. Acetylcholine increased phosphoinositide (PI) turnover in m1- and m3-transformed cells, but did not produce detectable changes in m2- and m4-transformed cells. In cells expressing m1 and m3 subtypes, ACh produced an initial outward K+ current, followed by a cationic current. In cells expressing m2 and m4 receptors, only the initial K+ current was detected. The outward currents were associated with a rise in intracellular Ca2+ as measured with Fura-2 or Indo-1, and were inhibited by chelating intracellular Ca2+ with external BAPTA-AM, or by external charybdotoxin or Ba2+: hence they were attributed to the activation of a Ca(2+)-dependent K+ current. However, the outward current produced in m2- and m4-transformed cells was blocked by pretreatment with 5 ng ml-1 Pertussis toxin (PTX), whereas that in m1- and m3-transformed cells was not. These results suggest that m2- and m4-receptors in transformed NL308 cells coupled to PTX-sensitive G-protein which is capable of mobilizing intracellular Ca2+ and activate IK(Ca), whereas m1 and m3 receptors activate a similar process through a different, PTX-insensitive G-protein.
Assuntos
Cálcio/metabolismo , Cálcio/farmacologia , Canais de Potássio/fisiologia , Receptores Muscarínicos/fisiologia , Acetilcolina/farmacologia , Animais , Bário/farmacologia , Quelantes/farmacologia , DNA/genética , Fibroblastos , Proteínas de Ligação ao GTP/metabolismo , Células Híbridas/efeitos dos fármacos , Células Híbridas/fisiologia , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Neuroblastoma , Toxina Pertussis , Canais de Potássio/efeitos dos fármacos , Regiões Promotoras Genéticas , Quinuclidinil Benzilato/metabolismo , Receptores Muscarínicos/efeitos dos fármacos , Receptores Muscarínicos/genética , Vírus 40 dos Símios/genética , Transfecção , Fatores de Virulência de Bordetella/farmacologiaRESUMO
1. The M-like current IK(M,ng) in differentiated NG108-15 mouse neuroblastoma x rat glioma hybrid cells has been studied using tight-seal, whole-cell patch-clamp recording. 2. When calculated from steady-state current-voltage curves, the conductance underlying IK(M,ng) showed a Boltzmann dependence on voltage with half-activation voltage Vo = -44 mV (in 3 mM [K+]) and slope factor (a) = 8.1 mV/e-fold increase in conductance. In 12 mM [K+] Vo = -38 mV and a = 6.9 mV. The deactivation reciprocal time constant accelerated with hyperpolarization with slope factor 17 mV/e-fold voltage change. 3. The reversal potential for deactivation tail currents varied with external [K+] as if PNa/PK were 0.005. 4. Steady-state current was increased on removing external Ca2+. In the presence of external Ca2+, reactivation of IK(M, ng) after a hyperpolarizing step was delayed. This delay was preceded by an inward Ca2+ current, and coincided with an increase in intracellular [Ca2+] as measured with Indo-1 fluorescence. Elevation of intracellular [Ca2+] with caffeine also reduced IK(M, ng). 5. IK(M, ng) was inhibited by external divalent cations in decreasing order of potency (mM IC50 in parentheses): Zn2+ (0.011) greater than Cu2+ (0.018) greater than Cd2+ (0.070) greater than Ni2+ (0.44) greater than Ba2+ (0.47) greater than Fe2+ (0.69) greater than Mn2+ (0.86) greater than Co2+ (0.92) greater than Ca2+ (5.6) greater than Mg2+ (16) greater than Sr2+ (33). This was not secondary to inhibition of ICa since: (i) inhibition persisted in Ca(2+)-free solution; (ii) La3+ did not inhibit IK(M, ng) at concentrations which inhibited ICa; and (iii) organic Ca2+ channel blockers were ineffective. Inhibition comprised both depression of the maximum conductance and a positive shift of the activation curve. Addition of Ca2+ (10 microM free [Ca2+]) or Ba2+ (1 mM total [Ba2+]) to the pipette solution did not significantly change IK(M, ng). 6. IK(M, ng) was reduced by 9-amino-1,2,3,4-tetrahydroacridine (IC50 8 microM) and quinine (30 microM) but was insensitive to tetraethylammonium (IC50 greater than 30 mM), 4-aminopyridine (greater than 10 mM), apamin (greater than 3 microM) or dendrotoxin (greater than 100 nM). 7. IK(M, ng) was inhibited by bradykinin (1-10 microM) or angiotensin II (1-10 microM), but not by the following other receptor agonists: acetylcholine (10 mM), muscarine (10 microM), noradrenaline (100 microM), adrenaline (100 microM), dopamine (100 microM), histamine (100 microM), 5-hydroxytryptamine (10 microM), Met-enkephalin (1 microM), glycine (100 microM), gamma-aminobutyric acid (100 microM) or baclofen (500 microM).(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Neurônios/metabolismo , Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Cálcio/farmacologia , Cátions Bivalentes/farmacologia , Glioma/metabolismo , Células Híbridas/efeitos dos fármacos , Células Híbridas/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Neuroblastoma/metabolismo , Neurônios/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , RatosRESUMO
Pharmacological blocking agents were used to assess the contributions of different K(+)-currents to spike-repolarization and early spike-afterhyperpolarization recorded in dissociated, tissue-cultured rat superior cervical sympathetic neurones using both patch-clamp and impalement microelectrode techniques. Effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA), in concentrations which selectively reduced the delayed rectifier current IK(DR) and Ca(2+)-activated K(+)-current IK(Ca, fast), respectively, indicated that IK(DR) made a significant contribution to both spike repolarization and spike afterhyperpolarization under all recording conditions, whereas the contribution of IK(Ca,fast) depended on the level of intracellular Ca(2+)-buffering. No evidence for a significant role for the transient current IK(A) could be adduced in these experiments.
Assuntos
Cádmio/farmacologia , Cálcio/farmacologia , Cloretos/farmacologia , Gânglios Simpáticos/fisiologia , Neurônios/fisiologia , Canais de Potássio/fisiologia , 4-Aminopiridina/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cloreto de Cádmio , Células Cultivadas , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Cinética , Neurônios/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Ratos , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
A new restriction endonuclease, SrfI has been isolated from an unidentified species of Streptomyces. SrfI recognizes the 8-bp palindrome, 5'-GCCCGGGC and cleaves double-stranded DNA after the third C in the sequence, producing blunt ends. SrfI is a rare-cutting enzyme and should therefore be useful for megabase mapping.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Oligonucleotídeos/química , Streptomyces/enzimologia , Adenoviridae/metabolismo , Clonagem Molecular , DNA Viral/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/isolamento & purificação , Especificidade por SubstratoRESUMO
IM is a voltage- and time-dependent K+ current that is suppressed by muscarinic receptor activation. IM augmentation following agonist washout was blocked by heavily buffering [Ca2+]i using BAPTA. Although IM is not primarily Ca2+ dependent, small increases in [Ca2+]i by photolysis of the "caged" Ca2+ chelator nitr-5 or by evoking action potentials augmented, while larger increases inhibited, IM. Raising [Ca2+]i for prolonged periods, by nitr-5 photolysis, reduced its sensitivity to agonist, leaving a poorly reversible response. These results suggest that IM can be regulated by physiologically relevant changes in [Ca2+]i, placing IM in a unique position to modulate cell excitability.
Assuntos
Cálcio/fisiologia , Membranas Intracelulares/fisiologia , Neurônios/fisiologia , Potássio/fisiologia , Sistema Nervoso Simpático/fisiologia , Animais , Soluções Tampão , Quelantes , Ácido Egtázico/farmacologia , Potenciais Evocados , Fotólise , Rana catesbeiana , Sistema Nervoso Simpático/citologiaRESUMO
1. Whole-cell recordings of M-currents and single-channel recordings have been made in cultured rat sympathetic ganglion (SCG) neurones using the patch clamp technique. 2. Muscarine caused a reduction in macroscopic M-current relaxations, induced by voltage steps, and a concomitant reduction in whole-cell current noise. Power spectra of the muscarine-sensitive component of current noise were fitted with two Lorentzian components corresponding, on average, to 162 and 15 ms. The longer time constant was very similar to that of deactivation tail currents measured at the same potential. 3. The single-channel conductance at -30 mV was estimated from power density spectra and whole-cell current-variance relationships to be 1-2 pS. 4. Putative single M-channels, activated by depolarization, were identified in cell-attached and outside-out patches from cultured SCG neurones. In particular, the ensemble average of a small amplitude channel (estimated to be ca4 pS in physiological [K+]) in a cell-attached patch, exhibited a similar time dependence to whole-cell M-current.
Assuntos
Gânglios Simpáticos/fisiologia , Canais Iônicos/fisiologia , Neurônios/fisiologia , Potássio/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Células Cultivadas , Cinética , Muscarina/farmacologia , Canais de Potássio/fisiologia , Ratos , Fatores de TempoRESUMO
THA (Tacrine) is an anticholinesterase drug reported to alleviate cognitive deficit in Alzheimer's disease. We have used rat isolated superior cervical sympathetic ganglia as a model mammalian cholinergic neural system to study effects of THA on cholinergic synaptic transmission and postsynaptic membrane currents. At 0.1 - 3 microM, THA augmented the postsynaptic depolarizations and inward clamp currents produced by acetylcholine but not by the cholinesterase-resistant analogue, DMPP. Higher concentrations depressed these responses to both acetylcholine and DMPP, and reduced the acetylcholine-induced increase in membrane current noise. At 1 microM, THA did not affect the amplitude or time-course of fast (nicotinic) excitatory postsynaptic currents (epscs) evoked by single orthodromic volleys, but higher concentrations induced a biphasic epsc decay. In contrast, low concentrations of THA (1 - 3 microM) greatly augmented and prolonged the muscarinic slow epsc evoked by repetitive orthodromic volleys: this effect was blocked by 1 microM atropine. Concentrations above 0.1 mM produced a membrane depolarization and inhibited a variety of membrane ionic currents, including voltage-gated Ca current and subsequent Ca-activated K currents, and voltage-gated M- and A-type K currents. It is concluded that the principal effect of THA is to inhibit cholinesterase, and that the main consequence of this is to augment and prolong the muscarinic slow epsc. In contrast, the nicotinic fast epsc is not increased but instead may be reduced through a nicotinic channel-blocking action. Although THA could also block several other ion channels the concentrations required were too high to contribute significantly to its principal pharmacological actions on ganglionic transmission.
RESUMO
1. Under voltage-clamp dissociated adult and foetal rat superior cervical ganglion (s.c.g.) cells exhibited a non-inactivating voltage- and time-dependent component of K+ current termed the M-current (IM). IM was detected and measured from the current decay during hyperpolarizing voltage steps applied from potentials where IM was pre-activated. 2. Neither the resting membrane current nor the amplitude of these current decay relaxations were reduced by omitting Ca from the bathing fluid, showing that the M-current was not a 'Ca-activated' K-current dependent on a primary Ca-influx. Concentrations of (+)-tubocurarine sufficient to block the slow Ca-activated K-current IAHP did not inhibit IM or antagonize the effect of muscarinic agonists on IM, showing that IM was not contaminated by IAHP. Tetraethylammonium (1 mM), which blocks the fast Ca-activated K-current IC, produced a small inhibition of IM. This was not due to contamination of IM by IC since muscarinic agonists did not consistently block IC. 3. The muscarinic agonists muscarine, oxotremorine, McN-A-343 and methacholine reversibly suppressed IM, resulting in an inward (depolarizing) current. The rank order of potency was: oxotremorine greater than or equal to muscarine greater than McN-A-343 greater than methacholine. 4. The suppression of IM by muscarine was similar in cultured cells derived from adult and foetal tissue to that seen in the intact ganglia. 5. IM-suppression by muscarine was inhibited by pirenzepine (Pz) and AF-DX 116 with mean pKB values of 7.53 +/- 0.13 (n = 3) and 6.02 +/- 0.13 (n = 4) respectively. 6. The suppression of IM by muscarinic agonists was not affected by gallamine (10-30 microM). 4-Diphenylacetoxy-N-methylpiperidine methiodide inhibited the response at 300 nM. 7. Pirenzepine inhibited the contractions of the guinea-pig isolated ileum produced by muscarine with a mean pKB of 6.37 +/- 0.03 (n = 8). 8. These results suggest that the receptors mediating suppression of the M-current accord with those designated pharmacologically as M1 and that these receptors reach maturity at a very early stage in the development of the rat s.c.g.