RESUMO
Patch-clamp recording was used to study rectifying K+ currents in myenteric neurons in short-term culture. In conditions that suppressed Ca2+ -activated K+ current, three kinds of voltage-activated K+ currents were identified by their voltage range of activation, inactivation, kinetics and pharmacology. These were A-type current, delayed outwardly rectifying current (I(K),dr) and inwardly rectifying current (I(K),ir). I(K),ir consisted of an instantaneous component followed by a time-dependent current that rapidly increased at potentials negative to -80 mV. Time-constant of activation was voltage-dependent with an e-fold decrease for a 31-mV hyperpolarization amounting to a decrease from 800 to 145 ms between -80 and -100 mV. I(K),ir did not inactivate. I(K),ir was abolished in K+ -free solution. Increases in external K+ increased I(K),ir conductance in direct relation to the square root of external K+ concentration. Activation kinetics were accelerated and the activation range shifted to more positive K+ equilibrium potentials. I(K),ir was suppressed by external Cs+ and Ba2+ in a concentration-dependent manner. Ca2+ and Mg+ were less effective than Ba2+. I(K),ir was unaffected by tetraethylammonium ions. I(K),dr was activated at membrane potentials positive to - 30 mV with an e-fold decrease in time-constant of activation from 145 to 16 ms between -20 and 30 mV. It was half-activated at 5 mV and fully activated at 50 mV. Inactivation was indiscernible during 2.5 s test pulses. I(K),dr was suppressed in a concentration-, but not voltage-dependent manner by either tetraethylammonium or 4-aminopyridine and was insensitive to Cs+. The results suggest that I(K),ir may be important in maintaining the high resting membrane potentials found in afterhyperpolarization-type enteric neurons. They also suggest importance of I(K),ir channels in augmentation of the large hyperpolarizing after-potentials in afterhyperpolarization-type neurons and the hyperpolarization associated with inhibitory postsynaptic potentials. I(K),dr in afterhyperpolarization-type enteric neurons has overall kinetics and voltage behaviour like delayed rectifier currents in other excitable cells where the currents can also be distinguished from A-type and Ca2+ -activated K+ current.
Assuntos
Intestino Delgado/inervação , Plexo Mientérico/metabolismo , Neurônios/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Animais , Cátions Bivalentes/farmacologia , Césio/farmacologia , Condutividade Elétrica , Cobaias , Plexo Mientérico/citologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Tetraetilamônio/farmacologiaRESUMO
1. Whole-cell patch clamp methods were used to analyse voltage-dependent calcium currents in cultured myenteric neurones enzymatically isolated from adult guinea-pig small intestine. 2. Activation of G-proteins by intracellular administration of GTP-gamma-S (100-200 microM in pipette) decreased the amplitude of high voltage activated Ca2+ current (ICa) by more than 50%. Residual ICa was activated more slowly and was non-inactivating during 500 ms test pulses when GTP-gamma-S was included in the pipette solution. 3. Inclusion of 500 microM GDP-beta-S in the patch pipettes increased the amplitude of ICa by over 30% without altering the voltage-dependency. 4. Extracellular application of 2-chloroadenosine suppressed ICa dose-dependently by reducing both transient and sustained components of the current. 5. Pretreatment of the neurones with cholera toxin or forskolin did not alter the actions of GTP-gamma-S or GDP-beta-S or 2-chloroadenosine. 6. The results suggest that high threshold calcium channels in myenteric neurones are influenced by G-proteins and that the inhibitory action of 2-chloroadenosine on ICa involves G-protein coupling of the adenosine receptors to the Ca2+ channel.
Assuntos
Adenosina/farmacologia , Canais de Cálcio/efeitos dos fármacos , Proteínas de Ligação ao GTP/efeitos dos fármacos , Guanosina Trifosfato/farmacologia , Plexo Mientérico/efeitos dos fármacos , Animais , Células Cultivadas , Cobaias , Intestino Delgado/efeitos dos fármacos , Técnicas de Patch-Clamp , Fatores de TempoRESUMO
Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to investigate changes in levels of cyclic 3',5'-adenosine monophosphate (cAMP) in response to stimulation of adenylate cyclase by forskolin and inhibition of phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX). A linear relation with a positive correlation coefficient greater than 0.98 was found between: (1) amount of cAMP and number of ganglia; (2) amount of protein and number of ganglia; (3) amount of DNA and amount of protein; (4) amount of DNA and number of ganglia. Basal levels of cAMP were 2.25 +/- 0.21 fmol per ganglion for 900 ganglia. Forskolin stimulated a dose-dependent increase in cAMP over a concentration range of 0.05 to 50 microM, with a level of 18.6 +/- 4.9 fmol/ganglion at 50 microM forskolin. The inactive forskolin analog 1,9-dideoxyforskolin did not elevate cAMP. Addition of IBMX to the incubation medium stimulated a dose-dependent increase in cAMP over a concentration range of 0.1-1000 microM, with a level of 17.58 +/- 3.38 fmol/ganglion at 1000 microM IBMX. Application of 1 mM IBMX strongly potentiated the stimulating action of forskolin on cAMP levels. Our results derived from direct determination of cAMP changes in small intestinal myenteric ganglia are consistent with existing electrophysiological evidence for second messenger function of cAMP in slow synaptic modulation of excitability in AH/Type 2 neurons of the enteric nervous system.
Assuntos
AMP Cíclico/metabolismo , Intestino Delgado/metabolismo , Plexo Mientérico/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Colforsina/análogos & derivados , Colforsina/farmacologia , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , Gânglios/efeitos dos fármacos , Gânglios/enzimologia , Gânglios/metabolismo , Cobaias , Técnicas In Vitro , Masculino , Proteínas do Tecido Nervoso/metabolismoRESUMO
This study was designed to test the hypothesis that stimulation of adenylate cyclase and elevation of cAMP is involved in the signal transduction process for substance P, calcitonin gene-related peptide, vasoactive intestinal peptide, cholecystokinin or gastrin releasing peptide in myenteric ganglia. Enzymatically dissociated ganglia from the myenteric plexus of the guinea-pig small intestine were used to study changes in levels of cAMP in response to application of the brain-gut peptides in the presence and absence of forskolin. Application of substance P and calcitonin gene-related peptide were found to increase intraganglionic cAMP in a dose-dependent fashion when a phosphodiesterase inhibitor was present. The ED50 values for substance P and calcitonin gene-related peptide were 5 microM and 0.75 microM, respectively. The presence of forskolin in the incubation medium resulted in significant upward shifts of the dose-response curves for both peptides. Neither vasoactive intestinal peptide, cholecystokinin nor gastrin releasing peptide stimulated increases in intraganglionic cAMP under the same experimental conditions used for substance P and calcitonin gene-related peptide.
Assuntos
AMP Cíclico/análise , Gânglios/efeitos dos fármacos , Plexo Mientérico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , 4-(3-Butoxi-4-metoxibenzil)-2-imidazolidinona/farmacologia , Animais , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Colecistocinina/farmacologia , AMP Cíclico/fisiologia , Relação Dose-Resposta a Droga , Gânglios/química , Gânglios/fisiologia , Peptídeo Liberador de Gastrina , Cobaias , Intestino Delgado/inervação , Masculino , Plexo Mientérico/química , Plexo Mientérico/fisiologia , Peptídeos/farmacologia , Substância P/farmacologia , Peptídeo Intestinal Vasoativo/farmacologiaAssuntos
Ferricianetos/farmacologia , Músculo Liso/efeitos dos fármacos , Nitroglicerina/farmacologia , Nitroprussiato/farmacologia , Animais , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Cobaias , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/citologia , Receptores de Neurotransmissores/efeitos dos fármacosAssuntos
Trifosfato de Adenosina/fisiologia , Apamina/farmacologia , Venenos de Abelha/farmacologia , Músculo Liso/fisiologia , Norepinefrina/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Colo/fisiologia , Cobaias , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/efeitos dos fármacosAssuntos
Histamina/farmacologia , Canais Iônicos/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ureter/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/metabolismo , Estimulação Elétrica , Cobaias , Técnicas In Vitro , Potássio/metabolismo , Sódio/metabolismoAssuntos
Trifosfato de Adenosina/farmacologia , Músculo Liso/efeitos dos fármacos , Norepinefrina/farmacologia , Potássio/fisiologia , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Cobaias , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , SoluçõesRESUMO
The action of bile salts, saponin and tween-80 on miniature end-plate potentials (MEPP) and electrotonic potentials (ETP) of the muscle fibre membrane was studied in frog Rana ridibunda. Bile salts in a concentration of 10(-4) g/ml increased the frequency and amplitude of MEPP. The input resistance of the muscle fibre membrane decreased under the action of these substances. At higher concentrations (to 10(-3) g/ml) the frequency of the MEPP initially increased, then these potentials were depressed and finally they completely disappeared. Tween-80 did not change noticeably spontaneous synaptic activity, and saponin inhibited it. In Ca-free solutions bile salt were less effective. It is suggested that bile salts change the structure of the presynaptic membrane and at the same time stimulate the release of acetylcholine form the nerve terminals.
Assuntos
Junção Neuromuscular/efeitos dos fármacos , Tensoativos/farmacologia , Animais , Ácidos e Sais Biliares/farmacologia , Cálcio/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Polissorbatos/farmacologia , Ranidae , Saponinas/farmacologiaRESUMO
Apamin is a toxic polypeptide extracted from bee venom. It has been considered as a neurotoxin with central action, but its low concentrations (10(-8)-10(-7) M) were shown to reversibly block the nonadrenergic inhibition and effects of externally applied ATP, noradrenaline and caffeine in smooth muscles of the gastrointestinal tract. All these processes are related to the activation of Ca-dependent potassium permeability. Current-clamp, voltage-clamp and patch-clamp experiments have also shown that apamin blocks specifically some types of these conductances in other tissues: skeletal muscles, mammalian neurons and neuroblastoma, hepatocytes. Nowadays apamin is the most specific but not a universal blocker of the Ca-activated potassium conductance.
Assuntos
Apamina/farmacologia , Venenos de Abelha/farmacologia , Cálcio/fisiologia , Canais de Potássio/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Condutividade Elétrica , Fígado/efeitos dos fármacos , Fígado/fisiologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Canais de Potássio/fisiologia , Relação Estrutura-AtividadeRESUMO
The patch-clamp method has been used to investigate the action of caffeine on the calcium current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum. Caffeine (10 mM) substantially inhibited ICa. This effect occurred in a biphasic manner and it was not due either to activation of additional ionic currents of opposite direction nor to inhibition of phosphodiesterase activity. It strongly depended upon the ethylenebis-(oxonitrilo)tetraacetate (EGTA) concentration in the pipette solution. When there was K+ in the pipette solution, application of caffeine evoked a transient Ca-dependent K+ current and an abrupt and transient increase in the frequency of channel openings. Such well-known blockers of Ca release as procaine and ruthenium red strongly decreased ICa. Ryanodine had only little effect on ICa, but application of caffeine in the presence of ryanodine led to a complete and irreversible inhibition of ICa. The results of experiments involving different EGTA concentrations and comparison of the time courses of all caffeine-induced phenomena clearly indicated that only the initial, transient component of the ICa inhibition by caffeine was related to a Ca-dependent inactivation of Ca channels, evoked as a result of Ca release from intracellular stores. The tonic component of ICa inhibition was probably due to a direct blocking action of caffeine on Ca channels.
Assuntos
Cafeína/farmacologia , Canais de Cálcio/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Músculo Liso/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/fisiologia , Separação Celular , Eletrofisiologia , Intestinos/citologia , Músculo Liso/citologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Fatores de TempoRESUMO
1. Late transient outward Ca(2+)-dependent K+ current (ILTO) correlated with Ca(2+)-induced Ca2+ release mechanism was studied in relation to the calcium inward current (ICa) in single isolated smooth muscle cells of the guinea-pig ileum using the whole-cell patch-clamp technique. 2. The voltage dependencies of peak ICa and ILTO were both bell shaped. However, the I-V curve of the outward current was shifted toward more positive potentials by about 60 mV in comparison to that for ICa. 3. Reduction in the external Ca2+ concentration resulted in a decrease of peak amplitude of both ICa and ILTO. However, caffeine-induced outward current was also decreased abruptly suggesting a rapid loss of stored Ca2+ upon lowering the external Ca2+ concentration. 4. Investigation of the relation of ILTO to partially inactivated ICa showed that inactivation of ICa by approximately 65, 80 or 84% of control (produced by prepulse to -20 mV for 2 s, shifting the holding potential to -20 mV for 30 s or by the ramp voltage command from -50 to +10 mV, respectively) was without detectable effect on the ILTO generation. 5. Bath application of the Ca2+ antagonist nifedipine (300 nM) inhibited ICa by 81% without affecting ILTO peak amplitude (92.0 +/- 5.6% of control in six cells). The mean concentration-response curve for ICa inhibition was sigmoidal with the apparent dissociation constant of 86.9 nM, whereas that for the ILTO had a characteristic sharp transition indicating a definite threshold of Ca2+ influx for ILTO generation. 6. Application of Ca(2+)-free external solution during 500 ms of the time when ICa peaked inhibited the current by about 76% whereas the ILTO during such an intervention remained virtually unchanged. 7. In double-pulse experiments, with conditioning and test pulses to +10 mV from -50 mV and an interpulse interval of 600 ms, most of the cells (about 80%) showed larger outward current at the test pulse suggesting continued Ca2+ release triggered by Ca2+ influx during a short (50-200 ms) depolarizing prepulse. The outward current could also be evoked at large positive potentials (presumably near the calcium equilibrium potential) where it did not occur normally by a prepulse to +10 mV for 50 ms. The charge transferred by Ca2+ current necessary to activate Ca2+ release in most of the cells was estimated to be from 6 to 20 pC. 8. The data are interpreted to suggest that the Ca(2+)-induced Ca2+ release mechanism operates in single ileal cells in a regenerative manner.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Cálcio/metabolismo , Contração Muscular/fisiologia , Músculo Liso/metabolismo , Animais , ATPases Transportadoras de Cálcio , Células Cultivadas , Feminino , Cobaias , Íleo/fisiologia , Masculino , Potenciais da Membrana/fisiologia , Músculo Liso/citologia , Músculo Liso/fisiologia , ATPase Trocadora de Sódio-Potássio/fisiologiaRESUMO
Nonadrenergic synaptic transmission in circular and longitudinal smooth muscles of caecum preexposed to K-free solution for 4-5 h has been studied by means of sucrose gap technique. In addition, the effects of noradrenaline (NA) and ATP on these muscles were investigated under these conditions. The action of the above substances was accompanied by depolarization and contraction. NA induced a decrease in the membrane resistance. Addition of 0.5 mM Ba2+ to K-free solution intensified the depolarization. 1 mM of Mn2+ blocked depolarization and contraction. Intramural stimulation produced noncholinergic e.j.p.s blocked by TTX. Addition of 0.5 mM Ba2+ increased their amplitude. A reversal potential of both NA-induced depolarization and e.j.p. was in the range of + 10 to + 20 mV. It is supposed that e.j.p.s and depolarization observed in response to ATP and NA action are due to an increase in calcium permeability of the membrane.
Assuntos
Ceco/fisiologia , Músculo Liso/fisiologia , Inibição Neural , Sistema Nervoso Simpático/fisiologia , Trifosfato de Adenosina/farmacologia , Fenômenos Biomecânicos , Ceco/citologia , Humanos , Junções Intercelulares , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Norepinefrina/farmacologia , Transmissão SinápticaRESUMO
1. Whole-cell membrane currents in voltage-clamped single isolated cells of longitudinal smooth muscle of guinea-pig ileum were studied at room temperature using patch pipettes filled with either high-K+ solution or high-Cs+ solution, to suppress K+ outward current, and containing 0.3 mM-EGTA. 2. In the presence of high-K+ solution in the pipette, membrane depolarization from the holding potential of -50 mV evoked an initial inward calcium current (ICa) followed by a large initial transient outward current and a sustained outward current with spontaneous oscillations superimposed. Prolonged depolarization above -20 mV produced a late transient outward current which reached a maximum (up to several nanoamps at +10 mV) within approximately 1 s and lasted several seconds. 3. The late outward current (ILTO) was voltage dependent and reversed at the EK (potassium equilibrium potential) in cells exposed to high-K+ external solution. It was blocked by TEA+ (tetraethylammonium) or Ba2+ applied externally (calculated Kd (dissociation constant) values were 0.67 and 4.43 mM, respectively) or by high-Cs+ solution perfusing the cell. The removal of extracellular Ca2+, application of Ca2+ channel blockers (3 mM-Co2+, 0.2 mM-Cd2+ or 1 microM-nifedipine) or perfusion of 5 mM-EGTA inside the cell also abolished the current. Thus, the current seems to be a Ca(2+)-activated K+ current. 4. There is a great discrepancy between the time course of the ICa and that of the late ILTO, which suggests that Ca2+ release from intracellular storage sites may contribute to the generation of the ILTO. 5. Bath application of caffeine (10 mM) during the development of ILTO enhanced the current. However, in the presence of caffeine ILTO was inhibited. Moderate inhibition of ICa by caffeine was also observed. 6. Ryanodine (5 microM) applied to the bathing solution completely inhibited ILTO within 3.5 min; however, it had no or little effect on the ICa. 7. Ruthenium Red (10 microM) completely blocked the ILTO and slightly and more slowly inhibited the ICa. 8. Increasing Mg2+ concentration in the pipette solution from 1 to 6 mM abolished the ILTO. 9. It was concluded that the ILTO was activated mainly by Ca2+ released from the intracellular storage sites following Ca2+ entry, presumably by a Ca(2+)-induced Ca2+ release mechanism.
Assuntos
Músculo Liso/fisiologia , Animais , Cafeína/farmacologia , Cálcio/metabolismo , Feminino , Cobaias , Íleo/fisiologia , Técnicas In Vitro , Magnésio/metabolismo , Masculino , Músculo Liso/efeitos dos fármacos , Potássio/metabolismo , Rutênio Vermelho/farmacologia , Rianodina/farmacologia , Compostos de Tetraetilamônio/farmacologiaRESUMO
Microelectrode method was applied to the study of the influence of sodium bile salts (BS), saponin and between-80 on the end-plate potentials (EPP) of m. sartorius of Rana ridibunda. Bile salts in a concentration of 10(-5) g/ml did not change these potentials significantly. With increase of the BS concentration up to 10(-4) g/ml the amplitude of EPP increased 1.5- 3 times. The action of these substances in a concentration up to 10(-3) g/ml caused similar, but more rapid, increase in the EPP amplitude, and then the amplitude decreased. Saponin and tween-80 were less efffective in their action on EPP, but evoked muscle contraction. It is suggested that an increase in the EPP amplitude as a result of the action of the mentioned agents was chiefly connected with stimulation of the acetylcholine release by the nerve terminals.
Assuntos
Potenciais da Membrana/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Tensoativos/farmacologia , Acetilcolina/metabolismo , Animais , Ácidos e Sais Biliares/farmacologia , Contração Muscular/efeitos dos fármacos , Polissorbatos/farmacologia , Ranidae , Saponinas/farmacologia , Estimulação QuímicaRESUMO
Inhibitory junctional currents (IJCs) were recorded under voltage clamp conditions in response to brief transmural stimulation of the circular muscle of the guinea pig colon using the double sucrose gap method in the presence of atropine. The time course of IJC decay was approximately exponential 100-150 ms after the peak value. The IJC amplitude depended linearly on the membrane potential with the reversal potential (-70 mV) near the potassium equilibrium potential. The time constant (tau) of the IJC decay depended exponentially on the membrane potential and became e-fold decreased when the membrane was hyperpolarized approximately by 120 mV. Varying the quantal content of IJC caused an increase of tau upon rising the amount or transmitter released and its decrease with the depression of IJC. Application of ATP (10(-3)M) caused a decrease of tau and IJC amplitude, while apamine reduced the amplitude of IJC without any changes in their time course. The results are discussed in terms of a buffered diffusion hypothesis supposing a cooperative action of transmitter released on junctional receptors.
Assuntos
Músculo Liso/fisiologia , Junção Neuromuscular/fisiologia , Transmissão Sináptica , Trifosfato de Adenosina/farmacologia , Animais , Apamina/farmacologia , Atropina/farmacologia , Cobaias , Técnicas In Vitro , Intestino Grosso/fisiologia , Potenciais da Membrana , Membranas Sinápticas/fisiologiaRESUMO
Fura 2 microfluorimetry was used to test the hypothesis that ATP acts at P1 and P2 purinoceptors to elevate cytosolic free Ca2+ concentrations [Ca2+]i) in calbindin-immunoreactive cultured myenteric neurons from adult guinea pig small intestine. Local "micro-puff" application of ATP or ATP(gamma)S caused an increase in [Ca2+]i in 99% of 200 multipolar neurons. The potency profile of agonists for the rise in [Ca2+]i was ATP(gamma)S = ATP >> ADP >> AMP, adenosine, 5'-(N-ethylcarboxamido)adenosine, and 2-chloro-N(6)-cyclopentyladenosine. Tetrodotoxin-sensitive synaptic transmission could contribute as much as 25% to the ATP response. The P1 antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked 50% of the peakATP Ca2+ response. P2 antagonists blocked the ATP response: pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid > reactive blue 2 > suramin. Suramin enhanced the ATP response in 27.5% of neurons. Some neurons (<15%) displayed distinct multiphasic Ca2+ signatures. About 54% of ATP-responsive neurons expressed calbindin. The data support the following hypotheses: 1) two distinct P2 purinoceptors are linked to the rise in [Ca2+]i in myenteric neurons; 2) purinergic Ca2+ signaling is not restricted to one neuronal phenotype; and 3) intraneuronal Ca2+ is not involved in adenosinergic hyperpolarization in AH/type 2 neurons.