RESUMEN
Fully grown oocytes of Xenopus laevis undergo resumption of the meiotic cycle when treated with the steroid hormone progesterone. Previous studies have shown that meiotic maturation results in profound downregulation of specific endogenous membrane proteins in oocytes. To determine whether the maturation impacts the functional properties of exogenously expressed membrane proteins, we used cut-open recordings from Xenopus oocytes expressing several types of Na(+) and K(+) channels. Treatment of oocytes with progesterone resulted in a downregulation of heterologously expressed Na(+) and K(+) channels without a change in the kinetics of the currents. The time course of progesterone-induced ion channel inhibition was concentration dependent. Complete elimination of Na(+) currents temporally coincided with development of germinal vesicle breakdown, while elimination of K(+) currents was delayed by approximately 2 h. Coexpression of human beta(1)-subunit with rat skeletal muscle alpha-subunit in Xenopus oocytes did not prevent progesterone-induced downregulation of Na(+) channels. Addition of 8-bromo-cAMP to oocytes or injection of heparin before progesterone treatment prevented the loss of expressed currents. Pharmacological studies suggest that the inhibitory effects of progesterone on expressed Na(+) and K(+) channels occur downstream of the activation of cdc2 kinase. The loss of channels is correlated with a reduction in Na(+) channel immunofluorescence, pointing to a disappearance of the ion channel-forming proteins from the surface membrane.
Asunto(s)
Oocitos/metabolismo , Bloqueadores de los Canales de Potasio , Progesterona/farmacología , Bloqueadores de los Canales de Sodio , Animales , Proteína Quinasa CDC2/fisiología , Membrana Celular/metabolismo , AMP Cíclico/fisiología , Citoplasma/metabolismo , Citoesqueleto/fisiología , Regulación hacia Abajo , Conductividad Eléctrica , Femenino , Inositol 1,4,5-Trifosfato/fisiología , Canales de Potasio/fisiología , Proteínas/metabolismo , Canales de Sodio/fisiología , XenopusRESUMEN
Natriuretic peptides inhibit the release and action of many hormones through cyclic guanosine monophosphate (cGMP), but the mechanism of cGMP action is unclear. In frog ventricular muscle and guinea-pig hippocampal neurons, cGMP inhibits voltage-activated Ca2+ currents by stimulating phosphodiesterase activity and reducing intracellular cyclic AMP; however, this mechanism is not involved in the action of cGMP on other channels or on Ca2+ channels in other cells. Natriuretic peptide receptors in the rat pituitary also stimulate guanylyl cyclase activity but inhibit secretion by increasing membrane conductance to potassium. In an electrophysiological study on rat pituitary tumour cells, we identified the large-conductance, calcium- and voltage-activated potassium channels (BK) as the primary target of another inhibitory neuropeptide, somatostatin. Here we report that atrial natriuretic peptide also stimulates BK channel activity in GH4C1 cells through protein dephosphorylation. Unlike somatostatin, however, the effect of atrial natriuretic peptide on BK channel activity is preceded by a rapid and potent stimulation of cGMP production and requires cGMP-dependent protein kinase activity. Protein phosphatase activation by cGMP-dependent kinase could explain the inhibitory effects of natriuretic peptides on electrical excitability and the antagonism of cGMP and cAMP in many systems.
Asunto(s)
Factor Natriurético Atrial/farmacología , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , Canales de Potasio/fisiología , Animales , Caribdotoxina , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , GMP Cíclico/farmacología , Éteres Cíclicos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Neoplasias Hipofisarias , Canales de Potasio/efectos de los fármacos , Proteínas Quinasas/metabolismo , Venenos de Escorpión/farmacología , Tetraetilamonio , Compuestos de Tetraetilamonio/farmacología , Tionucleótidos/farmacología , Células Tumorales CultivadasRESUMEN
1. Isolated, non-identified neurons were voltage clamped using the internal perfusion technique. 2. Ions of Ag+ (1-100 microM) introduced into the bathing solution activated a steady-state inward current (IAg) in the soma. The effect of Ag+ was reversible when the concentration of Ag+ was less than 75 microM or the time of application was shorter than 10 min. 3. IAg was observed both in the presence and absence of Na+ ions in the extracellular saline. It could also be activated when Cs+ ions were substituted for Na+ ions. 4. The current-voltage characteristics were linear in the voltage range -100 to 0 mV. The reversal potential in control saline was an average of 1.19 +/- 5.1 mV. 5. The application of Ag+ ions induces an elevation of intracellular free Ca2+ concentration by 10-20 times in both Ca(2+)-containing and Ca(2+)-free extracellular salines, as revealed by Fura-2 measurements. 6. Agents that increase the intracellular free Ca2+ concentration ([Ca2+]i), like thymol, caffeine and dinitrophenol, increased the amplitude of IAg. The effect was additive. Ruthenium Red, which blocks the release of Ca2+ from intracellular stores, decreased the Ag+ effect. 7. It is concluded that extracellularly applied Ag+ ions increase the cytoplasmic free Ca2+ concentration, which in turn activates non-specific cationic channels. 8. Ag+ ions in 1-10 microM concentration were able to decrease the voltage-activated Ca2+ current amplitude. This decrease, however, was due to the increase of [Ca2+]i which caused Ca(2+)-dependent inactivation.
Asunto(s)
Permeabilidad de la Membrana Celular/efectos de los fármacos , Neuronas/metabolismo , Plata/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Caracoles Helix , Conducción Nerviosa/efectos de los fármacos , Neuronas/efectos de los fármacosRESUMEN
1. The effect of external application of oxytocin on inward calcium current in dialyzed snail neurons has been investigated under clamp conditions. 2. External application of oxytocin in a dose-dependent manner (Kd 0.9 microM) inhibits inward calcium current in dialyzed neurons of the snail, Helix pomatia. 3. Inhibition of calcium current developed with the time constant of about 2 min. The degree of restoration of calcium current after oxytocin washout depends on duration of oxytocin action. 4. It has been suggested that inhibition of calcium current by oxytocin occurs in two stages, the initial one is more fast and reversible and the second one--more slow and irreversible. The participation of soluble second messengers in the inhibitory effect of oxytocin on calcium current is discussed.
Asunto(s)
Calcio/fisiología , Neuronas/fisiología , Oxitocina/farmacología , Animales , Calcio/metabolismo , AMP Cíclico/farmacología , Diálisis/métodos , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Electrofisiología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Caracoles Helix , Líquido Intracelular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Temperatura , Factores de Tiempo , Vasopresinas/farmacologíaRESUMEN
The model of calcium-channel gating described above, although almost certainly too simple, suggests a direct role for protein kinases and phosphatases in determining the kinetics of calcium channel gating on a subsecond time scale. In addition, it provides a unique perspective for understanding studies of calcium channel gating under widely different metabolic and pharmacological conditions. Although many of these effects may be specific to the dihydropyridine-sensitive or L-type calcium channel, they give an indication of the range of possibilities for integrating calcium-channel activity with cellular biochemistry.
Asunto(s)
Canales de Calcio/fisiología , Activación del Canal Iónico/fisiología , Proteínas Quinasas/metabolismo , Animales , Canales de Calcio/efectos de los fármacos , Dihidropiridinas/farmacología , Activación del Canal Iónico/efectos de los fármacos , Fosfoproteínas Fosfatasas/metabolismo , FosforilaciónRESUMEN
Effect of oxytocin and other peptides on the potential-activated calcium current was studied in neurons of the snail Helix pomatia under clamp conditions. It has been found that oxytocin inhibits the calcium current and ED50 of this process is about 9 x 10(-7) mol/l. Vasopressin has the same effect on the calcium current with ED50 about 8 x 10(-6) mol/l. Des-Gly-vasopressin exerts no noticeable effect on the calcium current. Possible mechanisms of the inhibitory effect of oxytocin on the calcium current are discussed.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Caracoles Helix/efectos de los fármacos , Neuronas/efectos de los fármacos , Oxitocina/farmacología , Animales , Canales de Calcio/fisiología , Depresión Química , Caracoles Helix/fisiología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/fisiologíaRESUMEN
The effect of microiontophoretic injection of AMP and cAMP on the calcium current was investigated in dialyzed snail neurons. It was found that microiontophoretic injection of AMP restores calcium current after its "washout" during the dialysis and enhances its stability. In this case the current-voltage characteristic was shifted by 10 mV in the depolarizing direction. Under these conditions the iontophoretic cAMP injection into a dialyzed neuron produced a reversible decrease of the calcium current.
Asunto(s)
Adenosina Monofosfato/farmacología , Canales de Calcio/efectos de los fármacos , AMP Cíclico/farmacología , Caracoles Helix/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Canales de Calcio/fisiología , Diálisis , Iontoforesis , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Microinyecciones , Neuronas/fisiologíaRESUMEN
The effects of different substances affecting some or other chains of cAMP metabolism on the properties of transmembrane current induced by cAMP injection have been studied in Helix pomatia neurons. It was found that preinjection of papaverine or adenosine triphosphate from a microelectrode into the neuron increases the cAMP-current amplitude. Injection of dibutyryl-cAMP does not induce transmembrane current by itself but results in a noticeable reversible inhibition of cAMP-current. Extracellular administration of trifluoperazine produces either an increase or a decrease of the cAMP-current amplitude in different cells but in both cases its removal restores the initial amplitude of cAMP-current. Furosemide has no appreciable effect on cAMP-current. An increase in intracellular Ca2+ concentration (by iontophoretic injection through a microelectrode, generation of a burst of action potentials, application of dinitrophenol, tetraphenylphosphonium or caffeine) considerably enhances the amplitude of cAMP-current. The amplitude of cAMP-current remains increased for many minutes after return of the intracellular Ca2+ level to its initial value. A possible physiological role of the observed effect is discussed.
Asunto(s)
AMP Cíclico/farmacología , Canales Iónicos/efectos de los fármacos , Conducción Nerviosa/efectos de los fármacos , Sodio/metabolismo , Adenosina Trifosfato/farmacología , Animales , Bucladesina/farmacología , Calcio/farmacología , GMP Cíclico/farmacología , Guanilil Imidodifosfato/farmacología , Caracoles Helix , Trifluoperazina/farmacologíaRESUMEN
Changes in inward calcium current observed on the background of postsynaptic currents have been studied in snail neurons under voltage clamp conditions. It is shown that when inhibitory or excitatory postsynaptic current developed, the calcium current reversibly decreased. Application of serotonin to the neuronal soma has also led to a reversible block of the calcium current. Possible cellular mechanisms involved in the changes under study and their putative physiological role are discussed.
Asunto(s)
Calcio/metabolismo , Permeabilidad de la Membrana Celular , Potenciales de la Membrana , Fenómenos Fisiológicos del Sistema Nervioso , Serotonina/farmacología , Sinapsis/fisiología , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Sistema Nervioso/efectos de los fármacos , Transmisión SinápticaRESUMEN
The effect of intracellular Ca2+ concentration increase on transmembrane current induced by cAMP injection into a neuron (cAMP-current) was studied on Helix pomatia neurons under voltage clamp conditions. The elevation of intracellular Ca2+ concentration was produced by iontophoretic injection of Ca2+ into the cytoplasm, generation of action potentials, addition of uncouplers of oxidative phosphorylation into the external solution resulting in Ca2+ efflux from mitochondria. It was found that elevation of the intracellular Ca2+ concentration increases the amplitude of the cAMP-current and in most cases its duration as well. It is suggested that membrane structures responsible for the cAMP-current have two centers of phosphorylation: cAMP-dependent and Ca2+-calmodulin-dependent. Possible role of the studied process in integration of signals on the intraneuronal level is discussed.
Asunto(s)
Calcio/fisiología , AMP Cíclico/farmacología , Ganglios/efectos de los fármacos , Animales , Calmodulina/fisiología , Membrana Celular/efectos de los fármacos , Dinitrofenoles/farmacología , Caracoles Helix , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/metabolismo , Compuestos Onio/farmacología , Compuestos Organofosforados/farmacologíaRESUMEN
3'5'-cAMP injected iontophoretically into identified neurons of the snail, Helix pomatia, induced depolarization of the membrane. Clamping the membrane potential revealed the appearance of a simultaneous inward transmembrane current ('cAMP-current') followed by a weaker outward current. External application of theophylline increased the amplitude of cAMP-current. Imidazole had an opposite effect on this current. Tolbutamide and lowering of temperature largely reduced its rate of rise and, correspondingly, its amplitude. Simultaneous removal of Na+ ions from external solution and addition of Cd2+ ions resulted in complete disappearance of the inward cAMP-current. Analysis of current-voltage characteristics of the cAMP-current at varying external concentrations of Na+, K+, Cl and Ca2+-ions has shown that the cAMP-current is due to an increase of membrane conductance to Na+, K+ and Ca2+ ions; a late component of the cAMP-current is associated with an increase of potassium conductance of the neuronal membrane induced, probably by Ca2+, influx. Besides the induction of stationary currents, cAMP injection also decreased the voltage- and time-dependent calcium currents reducing the maximum calcium conductance. After the end of injection, calcium currents restored their initial value in 1-2 min. An analogous decrease of the calcium current could be evoked by prolonged external application of theophylline. Possible mechanisms of intracellular effects of cAMP on electrical characteristics of the neuronal membrane are discussed.