RESUMEN
The control of Cl- conductance in rat parotid isolated acinar cells was studied by combined use of whole-cell recording and flash photolysis techniques. Cells were voltage-clamped either at a membrane potential of -40 mV or stepped between -85 mV and 0 mV. Bath-applied carbachol and noradrenaline evoked Cl- current at -85 mV and K+ current at 0 mV. Similar current activations resulted from the photolytic release of either inositol trisphosphate (InsP3) or Ca2+ by a brief near-UV flash. The peak amplitudes of the Cl- conductance (at -85 mV), measured relative to the K+ conductance (at 0 mV), evoked by application of carbachol, noradrenaline or direct manipulation of cytosolic free calcium ([Ca2+]i), were very similar, being 0.56 +/- 0.09 (mean +/- SEM, n = 9), 0.52 +/- 0.01 (n = 7) and 0.46 +/- 0.06 (n = 7). In contrast, the relative amplitude of the Cl- conductance evoked by InsP3 was much larger: 1.49 +/- 0.24 (n = 9). Neither bath application of isoprenaline nor photolysis of "caged" cAMP induced any detectable membrane current. The most probable interpretation of these results is that the observed activation of Cl- conductance by agonists can be explained by the elevation of [Ca2+]i alone. In addition, the present results provide further support for the previously reported suggestion that the Cl- channels and the Ca(2+)-release sites are co-localised [10].
Asunto(s)
Cloruros/fisiología , Glándula Parótida/fisiología , Agonistas Adrenérgicos/farmacología , Animales , Separación Celular , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Conductividad Eléctrica , Inositol 1,4,5-Trifosfato/análogos & derivados , Inositol 1,4,5-Trifosfato/farmacología , Luz , Masculino , Glándula Parótida/citología , Glándula Parótida/efectos de los fármacos , Ratas , Ratas Wistar , Receptores Muscarínicos/fisiologíaRESUMEN
1. The temporal relationship between cytosolic free Ca2+ concentration ([Ca2+]i) and activation of membrane current responses in single rat parotid acinar cells has been examined. Activation of muscarinic receptors by carbachol (CCh) at -40 mV (midway between EK and ECl under our experimental conditions) frequently evoked biphasic current responses, application of 2 microM CCh leading to rapid activation of an inward current followed by a slower outward current. 2. Photochemical release of inositol 1,4,5-trisphosphate (InsP3), from 'caged' InsP3, by a brief near-UV flash, evoked similar biphasic current responses at -40 mV. In contrast, elevation of [Ca2+]i by photolysis of the caged calcium compound nitr-5 at -40 mV activated only monophasic current responses. 3. These results can be explained by a model in which the InsP3-sensitive Ca2+ release sites are localized at the luminal pole of the cell, combined with a relative preponderance of Ca(2+)-activated Cl- channels at that pole, and a relative preponderance of Ca(2+)-activated K+ channels at the basal end.
Asunto(s)
Calcio/metabolismo , Glándula Parótida/metabolismo , Fotólisis , Animales , Carbacol/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Técnicas In Vitro , Inositol 1,4,5-Trifosfato/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Agonistas Muscarínicos/farmacología , Glándula Parótida/citología , Glándula Parótida/efectos de la radiación , Técnicas de Placa-Clamp , Ratas , Receptores Muscarínicos/efectos de los fármacos , Rayos UltravioletaRESUMEN
To understand the complex time course of cytosolic Ca2+ signalling evoked by hormones and neurotransmitters, it is necessary to know the kinetics of steps in the second-messenger cascade, particularly cooperative and inhibitory interactions between components that might give rise to periodic fluctuations. In the case of inositol trisphosphate (InsP3)-evoked Ca2+ release, fast perfusion studies with subcellular fractions or permeabilised cells can be made if sufficient homogeneous tissue is available. Single-cell studies can be made by combining whole-cell patch-clamp techniques and microspectrofluorimetry with flash photolytic release of InsP3 to give quantitative, time-resolved data of Ca2+ release from stores. A technical description is given here of flash photolysis of caged InsP3, and the results of fast perfusion and flash photolytic experiments are reviewed. Studies of kinetics of Ca2+ release have shown that the InsP3 receptor/channel is regulated first by positive and then by negative feedback by free cytosolic Ca2+ concentration, producing a pulse of Ca2+ release having properties that may be important in the spatial propagation of Ca2+ signals within and between cells. The properties of InsP3-evoked Ca2+ release in single cells differ between peripheral tissues, such as the liver, and Purkinje neurones of the cerebellum. Purkinje neurones need 20-50 times higher InsP3 concentrations and release Ca2+ to change the free cytosolic concentration 30 times faster and to higher peak concentrations than in liver. The InsP3 receptors in the two cell types appear to differ in apparent affinity, and the greater Ca2+ efflux from stores in Purkinje cells is probably due to a high receptor density.
Asunto(s)
Calcio/metabolismo , Hormonas/fisiología , Neurotransmisores/fisiología , Fotólisis , Animales , Calcio/fisiología , AMP Cíclico/fisiología , GMP Cíclico/fisiología , Citosol/metabolismo , Inositol 1,4,5-Trifosfato/fisiología , Luz , Hígado/metabolismo , Neuronas/metabolismoRESUMEN
1. Glutamate uptake into isolated, whole-cell patch-clamped glial cells was studied by monitoring the increase of cell fluorescence generated as glutamate and NAD(P) were converted into alpha-ketoglutarate and NAD(P)H by glutamate dehydrogenase. The current generated by the glutamate uptake carrier was recorded simultaneously. 2. L-Glutamate evoked an increase of cell fluorescence and an inward uptake current. L- and D-aspartate generated an uptake current but no fluorescence response, consistent with the amino acid specificity of glutamate dehydrogenase. 3. In the absence of external sodium the glutamate-evoked fluorescence response and uptake current were abolished, showing that there is no sodium-independent glutamate uptake across the cell membrane. 4. Varying the glutamate concentration altered both the fluorescence response and the uptake current. The fluorescence response saturated at a lower glutamate concentration than the uptake current, and depended in a Michaelis-Menten fashion on the uptake current. 5. The fluorescence response and the uptake current were reduced by membrane depolarization, and also by removal of intracellular potassium. 6. The dependence of the fluorescence response on uptake current when membrane potential was altered or intracellular potassium was removed was the same as that seen when the external glutamate concentration was altered. 7. These fluorescence studies show that glutamate uptake is inhibited by depolarization and by removal of intracellular potassium, consistent with the conclusion of earlier work in which uptake was monitored solely as a membrane current. The data are consistent with high-affinity electrogenic sodium- and potassium-dependent glutamate uptake with fixed stoichiometry being the only significant influx route for glutamate. Other possible interpretations of the data are also discussed.
Asunto(s)
Glutamatos/farmacocinética , NADP/metabolismo , Neuroglía/metabolismo , Retina/metabolismo , Potenciales de Acción/efectos de los fármacos , Ambystoma/metabolismo , Animales , Transporte Biológico Activo , Fluorescencia , Ácido Glutámico , Técnicas In Vitro , Líquido Intracelular/metabolismo , Potenciales de la Membrana , Modelos Biológicos , Potasio/metabolismo , Retina/citología , Sodio/farmacologíaRESUMEN
1. The changes in ciliary beat frequency (CBF) of human nasal respiratory epithelial cells were measured in vitro with a photometric technique following exposure to either 4-bromo-calcium ionophore A23187 (4-Br-A23187) or trifluoperazine (TFP), an inhibitor of calmodulin-sensitive calcium-dependent protein kinases. Changes in intracellular free calcium concentrations in response to 4-Br-A23187 were studied using a fluorescent dye (Fura-2). 2. Addition of 10(-5) M-4-Br-A23187 caused a time-dependent (P less than 0.01) rise in CBF. The increment in CBF was statistically significant 10 min after challenge (+10%; P less than 0.01) and was sustained for at least 1 h, with maximal stimulation after 40 min (+ 18%; P less than 0.01). 3. Exposure to 10(-5) M-4-Br-A23187 caused an immediate increase in intracellular free calcium concentration, which preceded the rise in CBF. 4. TFP (10(-4) M) caused a reduction of baseline CBF (-10%; P less than 0.01) and prevented the expected rise when the cells were subsequently exposed to 10(-5) M-4-Br-A23187. 5. We conclude that: (1) calcium ionophore stimulates the CBF of human respiratory cells; (2) this effect is mediated through a calmodulin-sensitive system, since it is abolished in the presence of TFP; (3) the same pathway appears to control the basal CBF of these cells, since TFP also decreases CBF.
Asunto(s)
Calcio/fisiología , Cilios/fisiología , Mucosa Nasal/fisiología , Adolescente , Adulto , Calcimicina/análogos & derivados , Calcimicina/farmacología , Calcio/análisis , Femenino , Humanos , Masculino , Persona de Mediana Edad , Mucosa Nasal/química , Factores de Tiempo , Trifluoperazina/farmacologíaRESUMEN
The relation between elevation of cytosolic free calcium and activation of membrane conductance has been studied in single acinar cells of the rat parotid. Outward and inward currents are activated by calcium elevation and oscillate in phase with oscillations of cytosolic calcium. The outward current results from activation of a large unit-conductance Ca2+ and voltage-dependent K+ channel, whereas the inward current is most likely carried predominantly by Cl-. Both these conductances have been previously described in exocrine cells. Buffering calcium at resting levels eliminated current responses to muscarinic agonists, suggesting that calcium is the only significant second messenger involved in the short-term control of this conductance by acetylcholine.
Asunto(s)
Calcio/metabolismo , Membrana Celular/fisiología , Canales Iónicos/fisiología , Glándula Parótida/fisiología , Animales , Citosol/metabolismo , Conductividad Eléctrica , Técnicas In Vitro , Cinética , Glándula Parótida/citología , Ratas , Sistemas de Mensajero Secundario , Transducción de SeñalRESUMEN
1. In single, dissociated, rat parotid acinar cells the muscarinic agonist carbachol evokes a rapid rise in cytosolic free calcium [( Ca2+]i), from near 100 nM to peak levels of up to 1 microM. In the continued presence of the agonist the response decays to a lower, maintained, level. 2. In most cells, at 22 degrees C, oscillations, with a mean frequency of 0.19 Hz, are superimposed upon this elevation of [Ca2+]i. In voltage-clamped cells oscillations of current occur in phase with the oscillations of [Ca2+]i. 3. The oscillations occur in voltage-clamped cells, and in the absence of extracellular Ca2+, indicating that neither voltage-gated processes, or an influx of Ca2+ is involved. 4. Oscillation frequency is independent of carbachol concentration, in the range 100 nM to 250 microM, and furthermore, shows no relationship to the mean level of [Ca2+]i during the oscillations. 5. Stimulation with the alpha-adrenergic agonist noradrenaline, in the presence of the beta-blocker propanolol, evokes oscillations having the same frequency as those evoked by carbachol. 6. The oscillations show a strong temperature dependence, the frequency increasing with a Q10 of 2.8. In contrast, the amplitude of the oscillations drops from a mean of 33% of the response amplitude at 22 degrees C, and below, to 6% at 33 degrees C. Above the latter temperature oscillations are not resolvable. 7. The phorbol esters, 12-O-tetradecanoyl-phorbol-13-acetate and 12,13-phorbol dibutyrate (1 microM), do not affect the response to carbachol at 22 degrees C, at which temperature the oscillations are of maximum amplitude. Diacylglycerol is, therefore, unlikely to be involved in oscillation generation in these cells. 8. These observations are consistent with a model in which a negative feed-back loop links [Ca2+]i to the mechanisms of Ca2+ elevation, possibly to the inositol 1,4,5-trisphosphate-sensitive Ca2+ release mechanism of the endoplasmic reticulum. If the feed-back path involved an enzymatic step, the slowing of this step at lowered temperatures could give rise to oscillations. At body temperature such a mechanism would act to ensure that [Ca2+]i was elevated in a regulated and dose-dependent manner.
Asunto(s)
Calcio/metabolismo , Carbacol/farmacología , Citosol/metabolismo , Norepinefrina/farmacología , Glándula Parótida/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Técnicas In Vitro , Masculino , Ésteres del Forbol/farmacología , Propranolol/farmacología , Ratas , Ratas Endogámicas , TemperaturaRESUMEN
A major function of glial cells in the central nervous system is to buffer the extracellular potassium concentration, [K+]o. A local rise in [K+]o causes potassium ions to enter glial cells, which have membranes that are highly permeable to K+; potassium then leaves the glial cells at other locations where [K+]o has not risen. We report here the first study of the individual ion channels mediating potassium buffering by glial cells. The patch-clamp technique was employed to record single channel currents in Müller cells, the radial glia of the vertebrate retina. Those cells have 94% of their potassium conductance in an endfoot apposed to the vitreous humour, causing K+ released from active retinal neurones to be buffered preferentially to the vitreous. Recordings from patches of endfoot and cell body membrane show that a single type of inward-rectifying K+ channel mediates potassium buffering at both cell locations. The non-uniform density of K+ conductance is due to a non-uniform distribution of one type of K+ channel, rather than to the cell expressing high conductance channels at the endfoot and low conductance channels elsewhere on the cell.
Asunto(s)
Canales Iónicos/fisiología , Potasio/metabolismo , Retina/fisiología , Células Ganglionares de la Retina/fisiología , Ambystoma , Animales , Potenciales de la Membrana , MicroelectrodosRESUMEN
Large voltage-dependent outward currents are recorded with the whole-cell patch-clamp technique from rat cultured astrocytes under conditions where an outward movement of potassium ions is excluded (either by blockage of the potassium channels pharmacologically or by replacement of the internal potassium by the impermeant large organic cation N-methyl-(+)-glucamine). The current, which is activated at potentials more positive than -40 to -50 mV, is normally carried by an inward movement of chloride ions. Its reversal potential is the same as the chloride equilibrium potential. With depolarization to +60 mV (for 225 ms) little or no inactivation of the current occurs: with depolarizations to +90 to +110 mV a time-dependent decay is seen. The current, which is often not marked immediately after formation of the whole-cell clamp, generally increases over a period of a few minutes to a maximum (after which it usually declines), as if some as yet unknown intracellular factor keeping the channels closed were being washed away from the membrane. The time course of this phenomenon is not affected by changing of the internal free calcium concentration (from 10(-8)M to 10(-6)M) or by an intracellular mixture of cyclic AMP (1 mM), ATP (4 mM) and Mg+ (2 mM). The conductance is slightly increased when the chloride of the bathing medium is replaced by bromide; is much reduced on replacement by methylsulphate, sulphate, isethionate, or acetate; and is virtually abolished on replacement by the large anion gluconate. The outward current is inhibited by the disulphonate stilbenes DIDS and SITS; this blocking action was initially partly reversible, although never completely so. It is suggested that the chloride conductance plays a role in the spatial buffering of potassium by astrocytes.
Asunto(s)
Astrocitos/fisiología , Cloruros/metabolismo , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/análogos & derivados , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-disulfónico/farmacología , Animales , Células Cultivadas , Conductividad Eléctrica , Gluconatos/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas EndogámicasRESUMEN
Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.
Asunto(s)
Fibroblastos/fisiología , Canales Iónicos/fisiología , 4-Aminopiridina , Aminopiridinas/farmacología , Proteínas Anfibias , Animales , Calcio/metabolismo , Proteínas Portadoras/análisis , Células Cultivadas , Cloruros/metabolismo , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Potasio/metabolismo , Conejos , Ratas , Ratas Endogámicas , Saxitoxina/farmacología , Compuestos de Tetraetilamonio/farmacología , Tetrodotoxina/farmacologíaRESUMEN
Patch-clamp recording from the plasmalemma of rat cultured astrocytes reveals the presence of both voltage-dependent sodium and voltage-dependent potassium conductances. These conductances are similar but not identical to the corresponding conductances in the axolemma. Whereas the h infinity relation of the sodium channels has the same voltage dependence as in the nodal axolemma, the peak current-voltage relation is shifted by about 30 mV along the voltage axis in the depolarizing direction. It is speculated that the glial cells synthesize sodium and potassium channels for later insertion into the axolemma of neighbouring axons. The astrocytes also express a plasmalemmal voltage-dependent anion conductance that is turned on at about -40 mV (that is, near the resting potential of the cultured astrocytes). The channels involved are large enough to be just permeable to glutamate but not to ascorbate. It is suggested that the conductance of this channel for chloride plays a physiological role in the spatial buffering of potassium by glial cells.
Asunto(s)
Astrocitos/fisiología , Canales Iónicos/fisiología , Animales , Astrocitos/metabolismo , Química Encefálica , Células Cultivadas , Corteza Cerebral/citología , Cloruros/fisiología , Potenciales de la Membrana , Ratas , Ratas Endogámicas , Saxitoxina/metabolismo , Veratridina/metabolismoRESUMEN
Calcium-activated channels, in the plasma membrane of rat cultured Schwann cells were studied in isolated 'inside-out' membrane patches. With identical (150 mM NaCl) solutions on either side of the membrane, a single channel conductance of 32 pS was calculated for inward current; the conductance was somewhat less for outward current. The channel is about equally permeable to sodium and potassium ions, but is not detectably permeable to either chloride or calcium. Under our experimental conditions the channel is activated by high (more than 10(-4) M) concentrations of calcium and is sensitive to voltage, channel activity increasing with membrane depolarization.
Asunto(s)
Calcio/farmacología , Canales Iónicos/efectos de los fármacos , Células de Schwann/metabolismo , Animales , Células Cultivadas , Canales Iónicos/metabolismo , Potenciales de la Membrana , Potasio/metabolismo , Ratas , Sodio/metabolismoRESUMEN
Anion-selective channels, with very large single unit conductances, are present in the cell membrane of rat cultured Schwann cells measured with the patch-clamp technique. In inside-out membrane patches, channels with a conductance of about 450 pS (in symmetrical 150 mM NaCl) were observed. These channels did not become active until several minutes after the cytoplasmic surface had been exposed to the bathing medium, suggesting that these channels may normally be kept in an inactive state by some as yet unknown internal factor. The channel opened over a relatively small potential range (-10 mV to +20 mV) and closed rapidly at more positive and more negative potentials with voltage-dependent kinetics. Although the channels showed a slight permeability towards small cations the major permeability was to anions. The order of permeability was: I greater than Br greater than Cl greater than methyl SO4 greater than SO4 greater than acetate = isethionate. Aspartate and glutamate were not detectably permeant. The physiological role of these channels remains unknown.
Asunto(s)
Aniones/metabolismo , Canales Iónicos/metabolismo , Células de Schwann/metabolismo , Animales , Permeabilidad de la Membrana Celular , Células Cultivadas , Conductividad Eléctrica , Estimulación Eléctrica , Cinética , Potenciales de la Membrana , Ratas , Ratas Endogámicas , Nervio Ciático , Nervio TrigéminoRESUMEN
The acetylcholine activated conductance of chick ciliary ganglion neurones grown in tissue culture was studied by the patch clamp method. Single channel currents at 30 degrees C had a conductance of 38-42 pS, a reversal potential near + 10 mV and an average open lifetime of 1.08 ms (range 0.74 - 1.54 ms) at the resting potential. The presence of a single component in the distributions of amplitudes and open lifetimes, and also in the noise spectrum of voltage clamp currents, suggests that acetylcholine channels have uniform characteristics in these cells. Evidence of a desensitised state of the receptor was obtained from the distribution of gap intervals and the decline of voltage clamp current. These properties are similar to those of acetylcholine channels at the vertebrate neuromuscular junction. However, two important differences were found. (a) The acetylcholine concentrations used here were 10-25 times higher than those required to produce a similar degree of channel activation at the endplate. (b) When the membrane was hyperpolarised the mean open lifetime of the channel showed no change or a slight reduction.
Asunto(s)
Acetilcolina/farmacología , Pollos/metabolismo , Ganglios Parasimpáticos/metabolismo , Canales Iónicos/efectos de los fármacos , Neuronas/metabolismo , Animales , Técnicas de Cultivo , Conductividad Eléctrica , Ganglios Parasimpáticos/citología , CinéticaRESUMEN
The culture of rat submandibular ganglion cells is described. The neurons can be distinguished from the non-neuronal cells in the cultures by their morphology. Recording with whole-cell voltage-clamp techniques indicates that the neurons have resting potentials of about -55 mV and that the kinetics of the ionic channels opened by locally perfused acetylcholine (ACh) are very similar to those previously observed in adult submandibular ganglion neurons. The major differences observed are that the recorded cell input impedances are much higher than those recorded with microelectrodes from adult neurons and that the sensitivity of the cultured neurons to ACh is much less than that of the adult neurons. Whether the latter is due to changed receptor properties or to the presence of fewer receptors is not known.
Asunto(s)
Acetilcolina/farmacología , Ganglios Parasimpáticos/efectos de los fármacos , Neuronas/efectos de los fármacos , Glándula Submandibular/inervación , Factores de Edad , Animales , Células Cultivadas , Femenino , Ganglios Parasimpáticos/citología , Ganglios Parasimpáticos/fisiología , Potenciales de la Membrana , Neuronas/citología , Neuronas/fisiología , RatasRESUMEN
The kinetics of the responses of rat submandibular ganglion neurones to nicotinic agonists were investigated by the analysis of agonist-induced membrane current noise. Two kinetic components are found in the responses of these cells to acetylcholine, carbachol, suberyldicholine, dimethyl-4-phenylpiperazinium and nicotine, in agreement with previous findings of two components in evoked synaptic currents in these cells. Small, though significant, differences in the relative amplitudes of the two kinetic components were observed in the spectra of noise evoked by the different agonists. In particular, the fast component in the response to carbachol was relatively larger with respect to the slow component than with any other agonist tested. No measurable dependence of the relative sizes of the two components on agonist concentration or membrane potential was observed. The evidence supports the hypothesis that the two kinetic components are the product of two independent channel populations for which the agonists tested show some slight differences in selectivity.