RESUMEN
Ambient measurements of PM1 aerosol chemical composition at Cabauw, the Netherlands, implicate higher ammonium concentrations than explained by the formation of inorganic ammonium salts. This additional particulate ammonium is called excess ammonium (eNH4). Height profiles over the Cabauw Experimental Site for Atmospheric Research (CESAR) tower, of combined ground based and airborne aerosol mass spectrometric (AMS) measurements on a Zeppelin airship show higher concentrations of eNH4 at higher altitudes compared to the ground. Through flights across the Netherlands, the Zeppelin based measurements furthermore substantiate eNH4 as a regional phenomenon in the planetary boundary layer. The excess ammonium correlates with mass spectral signatures of (di-)carboxylic acids, making a heterogeneous acid-base reaction the likely process of NH3 uptake. We show that this excess ammonium was neutralized by the organic fraction forming particulate organic ammonium salts. We discuss the significance of such organic ammonium salts for atmospheric aerosols and suggest that NH3 emission control will have benefits for particulate matter control beyond the reduction of inorganic ammonium salts.
RESUMEN
Sea spray is one of the largest natural aerosol sources and plays an important role in the Earth's radiative budget. These particles are inherently hygroscopic, that is, they take-up moisture from the air, which affects the extent to which they interact with solar radiation. We demonstrate that the hygroscopic growth of inorganic sea salt is 8-15% lower than pure sodium chloride, most likely due to the presence of hydrates. We observe an increase in hygroscopic growth with decreasing particle size (for particle diameters <150 nm) that is independent of the particle generation method. We vary the hygroscopic growth of the inorganic sea salt within a general circulation model and show that a reduced hygroscopicity leads to a reduction in aerosol-radiation interactions, manifested by a latitudinal-dependent reduction of the aerosol optical depth by up to 15%, while cloud-related parameters are unaffected. We propose that a value of κs=1.1 (at RH=90%) is used to represent the hygroscopicity of inorganic sea salt particles in numerical models.
RESUMEN
The human ether-a-go-go-related gene (herg) encodes a K+ current (IHERG) that plays a fundamental role in heart excitability by regulating the action potential repolarization (IKr); mutations of this gene are responsible for the chromosome 7-linked long QT syndrome (LQT2). In this report, we show that in a variety (n = 17) of tumor cell lines of different species (human and murine) and distinct histogenesis (neuroblastoma, rhabdomyosarcoma, adenocarcinoma, lung microcytoma, pituitary tumors, insulinoma beta-cells, and monoblastic leukemia), a novel K+ inward-rectifier current (IIR), which is biophysically and pharmacologically similar to IHERG, can be recorded with the patch-clamp technique. Northern blot experiments with a human herg cDNA probe revealed that both in human and murine clones the very high expression of herg transcripts can be quantified in at least three clearly identifiable bands, suggesting an alternative splicing of HERG mRNA. Moreover, we cloned a cDNA encoding for IIR from the SH-SY5Y human neuroblastoma. The sequence of this cDNA result was practically identical to that already reported for herg, indicating a high conservation of this gene in tumors. Consistently, the expression of this clone in Xenopus oocytes showed that the encoded K+ channel had substantially all of the biophysical and pharmacological properties of the native IIR described for tumor cells. In addition, in the tumor clones studied, IIR governs the resting potential, whereas it could not be detected either by the patch clamp or the Northern blot techniques in cells obtained from primary cell cultures of parental tissues (sensory neurons and myotubes), whose resting potential is controlled by the classical K+ anomalous rectifier current. This current substitution had a profound impact on the resting potential, which was markedly depolarized in tumors as compared with normal cells. These results suggest that IIR is normally only expressed during the early stages of cell differentiation frozen by neoplastic transformation, playing an important pathophysiological role in the regulatory mechanisms of neoplastic cell survival. In fact, because of its biophysical features, IIR, besides keeping the resting potential within the depolarized values required for unlimited tumor growth, could also appear suitable to afford a selective advantage in an ischemic environment.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Neoplasias/genética , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Transactivadores , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Fenómenos Biofísicos , Biofisica , Linaje de la Célula , Transformación Celular Neoplásica , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Humanos , Ratones , Datos de Secuencia Molecular , Músculos/fisiología , Neuroblastoma/genética , Neuronas/fisiología , Regulador Transcripcional ERG , Células Tumorales CultivadasRESUMEN
The expression pattern of K(+) currents is the principal regulator of electrical activity during development of the nervous and muscular system. We report here a study showing the expression pattern of HERG K(+) currents-encoding (erg) genes in various nervous and muscular tissues at different stages of quail embryo development.
Asunto(s)
Proteínas de Transporte de Catión , Regulación del Desarrollo de la Expresión Génica , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Codorniz/embriología , Codorniz/genética , Secuencia de Aminoácidos , Animales , Embrión no Mamífero/fisiología , Canales de Potasio Éter-A-Go-Go , Datos de Secuencia Molecular , Músculos/embriología , Sistema Nervioso/embriología , Alineación de SecuenciaRESUMEN
The human ether-a-go-go-related gene (herg) encodes a K+ current (I(HERG)) which plays a fundamental role in heart excitability and in neurons by contributing to action potential repolarization and to spike-frequency adaptation, respectively. In this paper we show that I(HERG), recorded in neuroblastoma cells and guinea-pig ventricular myocytes, was reversibly inhibited by the K(ATP) channel blocker glibenclamide (IC50 = 74 microM). The voltage and use dependence of glibenclamide blockade were also evaluated. Another sulfonylurea, glimepiride, had less effective results in blocking I(HERG). The findings of this study are relevant to the interpretation of glibenclamide effects on cellular electrophysiology and suggest that oral antidiabetic therapy with sulfonylureas may contribute to iatrogenic QT prolongation and related arrhythmias.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Gliburida/farmacología , Miocardio/metabolismo , Neuronas/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Compuestos de Sulfonilurea/farmacología , Transactivadores , Potenciales de Acción/efectos de los fármacos , Animales , Cromanos/farmacología , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Cobayas , Humanos , Concentración 50 Inhibidora , Ratones , Miocardio/citología , Neuroblastoma , Neuronas/efectos de los fármacos , Nisoldipino/farmacología , Piperidinas/farmacología , Canales de Potasio/metabolismo , Piridinas/farmacología , Ratas , Sulfonamidas/farmacología , Regulador Transcripcional ERG , Células Tumorales CultivadasRESUMEN
In the present study, the effect of the blockade of membrane calcium channels activated by intracellular Ca(2+) store depletion on basal and depolarization-induced [3H]norepinephrine ([3H]NE) release from SH-SY5Y human neuroblastoma cells was examined. The second-generation H(1) receptor blockers astemizole, terfenadine, and loratadine, as well as the first-generation compound hydroxyzine, inhibited [3H]NE release induced by high extracellular K(+) concentration ([K(+)](e)) depolarization in a concentration-dependent manner (the IC(50)s were 2.3, 1.7, 4.8, and 9.4 microM, respectively). In contrast, the more hydrophilic second-generation H(1) receptor blocker cetirizine was completely ineffective (0.1-30 microM). The inhibition of high [K(+)](e)-induced [3H]NE release by H(1) receptor blockers seems to be related to their ability to inhibit Ca(2+) channels activated by Ca(i)(2+) store depletion (SOCs). In fact, astemizole, terfenadine, loratadine, and hydroxyzine, but not cetirizine, displayed a dose-dependent inhibitory action on the increase in intracellular Ca(2+) concentrations ([Ca(2+)](i)) obtained with extracellular Ca(2+) reintroduction after Ca(i)(2+) store depletion with thapsigargin (1 microM), an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA) pump. The rank order of potency for SOC inhibition by these compounds closely correlated with their inhibitory properties on depolarization-induced [3H]NE release from SH-SY5Y human neuroblastoma cells. Nimodipine (1 microM) plus omega-conotoxin (100 nM) did not interfere with the present model for SOC activation. In addition, the inhibition of depolarization-induced [3H]NE release does not seem to be attributable to the blockade of the K(+) currents carried by the K(+) channels encoded by the human Ether-a-Gogo Related Gene (I(HERG)) by these antihistamines. In fact, whole-cell voltage-clamp experiments revealed that the IC(50) for astemizole-induced hERG blockade is about 300-fold lower than that for the inhibition of high K(+)-induced [3H]NE release. Furthermore, current-clamp experiments in SH-SY5Y cells showed that concentrations of astemizole (3 microM) which were effective in preventing depolarization-induced [3H]NE release were unable to interfere with the cell membrane potential under depolarizing conditions (100 mM [K(+)](e)), suggesting that hERG K(+) channels do not contribute to membrane potential control during exposure to elevated [K(+)](e). Collectively, the results of the present study suggest that, in SH-SY5Y human neuroblastoma cells, the inhibition of SOCs by some second-generation antihistamines can prevent depolarization-induced neurotransmitter release.
Asunto(s)
Canales de Calcio/metabolismo , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Antagonistas de los Receptores Histamínicos H1/farmacología , Norepinefrina/metabolismo , Canales de Potasio con Entrada de Voltaje , Receptores Histamínicos H1/metabolismo , Transactivadores , Astemizol/farmacología , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Cetirizina/farmacología , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Humanos , Hidroxizina/farmacología , Loratadina/farmacología , Neuroblastoma , Canales de Potasio/metabolismo , Terfenadina/farmacología , Regulador Transcripcional ERG , Tritio , Células Tumorales CultivadasRESUMEN
Galanin-like peptide (GALP) neurones participate in the metabolic control of reproduction and are targets of insulin and leptin regulation. Phosphoinositide 3-kinase (PI3K) is common to the signalling pathways utilised by both insulin and leptin. Therefore, we investigated whether PI3K signalling in neurones expressing GALP plays a role in the transcriptional regulation of the GALP gene and in the metabolic control of luteinising hormone (LH) release. Accordingly, we deleted PI3K catalytic subunits p110α and p110ß via conditional gene targeting (cKO) in mice (GALP-p110α/ß cKO). To monitor PI3K signalling in GALP neurones, these animals were also crossed with Cre-dependent FoxO1GFP reporter mice. Compared to insulin-infused control animals, the PI3K-Akt-dependent FoxO1GFP nuclear exclusion in GALP neurones was abolished in GALP-p110α/ß cKO mice. We next used food deprivation to investigate whether the GALP-neurone specific ablation of PI3K activity affected the susceptibility of the gonadotrophic axis to negative energy balance. Treatment did not affect LH levels in either sex. However, a significant genotype effect on LH levels was observed in females. By contrast, no genotype effect on LH levels was observed in males. A sex-specific genotype effect on hypothalamic GALP mRNA was observed, with fed and fasted GALP-p110α/ß cKO males having lower GALP mRNA expression compared to wild-type fed males. Finally, the effects of gonadectomy and steroid hormone replacement on GALP mRNA levels were investigated. Compared to vehicle-treated mice, steroid hormone replacement reduced mediobasal hypothalamus GALP expression in wild-type and GALP-p110α/ß cKO animals. In addition, within the castrated and vehicle-treated group and compared to wild-type mice, LH levels were lower in GALP-p110α/ß cKO males. Double immunofluorescence using GALP-Cre/R26-YFP mice showed androgen and oestrogen receptor co-localisation within GALP neurones. Our data demonstrate that GALP neurones are direct targets of steroid hormones and that PI3K signalling regulates hypothalamic GALP mRNA expression and LH levels in a sex-specific fashion.
Asunto(s)
Proteínas Quinasas Dependientes de 3-Fosfoinosítido/fisiología , Péptido Similar a Galanina/fisiología , Hipotálamo/metabolismo , Hormona Luteinizante/metabolismo , Neuronas/enzimología , Animales , Femenino , Péptido Similar a Galanina/biosíntesis , Hipoglucemiantes/farmacología , Insulina/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Orquiectomía , Fenotipo , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal/fisiologíaRESUMEN
Although it is widely believed that astrocytes lack excitability in adult tissue, primitive action potential-like responses have been elicited from holding potentials negative to -80 mV, in cultured and injury-induced gliotic rodent astrocytes and in human glia under pathological conditions such as glioblastomas and temporal lobe epilepsy. The present study was designed to investigate the properties of astrocytes (identified by immunoreactivity for glial fibrillary acidic protein) derived from multipotent human embryonic CNS stem cells and cultured for 12-25 days in differentiating conditions. We describe here for the first time that brief (1 ms) current pulses elicit spikes from a resting potential (VREST) of approximately -37 mV and, more interestingly, that spontaneous firing can be occasionally recorded in human astrocytes. A voltage-clamp study revealed that in these cells: (i) the half-inactivation of the tetrodotoxin (TTX)-sensitive Na+ channels is around VREST; (ii) the delayed rectifier K+ current is very small; (iii) the ever-present transient outward A-type K+ channels are paradoxically capable of inhibiting the action potentials elicited from a negative membrane potential (-55 to -60 mV); and (iv) inwardly rectifying currents are not present. The responses predicted from a simulation model are in agreement with the experiments. As suggested by recent studies, the decrease of Na+ channel expression and the changes of the electrophysiological properties during the postnatal maturation of the CNS seem to exclude the possibility that astrocytes may play an excitable role in adult tissue. Our data show that excitability and firing should be considered an intrinsic attribute of human astrocytes during CNS development. This is likely to have physiological importance because the role of astrocytes during development is different from the [K+]o-buffering role played in adult CNS, namely the glutamate release and/or the guiding of migrating neurons.
Asunto(s)
Potenciales de Acción/fisiología , Astrocitos/metabolismo , Sistema Nervioso Central/metabolismo , Células Madre/metabolismo , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Astrocitos/clasificación , Astrocitos/citología , Astrocitos/efectos de los fármacos , Recuento de Células , Tamaño de la Célula/fisiología , Células Cultivadas , Sistema Nervioso Central/citología , Sistema Nervioso Central/embriología , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo , Células Madre/citología , Células Madre/efectos de los fármacos , Tetrodotoxina/farmacologíaRESUMEN
1. The regular firing of a Hodgkin-Huxley neurone endowed with fast Na+ and delayed K+ channels can be converted into adapting firing by appending HERG (human eag-related gene) channels. 2. The computer model predictions were verified by studying the firing properties of F-11 DRG neurone x neuroblastoma hybrid cells induced to differentiate by long-term exposure to retinoic acid. These cells, which express HERG currents (IHERG), show clear spike-frequency adaptation of their firing when current clamped with long depolarizations. 3. In agreement with the prediction, the selective blocking of IHERG by class III antiarrhythmic drugs always led to the disappearance of the spike-frequency adaptation, and the conversion of adapting firing to regular firing. 4. It is proposed that, in addition to their role in the repolarization of the heart action potential, HERG channels may sustain a process of spike-frequency adaptation, and hence contribute to the control of burst duration in a way that is similar to that of the K+ currents, IAHP, IC and IM. In addition to the known cardiac arrhythmia syndrome (LQT2), genetic mutations or an altered HERG expression could lead to continuous hyperexcitable states sustained by the inability of nerve or endocrine cells to accommodate to repetitive stimuli. This might help in clarifying the pathogenesis of still undefined idiopathic familial epilepsies.
Asunto(s)
Adaptación Fisiológica/fisiología , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Transactivadores , Animales , Antiarrítmicos/farmacología , Neoplasias Encefálicas/metabolismo , Células Clonales , Simulación por Computador , Canal de Potasio ERG1 , Estimulación Eléctrica , Electrofisiología , Canales de Potasio Éter-A-Go-Go , Humanos , Potenciales de la Membrana/fisiología , Ratones , Modelos Neurológicos , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Canales de Potasio/genética , Ratas , Regulador Transcripcional ERGRESUMEN
The human ether-a-go-go-related genes (herg) are expressed in tissues other than heart and brain where the HERG K(+) channels are known to regulate the repolarization of the heart action potential and the neuronal spike-frequency accommodation. We provide evidence that herg1 transcripts are present in human pancreatic islets that were used to study both insulin secretion and electrical activity with radioimmunoassay and single cell perforated patch-clamp techniques, respectively. Glucose- and arginine-induced islets insulin secretion data suggested a net increase of release under perfusion with antiarrhythmic drugs known to selectively block HERG channels. Indeed we could routinely isolate a K(+) current that was recognized as biophysically and pharmacologically similar to the HERG current. An analysis of the glucose- and arginine-induced electrical activity (several applications during 30 min) in terms of firing frequency and putative insulin release was done in control and in the presence of selective blockers of HERG channels: the firing frequency and the release increased by 32% and 77%, respectively. It is concluded that HERG channels have a crucial role in regulating insulin secretion and firing of human beta-cells. This raises the possibility that some genetically characterized hyperinsulinemic diseases of unknown origin might involve mutations in the HERG channels.
Asunto(s)
Arginina/farmacología , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Transactivadores , Transportadoras de Casetes de Unión a ATP , Bencimidazoles/farmacología , Canal de Potasio ERG1 , Conductividad Eléctrica , Canales de Potasio Éter-A-Go-Go , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Canales KATP , Técnicas de Placa-Clamp , Potasio/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio de Rectificación Interna , Radioinmunoensayo , Reproducibilidad de los Resultados , Sulfanilamidas/farmacología , Tolbutamida/farmacología , Regulador Transcripcional ERGRESUMEN
Expression of four members of the KChIP family of potassium channel beta subunits was examined in canine heart. Only one member of the gene family, KChIP2, was expressed in heart. There was a steep gradient of KChIP2 mRNA expression across the canine ventricular free wall. KChIP2 mRNA was 25-fold more abundant in the epicardium than in the endocardium, and this gradient paralleled the gradient in transient outward current (Ito) expression. In contrast, Kv4.3 potassium channel alpha subunit mRNA was expressed at equal levels across the ventricular wall. There was no difference in the pharmacological sensitivity of epicardial and endocardial Ito channels to flecainide, suggesting that the current is produced by the same channel in the two tissues. A similar gradient of KChIP2 expression was found across the ventricular wall of human heart, but not rat heart. It is concluded that transcriptional regulation of the KChIP2 beta subunit gene, rather than the Kv4.3 [alpha] subunit gene, is the primary determinant regulating the transmural gradient of Ito expression in the ventricular free wall of canine and human heart.
Asunto(s)
Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Miocardio/metabolismo , Canales de Potasio con Entrada de Voltaje , Potasio/metabolismo , Proteínas Represoras , Animales , Cartilla de ADN , Perros , Electrofisiología , Endocardio/citología , Endocardio/metabolismo , Expresión Génica/fisiología , Ventrículos Cardíacos/metabolismo , Humanos , Técnicas In Vitro , Proteínas de Interacción con los Canales Kv , Fibras Musculares Esqueléticas/metabolismo , Miocardio/citología , Oocitos/fisiología , Canales de Potasio/genética , Canales de Potasio/metabolismo , ARN Mensajero/análisis , Ratas , Canales de Potasio Shal , XenopusRESUMEN
The modulation of inward K+ conductances was studied during neuronal differentiation of human SH-SY5Y neuroblastoma cells. Under standard culture conditions, these cells express the herg gene, and the HERG current is the main inward K+ current regulating their Vrest. After 10-20 days exposure to Retinoic Acid (RA), SH-SY5Y cells showed, in addition to HERG currents, a novel current characterized by inward rectification, dependence on the extracellular K+ concentration, and blockade by Cs+ and Ba2+, the main features of the IRK1 current. The appearance of this current is accompanied by a strong hyperpolarisation of Vrest. RT-PCR experiments confirmed that a transcript of the IRK1 (Kir 2.1) gene actually appears in SH-SY5Y cells treated for 10-20 days with RA. On the whole, data here presented demonstrate that RA-induced neuronal differentiation of neuroblastoma cells is accompanied by the switch from a HERG-driven to a IRK1-driven control of Vrest, similarly to what happens in normal differentiating neurons; however, in tumor cells, this switch does not imply the abolition of HERG channel expression.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Neuronas/efectos de los fármacos , Canales de Potasio de Rectificación Interna , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/biosíntesis , Transactivadores , Tretinoina/farmacología , Diferenciación Celular , Canal de Potasio ERG1 , Conductividad Eléctrica , Canales de Potasio Éter-A-Go-Go , Expresión Génica , Humanos , Neuroblastoma , Técnicas de Placa-Clamp , Canales de Potasio/genética , ARN Mensajero/análisis , Factores de Tiempo , Regulador Transcripcional ERG , Células Tumorales CultivadasRESUMEN
Changes in the resting potential (VREST) and in the underlying ionic conductances were measured by the patch-clamp technique in SH-SY5Y human neuroblastoma cells exposed to substrate-bound or soluble Laminin (bLN; sLN), as compared to integrin-independent substrates (polylysine (PL); bovine serum albumin (BSA)). While PL and BSA were ineffective, both forms of LN caused an early (5-15 min) activation of a peculiar type of Inwardly Rectifying K+ current (IIR) characterised by a voltage-dependent inactivation in the range of membrane potentials around -50/0 mV. IIR was blocked by Cs+ ions and by the antiarrhythmic drug E-4031, a specific inhibitor of the HERG-codified channels. In cells adherent to bLN, IIR potentiation (85%) persisted for 90-120 min and was accompanied by a similar, but transient, increase in the leakage conductance (GL). Successively, the persistence of a high IIR conductance and the decrease of GL progressively bring VREST from -12 to approximately -30 mV in about 120 min. On the other hand, in cells adherent to PL, exposure to sLN produced a similar but not persistent activation of IIR, without any increase in GL: this caused a rapid, transient hyperpolarisation of VREST. The effects of bLN and sLN were mimicked by antibodies raised against the integrin beta 1 subunit, suggesting a specific integrin-mediated mechanism. In fact, when bound to the culture dishes, these antibodies simultaneously activated the IIR and GL, whereas in soluble form they only activated IIR. Cells adherent to bLN emitted neurites, a process impaired by the block of IIR by E-4031 and Cs+. On the whole data suggest that the integrin-mediated activation of IIR plays a crucial role in the commitment to neuritogenesis of neuroblastoma cells, independently on the effects of this activation on VREST.
Asunto(s)
Laminina/farmacología , Potenciales de la Membrana/efectos de los fármacos , Neuritas/efectos de los fármacos , Canales de Potasio de Rectificación Interna , Canales de Potasio/efectos de los fármacos , Adhesión Celular , Conductividad Eléctrica , Electrofisiología , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Integrinas/fisiología , Activación del Canal Iónico , Morfogénesis/efectos de los fármacos , Neuroblastoma , Técnicas de Placa-Clamp , Células Tumorales CultivadasRESUMEN
Quail neural crest cells were cultured in a differentiative medium to study the inward K+ channel profile in neuronal precursors at various stages of maturation. Between 12 and 24 h of culture, neural crest-derived neurons displayed, in addition to the previously described outward depolarization-activated K+ currents, an inward current enhanced in high K+ medium. A biophysical and pharmacological analysis led us to conclude that this inward K+ current is identical to that previously demonstrated in mouse and human neuroblastoma cell lines (I[IR]). This current (quail I[IR] or ql[IR]), which is active at membrane potentials positive to -35 mV, was blocked by Cs+ and by class III antiarrhythmic drugs, thus resembling the K+ current encoded by the human ether-a-gò-gò-related gene (HERG). At later stages of incubation (>48 h), neural crest-derived neurons underwent morphological and biochemical differentiation and expressed fast Na+ currents. At this stage the cells lost qI[IR], displaying instead a classical inward rectifier K+ (IRK) current (quail I[IRK] = qI[IRK]). This substitution was reflected in the resting potential (VREST), which became hyperpolarized by >20 mV compared with the 24 h cells. Neurons were also harvested from peripheral ganglia and other derivatives originating from the migration of neural crest cells, viz. ciliary ganglia, dorsal root ganglia, adrenal medulla and sympathetic chain ganglia. After brief culture following harvesting from young embryos, ganglionic neurons always expressed qI(IR). On the other hand, when ganglia were explanted from older embryos (7-12 days), briefly cultured neurons displayed the IRK-like current. Again, in all the above derivatives the qI(IR) substitution by qI(IRK) was accompanied by a 20 mV hyperpolarization of VREST. Together, these data indicate that the VREST of normal neuronal precursors is sequentially regulated by HERG- and IRK-like currents, suggesting that HERG-like channels mark an immature and transient stage of neuronal differentiation, probably the same stage frozen in neuroblastomas by neoplastic transformation.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Cresta Neural/citología , Neuronas/citología , Canales de Potasio de Rectificación Interna , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Transactivadores , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Embrión de Pollo , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Ganglios Espinales/citología , Ganglios Simpáticos/citología , Regulación del Desarrollo de la Expresión Génica , Mesencéfalo/citología , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio/metabolismo , CodornizRESUMEN
ERG (ether-à-go-go-related gene) K+ channels are crucial in human heart physiology (h-ERG), but are also found in neuronal cells and are impaired in Drosophila 'seizure' mutants. Their biophysical properties include the relatively fast kinetics of the inactivation gate and much slower kinetics of the activation gate. In order to elucidate how the complex time- and voltage-dependent activation properties of ERG channels underlies distinct roles in excitability, we investigated different types of ERG channels intrinsically present in cells or heterologously expressed in mammalian cells or Xenopus oocytes. Voltage-dependent activation curves were highly dependent on the features of the eliciting protocols. Only very long preconditioning times produced true steady-state relationships, a fact that has been largely neglected in the past, hampering the comparison of published data on ERG channels. Beyond this technical aspect, the slow activation property of ERG can be responsible for unsuspected physiological roles. We found that around the midpoint of the activation curve, the time constant of ERG open-close kinetics is of the order of 10-15 s. During sustained trains of depolarizations, e.g. those produced in neuronal firing, this leads to the use-dependent accumulation of open-state ERG channels. Accumulation is not observed in a mutant with a fast activation gate. In conclusion, it is well established that other K+ channels (i.e. Ca2+-activated and M) control the spike-frequency adaptation, but our results support the notion that the purely voltage-dependent activation property of ERG channels would allow a slow inhibitory physiological role in rapid neuronal signalling.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Activación del Canal Iónico/fisiología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/genética , Canales de Potasio/metabolismo , Transactivadores , Animales , Canal de Potasio ERG1 , Estimulación Eléctrica , Electrofisiología , Canales de Potasio Éter-A-Go-Go , Ganglios Espinales/citología , Humanos , Riñón/citología , Leucemia , Potenciales de la Membrana/fisiología , Ratones , Mutagénesis/fisiología , Neuroblastoma , Oocitos/fisiología , Ratas , Regulador Transcripcional ERG , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/fisiología , XenopusRESUMEN
The modulation of herg gene and HERG currents (I(HERG)) was studied in SH-SY5Y neuroblastoma (NB) cells treated with all-trans-retinoic acid (RA) in the absence or presence of the neurotrophin brain-derived neurotrophic factor (BDNF). Both treatments produced a strong increase in the percentage of cells differentiated along the neuronal pathway, with an orientation to a cholinergic phenotype, while a minority of cells displayed a glial phenotype particularly evident after long-term exposure to the inducers. Differentiation of NB cells was accompanied by an increase in herg gene transcription, which attained its maximum after 6 days of treatment with RA and was not further increased by BDNF. This effect evidently reflected on HERG currents: In fact, RA produced an increase in HERG current density which was strongly potentiated by BDNF. Moreover, RA treatment affected the biophysical properties of I(HERG), inducing an increase in the deactivation time constant and a left shift of the activation curve. These effects were not substantially affected by BDNF. This modulation of I(HERG) influenced the value of the resting potential (V(REST)), which resulted significantly hyperpolarized in (RA with or without BDNF)-treated cells. Interestingly, these effects were absent in the glial population, which prevailed in cultures after long-term exposure to the inducers. On the whole, we demonstrate that besides expressing IRK currents, NB cells display another strategy to hyperpolarize their V(REST), based on the appropriate modulation of HERG currents. Different from what happens in normal neuroblast development, the latter are never lost by cancer cells despite the progression of these cells along the neuronal differentiative pathway, raising intriguing questions about the role of HERG currents in tumour behavior.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/farmacología , Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas de Neoplasias/fisiología , Neuroblastoma/patología , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/fisiología , Potasio/metabolismo , Transactivadores , Tretinoina/farmacología , Acetilcolina/metabolismo , Diferenciación Celular/efectos de los fármacos , Sinergismo Farmacológico , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Transporte Iónico/efectos de los fármacos , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Técnicas de Placa-Clamp , Canales de Potasio/biosíntesis , Canales de Potasio/genética , Regulador Transcripcional ERG , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
Using the patch-clamp technique, we analysed changes in the biophysical properties of HERG potassium channels in neuroblastoma cells long-term exposed to hypoxia. In this condition, HERG channels underwent a profound modulation that consisted of: (i) a slowing in open-close kinetics; (ii) a marked hyperpolarizing shift in voltage dependence of steady-state activation; and (iii) a slowing of the inactivation removal. The overall physiological impact of these changes in neuroblastoma cells is an increase in the HERG window current in the range of the resting potential (V(REST)), which varied between -40 and -30 mV. Such a current modulation is suitable to stabilise the resting potential (V(REST)) in hypoxic environments.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Hipoxia , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Transactivadores , Membrana Celular/química , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Humanos , Cinética , Potenciales de la Membrana , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Regulador Transcripcional ERG , Células Tumorales CultivadasRESUMEN
Toxins isolated from a variety of venoms are tools for probing the physiological function and structure of ion channels. The ether-a-go-go-related genes (erg) codify for the K+ channels (ERG), which are crucial in neurons and are impaired in human long-QT syndrome and Drosophila 'seizure' mutants. We have isolated a peptide from the scorpion Centruroides noxius Hoffmann that has no sequence homologies with other toxins, and demonstrate that it specifically inhibits (IC50=16+/-1 nM) only ERG channels of different species and distinct histogenesis. These results open up the possibility of investigating ERG channel structure-function relationships and novel pharmacological tools with potential therapeutic efficacy.
Asunto(s)
Proteínas de Transporte de Catión , Proteínas de Unión al ADN , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Venenos de Escorpión/química , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/toxicidad , Transactivadores , Potenciales de Acción/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Canal de Potasio ERG1 , Glándulas Endocrinas/efectos de los fármacos , Glándulas Endocrinas/metabolismo , Canales de Potasio Éter-A-Go-Go , Cobayas , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Miocardio/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Canales de Potasio/genética , Ratas , Venenos de Escorpión/genética , Escorpiones , Homología de Secuencia de Aminoácido , Regulador Transcripcional ERGRESUMEN
Integrin receptors have been demonstrated to mediate either "inside-to-out" and "outside-to-in" signals, and by this way are capable of regulating many cellular functions, such as cell growth and differentiation, cell migration, and activation. Among the various integrin-centered signaling pathways discovered so far, we demonstrated that the modulation of the electrical potential of the plasma membrane (V(REST)) is an early integrin-mediated signal, which is related to neurite emission in neuroblastoma cells. This modulation is sustained by the activation of HERG K(+) channels, encoded by the ether-à-go-go-related gene (herg). The involvement of integrin-mediated signaling is being discovered in the hemopoietic system: in particular, osteoclasts are generated as well as induced to differentiate by interaction of osteoclast progenitors with the stromal cells, through the involvement of integrin receptors. We studied the effects of cell interaction with the extracellular matrix protein fibronectin (FN) in a human leukemic preosteoclastic cell line (FLG 29.1 cells), which has been demonstrated to express HERG currents. We report here that FLG 29.1 cells indeed adhere to purified FN through integrin receptors, and that this adhesion induces an osteoclast phenotype in these cells, as evidenced by the appearance of tartrate-resistant acid phosphatase, as well as by the increased expression of CD51/alpha(v)beta(3) integrin and calcitonin receptor. An early activation of HERG current (I(HERG)), without any increase in herg RNA or modifications of HERG protein was also observed in FN-adhering cells. This activation is apparently sustained by the beta(1) integrin subunit activation, through the involvement of a pertussis-toxin sensitive G(i) protein, and appears to be a determinant signal for the up-regulation of alpha(v)beta(3) integrin, as well as for the increased expression of calcitonin receptor.
Asunto(s)
Proteínas de Transporte de Catión , Adhesión Celular , Proteínas de Unión al ADN , Fibronectinas/metabolismo , Integrina beta1/fisiología , Osteoclastos/metabolismo , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Receptores de Vitronectina/genética , Transactivadores , Anticuerpos Monoclonales/inmunología , Diferenciación Celular , Canal de Potasio ERG1 , Conductividad Eléctrica , Canales de Potasio Éter-A-Go-Go , Proteínas Filagrina , Humanos , Integrina beta1/inmunología , Leucemia , Osteoclastos/citología , Técnicas de Placa-Clamp , Canales de Potasio/genética , ARN Mensajero/biosíntesis , Receptores de Calcitonina/biosíntesis , Receptores de Calcitonina/genética , Receptores de Vitronectina/biosíntesis , Células Madre/citología , Células Madre/metabolismo , Regulador Transcripcional ERG , Células Tumorales Cultivadas , Regulación hacia ArribaRESUMEN
HERG K(+)channels, besides contributing to regulate cardiac and neuronal excitability, are preferentially expressed in tumour cell lines of different histogenesis, where their role in the development and maintenance of the neoplastic phenotype is under study. We show here that both herg gene and HERG protein are expressed with high frequency in primary human endometrial cancers, as compared to normal and hyperplastic endometrium. RT-PCR and immunohistochemistry, using specific anti-HERG antibodies developed in our laboratory, were applied to tissue specimens obtained from 18 endometrial cancers and 11 non-cancerous endometrial tissues. herg RNA and HERG protein are expressed in 67% and 82%, respectively, of cancerous, while in only 18% of non-cancerous tissues. In particular, no expression was found in endometrial hyperplasia. Moreover, electrophysiological experiments confirmed the presence of functioning HERG channels on the plasma membrane of tumour cells. On the whole, these data are the first demonstration of the presence of HERG channels in primary human neoplasias, and could candidate HERG as a potential tool capable of marking cancerous versus hyperplastic endometrial growth.