RESUMEN

Mutations in the presenilin genes cause the majority of early-onset familial Alzheimer's disease. Recently, presenilin mutations have been identified in patients with dilated cardiomyopathy (DCM), a common cause of heart failure and the most prevalent diagnosis in cardiac transplantation patients. However, the molecular mechanisms, by which presenilin mutations lead to either AD or DCM, are not yet understood. We have employed transgenic Drosophila models and optical coherence tomography imaging technology to analyze cardiac function in live adult Drosophila. Silencing of Drosophila ortholog of presenilins (dPsn) led to significantly reduced heart rate and remarkably age-dependent increase in end-diastolic vertical dimensions. In contrast, overexpression of dPsn increased heart rate. Either overexpression or silencing of dPsn resulted in irregular heartbeat rhythms accompanied by cardiomyofibril defects and mitochondrial impairment. The calcium channel receptor activities in cardiac cells were quantitatively determined via real-time RT-PCR. Silencing of dPsn elevated dIP3R expression, and reduced dSERCA expression; overexprerssion of dPsn led to reduced dRyR expression. Moreover, overexpression of dPsn in wing disc resulted in loss of wing phenotype and reduced expression of wingless. Our data provide novel evidence that changes in presenilin level leads to cardiac dysfunction, owing to aberrant calcium channel receptor activities and disrupted Wnt signaling transduction, indicating a pathogenic role for presenilin mutations in DCM pathogenesis.

Asunto(s)

Cardiomiopatías/genética , Cardiomiopatías/fisiopatología , Drosophila melanogaster/genética , Presenilinas/genética , Enfermedad de Alzheimer/genética , Animales , Animales Modificados Genéticamente , Western Blotting , Canales de Calcio/genética , Canales de Calcio/metabolismo , Proteínas de Drosophila/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Tomografía de Coherencia Óptica , Vía de Señalización Wnt/genética

RESUMEN

The syncytiotrophoblast of the human placenta arises from fusion of stem cells called cytotrophoblasts. The molecular mechanisms associated with cell fusion and syncytiation of cytotrophoblastic cells remain largely unknown. In the present study, we investigated the morphological and electrical properties of BeWo cells, a human choriocarcinoma-derived trophoblast cell model, with several features of the human cytotrophoblast. Cultured cells tended to cluster, but only fused into small, multinucleated syncytia in the presence of cAMP (72 h). The morphological features of both the actin and microtubular cytoskeletons indicated that within 72 h of constant exposure to cAMP, intracellular cortical actin cytoskeleton disappeared, which was the most prominent inducing factor of multi-nucleation. The presence of the cation channel protein, polycystin-2 (PC2), a TRP-type cation channel, associated with placental ion transport in term human syncytiotrophoblast, co-localised with acetylated tubulin in midbodies, but was found non-functional under any conditions. Different electrical phenotypes were observed among control BeWo cells, where only 26% (8 of 31 cells) displayed a voltage-dependent outwardly rectifying conductance. Most quiescent BeWo cells had, however, a low, slightly outwardly rectifying basal whole cell conductance. Acute exposure to intracellular cAMP (<15 min) increased the whole cell conductance by 122%, from 0.72 nS/cell to 1.60 nS/cell, and eliminated the voltage-regulated conductance. The encompassed evidence indicates that the early events in BeWo cell fusion and syncytiation occur by cAMP-associated changes in ionic conductance but not morphological changes associated to chronic exposure to the second messenger. This suggests a tight regulation, and important contribution of cation conductances in cytotrophoblastic cells prior to syncytiation.

Asunto(s)

Coriocarcinoma/fisiopatología , Trofoblastos/patología , Trofoblastos/fisiología , Neoplasias Uterinas/fisiopatología , Línea Celular Tumoral , Coriocarcinoma/patología , AMP Cíclico/farmacología , Citoesqueleto/metabolismo , Citoesqueleto/patología , Electrofisiología , Femenino , Humanos , Inmunohistoquímica , Potenciales de la Membrana/efectos de los fármacos , Embarazo , Neoplasias Uterinas/patología

RESUMEN

Pregnancy is often associated with oxidative stress (OS) and lower antioxidant defences, which are both implicated in the pathophysiology of preeclampsia, free radical-induced birth defects, and abortions, as well as gestational diabetes mellitus (GDM), where products of lipid peroxidation are increased. The molecular target(s) of increased oxygen free radicals and consequent lipid peroxidation in the human placenta remains ill defined. The human syncytiotrophoblast (hST) expresses abundant polycystin-2 (PC2, TRPP2), a TRP-type Ca(2+)-permeable non-selective cation channel. Here, we explored the effect of reactive oxygen species (ROS) on PC2 channel activity of term hST. Apical membranes of the hST were reconstituted in a lipid bilayer chamber. Addition of either hydrogen-peroxide (H(2)O(2)) or tert-butyl hydroperoxide (tBHP) to the cis chamber (intracellular side) rapidly and completely inhibited PC2-mediated cation channel activity in reconstituted hST vesicles. A dose-response titration with increasing concentrations of H(2)O(2) gave an IC(50)=131 nM. The effect of H(2)O(2) on the isolated protein from in vitro transcribed/translated material was significantly different. H(2)O(2) inhibited PC2 cation channel activity, with a much lower affinity (IC(50)=193 microM). To correlate these findings with H(2)O(2)-induced lipid peroxidation, TBARS where measured in hST apical membranes incubated with H(2)O(2). Increased TBARS by exposure of hST apical membranes to H(2)O(2) (625 microM) returned to control value in the presence of catalase (167 microg/ml). Taken together these data indicate that ROS affect PC2 channel function by targetting both membrane lipids and the channel protein. Thus, OS in human pregnancy may be linked to dysregulation of channels such as PC2, which allow the transport of Ca(2+) into the placenta. Oxidative complications in pregnancy may implicate dysfunctional cation transfer between mother and fetus.

Asunto(s)

Especies Reactivas de Oxígeno/farmacología , Canales Catiónicos TRPP/antagonistas & inhibidores , Nacimiento a Término/metabolismo , Trofoblastos/efectos de los fármacos , Electrofisiología , Femenino , Humanos , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio/fisiología , Embarazo , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Canales Catiónicos TRPP/metabolismo , Trofoblastos/metabolismo , terc-Butilhidroperóxido/farmacología

RESUMEN

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), which is caused by mutations in polycystins 1 (PC1) and 2 (PC2), is one of the most commonly inherited renal diseases, affecting ~1 : 1000 Caucasians. MATERIALS AND METHODS: We screened Greek ADPKD patients with the denaturing gradient gel electrophoresis (DGGE) assay and direct sequencing. RESULTS: We identified a patient homozygous for a nucleotide change c.1445T > G, resulting in a novel homozygous substitution of the non-polar hydrophobic phenylalanine to the polar hydrophilic cysteine in exon 6 at codon 482 (p.F482C) of the PKD2 gene and a de-novo PKD1 splice-site variant IVS21-2delAG. We did not find this PKD2 variant in a screen of 280 chromosomes of healthy subjects, supporting its pathogenicity. The proband's parents did not have the PKD1 mutation. Real-time PCR of the PKD2 transcript from a skin biopsy revealed 20-fold higher expression in the patient than in a healthy subject and was higher in the patient's peripheral blood mononuclear cells (PBMCs) than in those of her heterozygote daughter and a healthy subject. The greater gene expression was also supported by Western blotting. Inner medullar collecting duct (IMCD) cells transfected with the mutant PKD2 mouse gene presented a perinuclear and diffuse cytoplasmic localization compared with the wild type ER localization. Patch-clamping of PBMCs from the p.F482C homozygous and heterozygous subjects revealed lower polycystin-2 channel function than in controls. CONCLUSIONS: We report for the first time a patient with ADPKD who is heterozygous for a de novo PKD1 variant and homozygous for a novel PKD2 mutation.

Asunto(s)

Riñón Poliquístico Autosómico Dominante/genética , Proteínas/genética , Animales , Células Cultivadas , Análisis Mutacional de ADN , Electroforesis/métodos , Femenino , Homocigoto , Humanos , Masculino , Ratones , Mutación , Reacción en Cadena de la Polimerasa , Canales Catiónicos TRPP

Asunto(s)

Transducción de Señal/fisiología , Trofoblastos/metabolismo , Calcineurina/metabolismo , Señalización del Calcio , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Femenino , Humanos , Sistema de Señalización de MAP Quinasas , Factores de Transcripción NFATC , Proteínas Nucleares/metabolismo , Embarazo , Factor de Transcripción STAT3 , Transactivadores/metabolismo , Factores de Transcripción/metabolismo

RESUMEN

We investigate the conditions enabling actin filaments to act as electrical transmission lines for ion flows along their lengths. We propose a model in which each actin monomer is an electric element with a capacitive, inductive, and resistive property due to the molecular structure of the actin filament and viscosity of the solution. Based on Kirchhoff's laws taken in the continuum limit, a nonlinear partial differential equation is derived for the propagation of ionic waves. We solve this equation in two different regimes. In the first, the maximum propagation velocity wave is found in terms of Jacobi elliptic functions. In the general case, we analyze the equation in terms of Fisher-Kolmogoroff modes with both localized and extended wave characteristics. We propose a new signaling mechanism in the cell, especially in neurons.

Asunto(s)

Citoesqueleto de Actina/fisiología , Actinas/química , Modelos Teóricos , Transporte Iónico/fisiología

Asunto(s)

Transducción de Señal , Canales Catiónicos TRPP/metabolismo , Actinina/farmacología , Actinas/metabolismo , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/metabolismo , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/metabolismo , Modelos Biológicos , Presión , Transducción de Señal/efectos de los fármacos , Trofoblastos/efectos de los fármacos , Trofoblastos/metabolismo

RESUMEN

The heart must function from the moment of its embryonic assembly, but the molecular underpinnings of the first heart beat are not known, nor whether function determines form at this early stage. Here, we find by positional cloning that the embryonic lethal island beat (isl) mutation in zebrafish disrupts the alpha1 C L-type calcium channel subunit (C-LTCC). The isl atrium is relatively normal in size, and individual cells contract chaotically, in a pattern resembling atrial fibrillation. The ventricle is completely silent. Unlike another mutation with a silent ventricle, isl fails to acquire the normal number of myocytes. Thus, calcium signaling via C-LTCC can regulate heart growth independently of contraction, and plays distinctive roles in fashioning both form and function of the two developing chambers.

Asunto(s)

Canales de Calcio Tipo L/química , Canales de Calcio Tipo L/fisiología , Corazón/embriología , Alelos , Secuencia de Aminoácidos , Animales , Fibrilación Atrial , Calcio/metabolismo , Biblioteca de Genes , Hibridación in Situ , Microscopía Electrónica , Modelos Biológicos , Modelos Genéticos , Datos de Secuencia Molecular , Mutación , Miocardio/citología , Miocardio/metabolismo , Páncreas/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Homología de Secuencia de Aminoácido , Transducción de Señal , Factores de Tiempo , Pez Cebra

RESUMEN

Defects in polycystin-2, a ubiquitous transmembrane glycoprotein of unknown function, is a major cause of autosomal dominant polycystic kidney disease (ADPKD), whose manifestation entails the development of fluid-filled cysts in target organs. Here, we demonstrate that polycystin-2 is present in term human syncytiotrophoblast, where it behaves as a nonselective cation channel. Lipid bilayer reconstitution of polycystin-2-positive human syncytiotrophoblast apical membranes displayed a nonselective cation channel with multiple subconductance states, and a high perm-selectivity to Ca2+. This channel was inhibited by anti-polycystin-2 antibody, Ca2+, La3+, Gd3+, and the diuretic amiloride. Channel function by polycystin-2 was confirmed by patch-clamping experiments of polycystin-2 heterologously infected Sf9 insect cells. Further, purified insect cell-derived recombinant polycystin-2 and in vitro translated human polycystin-2 had similar ion channel activity. The polycystin-2 channel may be associated with fluid accumulation and/or ion transport regulation in target epithelia, including placenta. Dysregulation of this channel provides a mechanism for the onset and progression of ADPKD.

Asunto(s)

Canales de Calcio/genética , Proteínas de la Membrana/genética , Mutación , Riñón Poliquístico Autosómico Dominante/genética , Animales , Anticuerpos/farmacología , Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , Línea Celular , Membrana Celular/fisiología , Femenino , Gadolinio/farmacología , Humanos , Lantano/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/fisiología , Placenta/fisiología , Embarazo , Biosíntesis de Proteínas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Spodoptera , Canales Catiónicos TRPP , Transfección , Trofoblastos/fisiología

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the superfamily of ATP-binding cassette (ABC) transporters, also known as traffic ATPases. Recent studies from our laboratory determined that various members of the ABC family of transport proteins mediate the electrodiffusional movement of the nucleotide ATP. In this report, evidence for the movement of cellular nucleotides by the ABC transporter CFTR and related molecules, including P-glycoproteins (Pgp), is reviewed. The wild-type mdr1 gene product, Pgp, enables the spontaneous release of cellular ATP. However, single amino acid substitutions in both nucleotide-binding sites render a dysfunctional Pgp, whose function can only be reversed by voltage activation. This report includes data indicating that reconstitution of highly purified CFTR from human epithelial origin enables the permeation of both Cl and ATP. The relevance of the ABC domains in ATP transport is also explored, and the hypothesis is forwarded that improper ATP transport by a dysfunctional CFTR is a relevant factor in cystic fibrosis.

Asunto(s)

Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/metabolismo , Animales , Difusión , Humanos , Técnicas de Placa-Clamp

RESUMEN

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion-selective channel whose dysfunction leads to the onset of cystic fibrosis. CFTR activation is normally elicited by stimulation of the cAMP pathway, which effects protein kinase A activation. However, previous studies from our laboratory indicate that the actin cytoskeleton is also required for a proper CFTR function. In this report, the regulatory role of actin filament organization in the activation of CFTR was explored. Maneuvers to modify the steady-state organization of actin filaments elicit the activation of CFTR in the absence of a functional cAMP pathway. Partial disruption of the actin cytoskeleton of CFTR-expressing cells with cytochalasin D (CD) induced CFTR activation in the absence of an activated PKA. Similar findings were obtained by intracellular dialysis with the actin-severing protein gelsolin. However, extended treatment with CD leading to the collapse of the actin cytoskeleton rendered CFTR completely insensitive to direct PKA activation. cAMP activation of CFTR was also found to be dysfunctional in cells lacking the actin-crosslinking protein ABP-280, which was recovered after dialysis of the cells with filamin, a homologue of ABP-280. The present data indicate that an organized actin network is required for the proper cAMP-dependent activation of CFTR. The possibility is also explored that actin must be directly associated with CFTR to elicit its activation, further suggesting that this channel protein may bind actin as well.

Asunto(s)

Citoesqueleto de Actina/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Adenocarcinoma , Secuencia de Aminoácidos , Animales , Proteínas Contráctiles/química , Proteínas Contráctiles/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Citocalasina D/farmacología , Femenino , Filaminas , Gelsolina/metabolismo , Humanos , Neoplasias Mamarias Animales , Melanoma , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Datos de Secuencia Molecular , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Células Tumorales Cultivadas

RESUMEN

Recent studies have shown that the cystic fibrosis transmembrane conductance regulator (CFTR), an ATP-binding cassette (ABC) transporter whose mutations are responsible for cystic fibrosis (CF), permeates ATP. However, little information is available concerning extracellular ATP concentrations in CF patients. Thus, the goal of this preliminary study was to determine the circulating levels of plasma ATP in CF patients. Circulating levels of plasma ATP were determined by the luciferin-luciferase assay in both CF patients and healthy volunteer control subjects. The two groups were compared using an analysis of variance. CF genotype and age, which ranged from 7 to 56 years, were also used to compare data by single-blind analysis. With comparable sample numbers, CF patients had statistically higher levels of circulating ATP (34%, P<0.01) when compared by analysis of covariance with the age of the subjects as the cofactor. The CF patients bearing the DeltaF508 genotype had a 54% (n=33, P<0.01) higher plasma ATP concentration compared to controls, while patients bearing other CF genotypes were similar to controls (n=10, P<0.4). We conclude that CF patients have higher circulating levels of ATP when compared to controls. Increased levels of plasma ATP, which is an important autocrine/paracrine hormone in many cell types, may be associated with chronic manifestations of the disease.

Asunto(s)

Adenosina Trifosfato/sangre , Fibrosis Quística/sangre , Adolescente , Adulto , Factores de Edad , Análisis de Varianza , Niño , Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Genotipo , Homocigoto , Humanos , Persona de Mediana Edad , Mutación , Reproducibilidad de los Resultados

RESUMEN

The molecular mechanisms associated with intracellular ATP release by the heart are largely unknown. In this study the luciferin-luciferase assay and patch-clamp techniques were used to characterize the pathways responsible for ATP release in neonatal rat cardiac myocytes (NRCM). Spontaneous ATP release by NRCM was significantly increased after cAMP stimulation under physiological conditions. cAMP stimulation also induced an anion-selective electrodiffusional pathway that elicited linear, diphenylamine-2-carboxylate (DPC)-inhibitable Cl(-) currents in either symmetrical MgCl(2) or NaCl. ATP, adenosine 5'-O-(3-thiotriphosphate), and the ATP derivatives ADP and AMP, permeated this pathway; however, GTP did not. The cAMP-induced ATP currents were inhibited by DPC and glibenclamide and by a monoclonal antibody raised against the R domain of the cystic fibrosis transmembrane conductance regulator (CFTR). The channel-like nature of the cAMP-induced ATP-permeable pathway was also determined by assessing protein kinase A-activated single channel Cl(-) and ATP currents in excised inside-out patches of NRCM. Single channel currents were inhibited by DPC and the anti-CFTR R domain antibody. Thus the data in this report demonstrate the presence of a cAMP-inducible electrodiffusional ATP transport mechanism in NRCM. Based on the pharmacology, patch-clamping data, and luminometry studies, the data are most consistent with the role of a functional CFTR as the anion channel implicated in cAMP-activated ATP transport in NRCM.

Asunto(s)

Adenosina Trifosfato/metabolismo , Animales Recién Nacidos/metabolismo , AMP Cíclico/fisiología , Miocardio/metabolismo , Adenosina Trifosfato/fisiología , Animales , Anticuerpos/farmacología , Permeabilidad de la Membrana Celular , Células Cultivadas , Senescencia Celular/fisiología , Canales de Cloruro/fisiología , Cloruros/fisiología , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Conductividad Eléctrica , Miocardio/citología , Ratas

RESUMEN

In this study, patch-clamp techniques were applied to cultured neonatal mouse cardiac myocytes (NMCM) to assess the contribution of cAMP stimulation to the anion permeability in this cell model. Addition of either isoproterenol or a cocktail to raise intracellular cAMP increased the whole cell currents of NMCM. The cAMP-dependent conductance was largely anionic, as determined under asymmetrical (low intracellular) Cl(-) conditions and symmetrical Cl(-) in the presence of various counterions, including Na(+), Mg(2+), Cs(+), and N-methyl-D-glucamine. Furthermore, the cAMP-stimulated conductance was also permeable to ATP. The cAMP-activated currents were inhibited by diphenylamine-2-carboxylate, glibenclamide, and an anti-cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody. The anti-CFTR monoclonal antibody failed, however, to inhibit an osmotically activated anion conductance, indicating that CFTR is not linked to osmotically stimulated currents in this cell model. Immunodetection studies of both neonatal mouse heart tissue and cultured NMCM revealed that CFTR is expressed in these preparations. The implication of CFTR in the cAMP-stimulated Cl(-)- and ATP-permeable conductance was further verified with NMCM of CFTR knockout mice [cftr(-/-)] in which cAMP stimulation was without effect on the whole cell currents. In addition, stimulation with protein kinase A and ATP induced Cl(-)-permeable single-channel activity in excised, inside-out patches from control, but not cftr(-/-) NMCM. The data in this report indicate that cAMP stimulation of NMCM activates an anion-permeable conductance with functional properties similar to those expected for CFTR, thus suggesting that CFTR may be responsible for the cAMP-activated conductance. CFTR may thus contribute to the permeation and/or regulation of Cl(-)- and ATP-permeable pathways in the developing heart.

Asunto(s)

Cloruros/metabolismo , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Miocardio/química , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Adenosina Trifosfato/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Cardiotónicos/farmacología , Células Cultivadas , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/aislamiento & purificación , Conductividad Eléctrica , Femenino , Expresión Génica/fisiología , Gliburida/farmacología , Hipoglucemiantes/farmacología , Isoproterenol/farmacología , Cloruro de Magnesio/farmacocinética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/fisiología , Miocardio/citología , Presión Osmótica , Técnicas de Placa-Clamp , Pruebas de Precipitina , Embarazo , ortoaminobenzoatos/farmacología

RESUMEN

Previous studies have indicated a role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel. However, the exact molecular nature of this regulation is still largely unknown. In this report human epithelial CFTR was expressed in human melanoma cells genetically devoid of the filamin homologue actin-cross-linking protein ABP-280 [ABP(-)]. cAMP stimulation of ABP(-) cells or cells genetically rescued with ABP-280 cDNA [ABP(+)] was without effect on whole cell Cl(-) currents. In ABP(-) cells expressing CFTR, cAMP was also without effect on Cl(-) conductance. In contrast, cAMP induced a 10-fold increase in the diphenylamine-2-carboxylate (DPC)-sensitive whole cell Cl(-) currents of ABP(+)/CFTR(+) cells. Further, in cells expressing both CFTR and a truncated form of ABP-280 unable to cross-link actin filaments, cAMP was also without effect on CFTR activation. Dialysis of ABP-280 or filamin through the patch pipette, however, resulted in a DPC-inhibitable increase in the whole cell currents of ABP(-)/CFTR(+) cells. At the single-channel level, protein kinase A plus ATP activated single Cl(-) channels only in excised patches from ABP(+)/CFTR(+) cells. Furthermore, filamin alone also induced Cl(-) channel activity in excised patches of ABP(-)/CFTR(+) cells. The present data indicate that an organized actin cytoskeleton is required for cAMP-dependent activation of CFTR.

Asunto(s)

Actinas/fisiología , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Citoesqueleto/fisiología , Aniones/farmacocinética , Bromo/farmacocinética , Cloro/farmacocinética , Proteínas Contráctiles/genética , Proteínas Contráctiles/farmacología , Reactivos de Enlaces Cruzados/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/farmacología , Diálisis , Filaminas , Expresión Génica/fisiología , Gluconatos/farmacocinética , Humanos , Yodo/farmacocinética , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Melanoma , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/farmacología , Técnicas de Placa-Clamp , Transfección , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/enzimología

RESUMEN

The actin cytoskeleton is an important contributor to the modulation of the cell function. However, little is known about the regulatory role of this supermolecular structure in the membrane events that take place in the heart. In this report, the regulation of cardiac myocyte function by actin filament organization was investigated in neonatal mouse cardiac myocytes (NMCM) from both wild-type mice and mice genetically devoid of the actin filament severing protein gelsolin (Gsn-/-). Cardiac L-type calcium channel currents (I(Ca)) were assessed using the whole cell voltage-clamp technique. Addition of the actin filament stabilizer phalloidin to wild-type NMCM increased I(Ca) by 227% over control conditions. The basal I(Ca) of Gsn-/- NMCM was 300% higher than wild-type controls. This increase was completely reversed by intracellular perfusion of the Gsn-/- NMCM with exogenous gelsolin. Further, cytoskeletal disruption of either Gsn-/- or phalloidin-dialyzed wild-type NMCM with cytochalasin D (CD) decreased the enhanced I(Ca) by 84% and 87%, respectively. The data indicate that actin filament stabilization by either a lack of gelsolin or intracellular dialysis with phalloidin increase I(Ca), whereas actin filament disruption with CD or dialysis of Gsn-/- NMCM with gelsolin decrease I(Ca). We conclude that cardiac L-type calcium channel regulation is tightly controlled by actin filament organization. Actin filament rearrangement mediated by gelsolin may contribute to calcium channel inactivation.

Asunto(s)

Actinas/metabolismo , Canales de Calcio Tipo L/metabolismo , Gelsolina/genética , Miocardio/metabolismo , Actinas/análisis , Animales , Animales Recién Nacidos , Calcio/metabolismo , Células Cultivadas , Citocalasina D/farmacología , Gelsolina/metabolismo , Expresión Génica/fisiología , Cinética , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Miocardio/química , Miocardio/citología , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Técnicas de Placa-Clamp , Faloidina/farmacología

RESUMEN

The alpha subunit of the stimulatory heterotrimeric G protein (Gsalpha) is critical for the beta-adrenergic receptor activation of the cAMP messenger system. The role of Gsalpha in regulating cardiac Ca2+ channel activity, however, remains controversial. Cultured neonatal cardiac myocytes from transgenic mice overexpressing cardiac Gsalpha were used to assess the role of Gsalpha on the whole-cell Ca2+ currents (ICa). Cardiac myocytes from transgenic mice had a 490% higher peak ICa compared with those of either wild-type controls or Gsalpha-nonexpressing littermates. The effect of Gsalpha overexpression was mimicked by intracellular dialysis of wild-type cardiac myocytes with GTPgammaS-activated Gsalpha. This effect was not mediated by protein kinase A activation as intracellular perfusion with a protein kinase A inhibitor rendered the same degree of activation in either transgenic or wild-type myocytes also dialyzed with activated Gsalpha. The data indicate that Gsalpha overexpression is associated with a constitutive enhancement of ICa which is independent of the cAMP pathway and activation of endogenous adenylyl cyclase.

Asunto(s)

Adenilil Ciclasas/metabolismo , Canales de Calcio/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Miocardio/metabolismo , Animales , Animales Recién Nacidos , Canales de Calcio Tipo L , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Femenino , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Cinética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocardio/citología , Embarazo , Transducción de Señal

RESUMEN

Expression of the cystic fibrosis transmembrane conductance regulator (CFTR), and of at least one other member of the ATP-binding cassette family of transport proteins, P-glycoprotein, is associated with the electrodiffusional movement of the nucleotide ATP. Evidence directly implicating CFTR expression with ATP channel activity, however, is still missing. Here it is reported that reconstitution into a lipid bilayer of highly purified CFTR of human epithelial origin enables the permeation of both Cl- and ATP. Similar to previously reported data for in vivo ATP current of CFTR-expressing cells, the reconstituted channels displayed competition between Cl- and ATP and had multiple conductance states in the presence of Cl- and ATP. Purified CFTR-mediated ATP currents were activated by protein kinase A and ATP (1 mM) from the "intracellular" side of the molecule and were inhibited by diphenylamine-2-carboxylate, glibenclamide, and anti-CFTR antibodies. The absence of CFTR-mediated electrodiffusional ATP movement may thus be a relevant component of the pleiotropic cystic fibrosis phenotype.

Asunto(s)

Adenosina Trifosfato/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Animales , Transporte Biológico , Bloqueadores de los Canales de Calcio/metabolismo , Línea Celular , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Difusión , Conductividad Eléctrica , Humanos , Membrana Dobles de Lípidos/metabolismo , Magnesio/metabolismo , Modelos Moleculares , Proteínas Recombinantes/metabolismo , Spodoptera , ortoaminobenzoatos/metabolismo

RESUMEN

The actin cytoskeleton is an important contributor to the integrity of cellular shape and responses in neurons. However, the molecular mechanisms associated with functional interactions between the actin cytoskeleton and neuronal ion channels are largely unknown. Whole-cell and single channel recording techniques were thus applied to identified retinal bipolar neurons of the tiger salamander (Ambystoma tigrinum) to assess the role of acute changes in actin-based cytoskeleton dynamics in the regulation of voltage-gated ion channels. Disruption of endogenous actin filaments after brief treatment (20-30 min) with cytochalasin D (CD) activated voltage-gated K+ currents in bipolar cells, which were largely prevented by intracellular perfusion with the actin filament-stabilizer agent, phalloidin. Either CD treatment under cell-attached conditions or direct addition of actin to excised, inside-out patches of bipolar cells activated and/or increased single K+ channels. Thus, acute changes in actin-based cytoskeleton dynamics regulate voltage-gated ion channel activity in bipolar cells.

Asunto(s)

Actinas/fisiología , Citoesqueleto/fisiología , Canales Iónicos/fisiología , Células Fotorreceptoras/fisiología , Células Ganglionares de la Retina/fisiología , Ambystoma , Animales , Citocalasina D/farmacología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/ultraestructura , Homeostasis , Técnicas In Vitro , Potenciales de la Membrana , Técnicas de Placa-Clamp , Faloidina/farmacología , Células Fotorreceptoras/citología , Células Fotorreceptoras/efectos de los fármacos , Canales de Potasio/efectos de los fármacos , Canales de Potasio/fisiología , Células Ganglionares de la Retina/ultraestructura , Factores de Tiempo

Asunto(s)

Actinas/metabolismo , Actinas/ultraestructura , Corteza Renal/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Cinética , Fosforilación , Ratas