RESUMEN
Cystic fibrosis (CF) is one of the most common inherited diseases in the white population. The disease results from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR). How this gene defect leads to the clinical manifestations of the disease, however, is not entirely clear. CFTR functions as a Cl(-) channel in the apical membrane of most secretory epithelia, including biliary epithelial cells, or cholangiocytes. In cholangiocytes, CFTR appears to be an important determinant of biliary secretion and bile flow. Additionally, recent evidence suggests that CFTR regulates other membrane transporters, channels, and proteins. Improving life expectancy has led to an increasing recognition of hepatobiliary complications from CF. The true prevalence of CF liver disease is unknown, but may affect up to 17-25% of CF patients. Clinical manifestations include hepatic steatosis, neonatal cholestasis, focal nodular cirrhosis, multilobular cirrhosis, and biliary tract complications. Why only a subset of CF patients develops severe liver disease and others with the same genotype do not is one of the many scientific curiosities of this disease. This review focuses on the function of CFTR in cholangiocytes with emphasis on ductular bile formation as well as the clinical consequences of abnormal CFTR, namely CF-associated liver disease. Data on the pathogenesis, prevalence, clinical course, and treatment of CF liver disease will be reviewed.
Asunto(s)
Conductos Biliares/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Fibrosis Quística/complicaciones , Hepatopatías/fisiopatología , Niño , Preescolar , Humanos , Lactante , Hepatopatías/etiología , Hepatopatías/terapiaRESUMEN
The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1 (MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular ATP, increases in cell volume increased ATP release approximately 3-fold. The MDR1 inhibitors cyclosporine A (10 microm) and verapramil (10 microm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased ATP-dependent volume-sensitive Cl- current density from -33.1 +/- 12.5 pA/pF to -2.0 +/- 0.3 pA/pF (-80 mV, p < or = 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator of other cellular ATP transport pathways.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Permeabilidad de la Membrana Celular/fisiología , Cloruros/metabolismo , Células 3T3/citología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/inmunología , Adenosina Trifosfato/antagonistas & inhibidores , Animales , Anticuerpos/inmunología , Anticuerpos/farmacología , Carcinoma Hepatocelular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Células Cultivadas/citología , Ciclosporina/farmacología , Humanos , Ratones , Ratas , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismo , Verapamilo/farmacología , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
In hepatocytes, Na+ influx through nonselective cation (NSC) channels represents a key point for regulation of cell volume. Under basal conditions, channels are closed, but both physiologic and pathologic stimuli lead to a large increase in Na+ and water influx. Since osmotic stimuli also activate mitogen-activated protein (MAP) kinase pathways, we have examined regulation of Na+ permeability and cell volume by MAP kinases in an HTC liver cell model. Under isotonic conditions, there was constitutive activity of p38 MAP kinase that was selectively inhibited by SB203580. Decreases in cell volume caused by hypertonic exposure had no effect on p38, but increases in cell volume caused by hypotonic exposure increased p38 activity tenfold. Na+ currents were small when cells were in isotonic media but could be increased by inhibiting constitutive p38 MAP kinase, thereby increasing cell volume. To evaluate the potential inhibitory role of p38 more directly, cells were dialyzed with recombinant p38alpha and its upstream activator, MEK-6, which substantially inhibited volume-sensitive currents. These findings indicate that constitutive p38 activity contributes to the low Na+ permeability necessary for maintenance of cell volume, and that recombinant p38 negatively modulates the set point for volume-sensitive channel opening. Thus, functional interactions between p38 MAP kinase and ion channels may represent an important target for modifying volume-sensitive liver functions.
Asunto(s)
Hígado/citología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Sodio/metabolismo , Animales , Permeabilidad de la Membrana Celular , Tamaño de la Célula , Inhibidores Enzimáticos/farmacología , Imidazoles/farmacología , Transporte Iónico , Hígado/enzimología , Hígado/metabolismo , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Concentración Osmolar , Piridinas/farmacología , Ratas , Células Tumorales Cultivadas , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Canales de Cloruro/metabolismo , Hepatocitos/metabolismo , Homeostasis/fisiología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Anticuerpos/administración & dosificación , Canales de Cloruro CLC-2 , Carcinoma Hepatocelular/patología , Línea Celular , Membrana Celular , Tamaño de la Célula/efectos de los fármacos , Tamaño de la Célula/fisiología , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/genética , Cloruros/metabolismo , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Microinyecciones , Técnicas de Placa-Clamp , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) and NHE3 Kinase A regulatory protein (E3KARP) are membrane-cytoskeleton linking proteins that utilize 2 PSD-95/DIg/ZO-1 (PDZ) domains and an ERM binding site to coordinate cyclic adenosine monophosphate (cAMP)-regulated ion transport in a number of distinct epithelia. ERM family members serve to anchor EBP50 and E3KARP to the actin cytoskeleton and sequester protein kinase A (PKA) to these protein complexes. In hepatocytes and cholangiocytes, the epithelial cells of the bile secretory unit, cAMP-activated PKA stimulates secretion and bile formation, but the molecular mechanisms, including the potential contribution of EBP50 and E3KARP, remain undetermined. The present studies evaluated the comparative expression and localization of EBP50 and E3KARP in rat hepatocytes and cholangiocytes. Complementary DNAs encoding rat EBP50 and E3KARP were identified by reverse transcription-polymerase chain reaction in both epithelial cell types and subsequently sequenced. Northern and Western analysis showed the presence of EBP50 messenger RNA and protein in both hepatocytes and cholangiocytes. Confocal immunofluorescence revealed EBP50 was concentrated at the apical domain of both cell types. E3KARP was also expressed in cholangiocytes but had a distinct cytoplasmic/nuclear distribution. In dominant-negative transfection studies, patch clamp analysis of Mz-ChA1 cholangiocarcinoma cells showed that expression of the PDZ1 domain of EBP50 selectively decreased the endogenous cAMP-mediated Cl secretory response. The apical expression of EBP50, presence of specific ERM proteins, and functional effects of PDZ1 expression on cholangiocyte secretion suggest EBP50 is positioned to contribute to the organization and regulation of bile secretory proteins in both hepatocytes and cholangiocytes.
Asunto(s)
Proteínas Portadoras/metabolismo , Hígado/metabolismo , Fosfoproteínas/metabolismo , Intercambiadores de Sodio-Hidrógeno , Secuencia de Aminoácidos/genética , Animales , Secuencia de Bases/genética , Conductos Biliares/química , Conductos Biliares/metabolismo , Proteínas Portadoras/genética , Membrana Celular/metabolismo , Cloruros/antagonistas & inhibidores , Cloruros/metabolismo , AMP Cíclico/fisiología , Proteínas del Citoesqueleto/metabolismo , ADN Complementario/genética , Células Epiteliales/metabolismo , Expresión Génica , Hepatocitos/metabolismo , Hígado/citología , Masculino , Datos de Secuencia Molecular , Fosfoproteínas/genética , Estructura Terciaria de Proteína/genética , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
BACKGROUND/AIMS: Purinergic signaling potentially contributes to many liver functions. Therefore, the purpose of these studies was to characterize adenosine 5'-triphosphate (ATP) release from human hepatocytes, and to determine the role of extracellular ATP in the autocrine regulation of Cl- permeability and cell volume homeostasis. METHODS: Release of ATP (luciferase-luciferin assay), Cl- currents (whole-cell patch clamp), and cell volume (Coulter Multisizer) were measured in human hepatocytes within 12 h of isolation. RESULTS: Hepatocyte swelling increased bioluminescence from basal values of 11.21+/-0.45 to 178.29+/-44.49 and 492.15+/-89.41 arbitrary light units following 20 and 40% buffer dilutions, respectively (p<0.001), representing an increase in extracellular ATP from approximately 10 to >300 nM. Whole-cell Cl- currents activated during exposure to hypotonic buffer (15% less mosmol, 126.34+/-36.49 pA/pF) and ATP (10 microM, 71.92+/-15.48 pA/pF) exhibited outward rectification, time-dependent inactivation at depolarizing potentials, and sensitivity to the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). Removal of extracellular ATP (apyrase) prevented volume-sensitive current activation. Exposure to hypotonic buffer (30% less mosmol) increased mean relative volume to 1.092+/-0.004 by 2.5 min, and volume recovery (1.019+/-0.002 by 30 min) was abolished by NPPB, apyrase, and the P2 receptor antagonist suramin. CONCLUSIONS: These findings indicate that human hepatocytes exhibit constitutive and volume-dependent ATP release, which is a critical determinant of membrane Cl- permeability and cell volume regulation. ATP release may represent an extracellular signaling pathway that couples the cellular hydration state to important hepatic functions.
Asunto(s)
Hígado/fisiología , Receptores Purinérgicos P2/fisiología , Transducción de Señal , Adenosina Trifosfato/fisiología , Calcio/fisiología , Tamaño de la Célula/fisiología , Células Cultivadas , Humanos , Transporte Iónico , Hígado/citología , Técnicas de Placa-ClampRESUMEN
BACKGROUND & AIMS: Oxidative stress leads to a rapid initial loss of liver cell volume, but the adaptive mechanisms that serve to restore volume have not been defined. This study aimed to evaluate the functional interactions between oxidative stress, cell volume recovery, and membrane ion permeability. METHODS: In HTC rat hepatoma cells, oxidative stress was produced by exposure to H(2)O(2) or D-alanine plus D-amino acid oxidase (40 U/mL). RESULTS: Oxidative stress resulted in a rapid decrease in relative cell volume to 0.85 +/- 0.06. This was followed by an approximately 100-fold increase in membrane cation permeability and partial volume recovery to 0.97 +/- 0.05 of original values. The volume-sensitive conductance was permeable to Na(+) approximately K(+) >> Tris(+), and whole-cell current density at -80 mV increased from -1.2 pA/pF at 10(-5) mol/L H(2)O(2) to -95.1 pA/pF at 10(-2) mol/L H(2)O(2). The effects of H(2)O(2) were completely inhibited by dialysis of the cell interior with reduced glutathione, and were markedly enhanced by inhibition of glutathione synthase. CONCLUSIONS: These findings support the presence of dynamic functional interactions between cell volume, oxidative stress, and membrane Na(+) permeability. Stress-induced Na(+) influx may represent a beneficial adaptive response that partially restores cell volume over short periods, but sustained cation influx could contribute to the increase in intracellular [Na(+)] and [Ca(2+)] associated with cell injury and necrosis.
Asunto(s)
Permeabilidad de la Membrana Celular/fisiología , Neoplasias Hepáticas Experimentales/fisiopatología , Estrés Oxidativo , Sodio/metabolismo , Alanina/farmacología , Animales , Calcio/metabolismo , Catalasa/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula , Citosol/metabolismo , D-Aminoácido Oxidasa/metabolismo , D-Aminoácido Oxidasa/farmacología , Glutatión/farmacología , Peróxido de Hidrógeno/farmacología , Soluciones Hipertónicas , Cinética , Neoplasias Hepáticas Experimentales/patología , Estrés Oxidativo/efectos de los fármacos , Técnicas de Placa-Clamp , Ratas , Especies Reactivas de Oxígeno/metabolismo , Células Tumorales CultivadasRESUMEN
Extracellular ATP functions as an important autocrine and paracrine signal that modulates a broad range of cell and organ functions through activation of purinergic receptors in the plasma membrane. Because little is known of the cellular mechanisms involved in ATP release, the purpose of these studies was to evaluate the potential role of the lanthanide Gd(3+) as an inhibitor of ATP permeability and to assess the physiological implications of impaired purinergic signaling in liver cells. In rat hepatocytes and HTC hepatoma cells, increases in cell volume stimulate ATP release, and the localized increase in extracellular ATP increases membrane Cl(-) permeability and stimulates cell volume recovery through activation of P(2) receptors. In cells in culture, spontaneous ATP release, as measured by a luciferin-luciferase-based assay, was always detectable under control conditions, and extracellular ATP concentrations increased 2- to 14-fold after increases in cell volume. Gd(3+) (200 microM) inhibited volume-sensitive ATP release by >90% (P < 0.001), inhibited cell volume recovery from swelling (P < 0.01), and uncoupled cell volume from increases in membrane Cl(-) permeability (P < 0.01). Moreover, Gd(3+) had similar inhibitory effects on ATP release from other liver and epithelial cell models. Together, these findings support an important physiological role for constitutive release of ATP as a signal coordinating cell volume and membrane ion permeability and suggest that Gd(3+) might prove to be an effective inhibitor of ATP-permeable channels once they are identified.
Asunto(s)
Adenosina Trifosfato/metabolismo , Antiinflamatorios/farmacología , Gadolinio/farmacología , Receptores Purinérgicos/fisiología , Transducción de Señal/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/farmacología , Comunicación Autocrina/fisiología , Canales de Calcio/fisiología , Carcinoma Hepatocelular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Canales de Cloruro/fisiología , Células Epiteliales/química , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Ácido Flufenámico/farmacología , Soluciones Hipotónicas/farmacología , Soluciones Isotónicas/farmacología , Neoplasias Hepáticas , Comunicación Paracrina/fisiología , Ratas , Células Tumorales Cultivadas/química , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/metabolismo , Equilibrio Hidroelectrolítico/fisiologíaRESUMEN
OBJECTIVE: To prospectively evaluate the biochemical status of vitamins A, D, and E in children with cystic fibrosis (CF). SUBJECTS: A total of 127 infants identified by the Colorado CF newborn screening program. DESIGN: Vitamin status (serum retinol, 25-hydroxy vitamin D, ratio of alpha-tocopherol/total lipids) and serum albumin were assessed at diagnosis (4 to 8 weeks), ages 6 months, 12 months, and yearly thereafter, to age 10 years. RESULTS: Deficiency of 1 or more vitamins was present in 44 (45.8%) of 96 patients at age 4 to 8 weeks as follows: vitamin A 29.0%, vitamin D 22.5%, and vitamin E 22.8%. Of these patients with initial deficiency, the percent that was deficient at 1 or more subsequent time points, despite supplementation, was vitamin A 11.1%, vitamin D 12.5%, and vitamin E 57.1%. Of the initial patients with vitamin sufficiency, the percent who became deficient at any time during the 10-year period was as follows: vitamin A 4.5%, vitamin D 14.4%, and vitamin E 11.8%. The percent of patients deficient for 1 or more vitamins ranged from 4% to 45% for any given year. CONCLUSIONS: Despite supplementation with standard multivitamins and pancreatic enzymes, the sporadic occurrence of fat-soluble vitamin deficiency and persistent deficiency is relatively common. Frequent and serial monitoring of the serum concentrations of these vitamins is therefore essential in children with CF.
Asunto(s)
Fibrosis Quística/metabolismo , Deficiencia de Vitamina A/epidemiología , Deficiencia de Vitamina D/epidemiología , Deficiencia de Vitamina E/epidemiología , Niño , Preescolar , Fibrosis Quística/diagnóstico , Fibrosis Quística/tratamiento farmacológico , Estudios de Seguimiento , Fármacos Gastrointestinales/uso terapéutico , Humanos , Lactante , Recién Nacido , Tamizaje Neonatal , Pancrelipasa/uso terapéutico , Estudios Prospectivos , Factores de Tiempo , Vitamina A/sangre , Deficiencia de Vitamina A/diagnóstico , Vitamina D/sangre , Deficiencia de Vitamina D/diagnóstico , Vitamina E/sangre , Deficiencia de Vitamina E/diagnóstico , Vitaminas/uso terapéuticoRESUMEN
ATP stimulates Cl(-) secretion and bile formation by activation of purinergic receptors in the apical membrane of cholangiocytes. The purpose of these studies was to determine the cellular origin of biliary ATP and to assess the regulatory pathways involved in its release. In Mz-Cha-1 human cholangiocarcinoma cells, increases in cell volume were followed by increases in phophoinositide (PI) 3-kinase activity, ATP release, and membrane Cl(-) permeability. PI 3-kinase signaling appears to play a regulatory role because ATP release was inhibited by wortmannin or LY294002 and because volume-sensitive current activation was inhibited by intracellular dialysis with antibodies to the 110 kDa-subunit of PI 3-kinase. Similarly, in intact normal rat cholangiocyte monolayers, increases in cell volume stimulated luminal Cl(-) secretion through a wortmannin-sensitive pathway. To assess the role of PI 3-kinase more directly, cells were dialyzed with the synthetic lipid products of PI 3-kinase. Intracellular delivery of phosphatidylinositol 3, 4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate activated Cl(-) currents analogous to those observed following cell swelling. Taken together, these findings indicate that volume-sensitive activation of PI 3-kinase and the generation of lipid messengers modulate cholangiocyte ATP release, Cl(-) secretion, and, hence, bile formation.
Asunto(s)
Adenosina Trifosfato/metabolismo , Conductos Biliares/fisiología , Permeabilidad de la Membrana Celular , Cloruros/metabolismo , Células Epiteliales/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Androstadienos/farmacología , Animales , Neoplasias de los Conductos Biliares , Conductos Biliares/citología , Conductos Biliares Intrahepáticos , Transporte Biológico , Células Cultivadas , Colangiocarcinoma , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/citología , Homeostasis , Humanos , Soluciones Hipotónicas , Cinética , Potenciales de la Membrana/efectos de los fármacos , Morfolinas/farmacología , Ratas , Transducción de Señal , Células Tumorales Cultivadas , WortmaninaRESUMEN
P2Y receptor stimulation increases membrane Cl- permeability in biliary epithelial cells, but the source of extracellular nucleotides and physiological relevance of purinergic signaling to biliary secretion are unknown. Our objectives were to determine whether biliary cells release ATP under physiological conditions and whether extracellular ATP contributes to cell volume regulation and transepithelial secretion. With the use of a sensitive bioluminescence assay, constitutive ATP release was detected from human Mz-ChA-1 cholangiocarcinoma cells and polarized normal rat cholangiocyte monolayers. ATP release increased rapidly during cell swelling induced by hypotonic exposure. In Mz-ChA-1 cells, removal of extracellular ATP (apyrase) and P2 receptor blockade (suramin) reversibly inhibited whole cell Cl- current activation and prevented cell volume recovery during hypotonic stress. Moreover, exposure to apyrase induced cell swelling under isotonic conditions. In intact normal rat cholangiocyte monolayers, hypotonic perfusion activated apical Cl- currents, which were inhibited by addition of apyrase and suramin to bathing media. These findings indicate that modulation of ATP release by the cellular hydration state represents a potential signal coordinating cell volume with membrane Cl- permeability and transepithelial Cl- secretion.
Asunto(s)
Adenosina Trifosfato/metabolismo , Conductos Biliares/metabolismo , Cloruros/metabolismo , Animales , Comunicación Autocrina/fisiología , Conductos Biliares/citología , Línea Celular , Permeabilidad de la Membrana Celular/fisiología , Células Cultivadas , Canales de Cloruro/metabolismo , Células Epiteliales/citología , Células Epiteliales/metabolismo , Espacio Extracelular/metabolismo , Homeostasis/fisiología , Humanos , Canales Iónicos/metabolismo , Ratas , Receptores Purinérgicos P2/fisiologíaAsunto(s)
Morfogénesis/genética , Transactivadores , Vertebrados/anatomía & histología , Vertebrados/embriología , Animales , Anomalías Congénitas/genética , Expresión Génica , Genes , Glicoproteínas/genética , Proteínas Hedgehog , Humanos , Proteínas/genética , Factor de Crecimiento Transformador betaRESUMEN
Acute hepatic failure caused by primary Epstein-Barr virus (EBV) infection has been reported in the literature in 16 cases, with an overall mortality of 87%. We report a case of fulminant hepatic failure in an immunocompetent young girl caused by primary EBV infection that was treated by orthotopic liver transplantation. After transplantation she has been treated with low-dose immunosuppression, a pooled gammaglobulin preparation containing anti-EBV antibodies, and anti-viral therapy. The patient is presently doing well 2 years after transplantation without evidence of clinical EBV infection, primary immunodeficiency, or lymphoproliferative disease.
Asunto(s)
Infecciones por Virus de Epstein-Barr/complicaciones , Encefalopatía Hepática/etiología , Encefalopatía Hepática/cirugía , Hepatitis Viral Humana/virología , Herpesvirus Humano 4 , Trasplante de Hígado , Antivirales/uso terapéutico , Femenino , Humanos , Inmunosupresores/uso terapéutico , Lactante , Reacción en Cadena de la Polimerasa , gammaglobulinas/uso terapéuticoRESUMEN
Regulated changes in cell volume represent a signal that modulates a broad range of cell and organ functions. In HTC hepatoma cells, increases in volume are coupled to membrane ion permeability through a pathway involving (i) ATP efflux, (ii) autocrine stimulation of P2 receptors, and (iii) increases in anion permeability and Cl- efflux, contributing to recovery of volume toward basal values. Based on recent evidence that cell volume increases also stimulate phosphoinositide kinases, the purpose of these studies was to determine if phosphatidylinositol 3-kinase (PI 3-kinase) modulates these pathways. Exposure of cells to hypotonic buffer (20 or 40% less NaCl) caused an initial increase in cell volume and stimulated a rapid increase in ATP release. Subsequent opening of Cl- channels was followed by recovery of cell volume toward basal values, despite the continuous presence of hypotonic buffer. Inhibition of PI 3-kinase with wortmannin (Ki = 3 nM) significantly inhibited both the rate of volume recovery and activation of Cl- currents; similar results were obtained with LY294002 (10 microM). Additionally, current activation was inhibited by intracellular dialysis with antibodies specific for the 110-kDa catalytic subunit of PI 3-kinase. Since release of ATP is a critical element in the volume-regulatory pathway, the role of PI 3-kinase on volume-stimulated ATP release was assessed. Both wortmannin and LY294002 decreased basal and volume-stimulated ATP permeability but had no effect on the current response to exogenous ATP (10 microM). These findings indicate that PI 3-kinase plays a significant role in regulation of cell volume and suggest that the effects are mediated in part through modulation of cellular ATP release.
Asunto(s)
Adenosina Trifosfato/metabolismo , Tamaño de la Célula/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Androstadienos/farmacología , Animales , Anticuerpos/farmacología , Carcinoma Hepatocelular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Cloruros/farmacocinética , Cromonas/farmacología , Electrofisiología , Inhibidores Enzimáticos/farmacología , Soluciones Hipotónicas/farmacología , Canales Iónicos/fisiología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas , Células Tumorales Cultivadas , WortmaninaRESUMEN
In a model liver cell line, recovery from swelling is mediated by a sensitive autocrine pathway involving conductive release of ATP, P2 receptor stimulation, and opening of membrane Cl- channels (Wang, Y., Roman, R. M., Lidofsky, S. D., and Fitz, J. G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12020-12025). However, the mechanisms coupling changes in cell volume to ATP release are not known. Based on evidence that certain ATP-binding cassette (ABC) proteins may function as ATP channels or channel regulators, we evaluated the potential role of ABC proteins by comparing ATP release and volume regulation in rat HTC and HTC-R hepatoma cells, the latter of which overexpress Mdr proteins. In both cell types, Cl- current activation (ICl-swell) and volume recovery following swelling were dependent on conductive ATP efflux. The rate of volume recovery was approximately 6-fold faster in HTC-R cells compared with HTC cells. This effect is likely due to enhanced ABC protein-dependent ATP release since (i) ICl-swell and cell volume recovery were eliminated by inhibition of P-glycoprotein transport (20 microM verapamil and 15 microM cyclosporin A); (ii) swelling-induced Cl- current density was similar in both cell types (approximately -50 pA/pF; not significant); and (iii) ATP conductance measured by whole-cell techniques was increased approximately 3-fold in HTC-R cells compared with HTC cells. Moreover, HTC-R cells exhibited enhanced survival during hypotonic stress. By modulating ATP release, hepatic ABC proteins may play a key role in the cellular pathways coupling changes in cell volume to ion permeability and secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Hígado/citología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Transporte Biológico , Tamaño de la Célula/fisiología , Células Cultivadas , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Neoplasias Hepáticas Experimentales/patología , RatasRESUMEN
To identify behaviors associated with the onset of gastroesophageal reflux episodes in infants both systematically and prospectively, each of 10 patients (aged 2 to 32 weeks) was studied during 2 hours of intraluminal esophageal pH probe monitoring, using a split-screen audiovisual recording technique. Videotape analysis of eight infants who had scoreable reflux events revealed six discrete behaviors closely associated temporally (P < .001 to < .05) with the onset of reflux events: "discomfort" (crying or frowning), "emission" (of liquid or gas, i.e., regurgitation, drooling, or burping), yawning, stridor, stretching, and mouthing. Three behaviors (hiccuping, sneezing, and thumb-sucking) were infrequent but were significantly associated with onset of reflux events in one or two patients each. A tenth behavior, coughing or gagging, was significantly associated with onset of reflux events in two patients, but not in the rest, despite relatively frequent occurrence. Exploration of temporal relations between reflux and each behavior suggested that discomfort, emission, mouthing, and cough-gag may have caused reflux episodes, and that all 10 of the behaviors may have been caused by reflux episodes. These findings and a "quiet period" immediately preceding episodes in six of the infants suggest interesting pathophysiologic mechanisms in infants which require further evaluation.