RESUMEN
In normal airway epithelium, the cystic fibrosis transmembrane conductance regulator (CFTR) transports Cl(-) ions to the apical surface of the epithelium paralleled by the flow of water through transcellular and paracellular pathways. The hypothesis was tested whether CFTR not only regulates the transcellular but also the paracellular shunt pathway. Therefore, we performed measurements of transepithelial electrical resistance (TER) and paracellular (14)C-mannitol permeability in wtCFTR (16HBE14o(-)) and delF508-CFTR (CFBE41o(-)) expressing human bronchial epithelial cells. Under resting conditions, CFBE41o(-) cell monolayers exhibit a higher paracellular permeability and lower TER as compared to 16HBE14o(-) monolayers. Stimulation of CFTR by cAMP induces opposite effects in the two cell lines. 16HBE14o(-) monolayers show a sharp decrease of TER, in parallel with a concomitant increase of paracellular permeability. The change in paracellular permeability is mediated by a myosin II dependent mechanism because it can be blocked by the myosin light chain kinase inhibitor ML-7. In contrast, CFBE41o(-) cells respond to cAMP stimulation with a decrease of paracellular permeability, paralleled by slight increase of TER. We conclude that stimulation of wtCFTR increases vectorial transcellular salt transport and, simultaneously, the paracellular permeability allowing water to follow through the paracellular pathway. In contrast, in CF epithelium cAMP stimulation increases neither vectorial salt transport nor paracellular permeability which is likely to contribute to the CF pulmonary phenotype. Taken together, our results link CFTR dysfunction to an improper regulation of the paracellular transport route.
Asunto(s)
Bronquios/citología , Permeabilidad de la Membrana Celular/fisiología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/metabolismo , Azepinas/farmacología , Línea Celular , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Células Epiteliales/citología , Células Epiteliales/fisiología , Humanos , Manitol/metabolismo , Mutación , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Quinasa de Cadena Ligera de Miosina/metabolismo , Naftalenos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Uniones Estrechas/fisiologíaRESUMEN
It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o- cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o- cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o- and also in CFBE41o- cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o- cells and CFBE41o- cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o- cell monolayers. We observed that 16HBE14o- cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o- and its overexpressing clones. Consequently, 16HBE14o- cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in 'healthy' 16HBE14o- cells compared to 'cystic fibrosis' CFBE41o- cells. We found that claudin-3 expression was considerably stronger in 16HBE14o- cells than in the three CFBE41o- cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o- cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.