RESUMEN
Bacteria affect the respiratory epithelium, which is covered by airway surface liquid (ASL) and mucus. Ion concentrations in the ASL are determined by the cystic fibrosis transmembrane conductance regulator (CFTR) and the epithelial Na(+) channel (ENaC). Neonatal sepsis is a major risk factor for subsequent pulmonary disease in preterm newborns. Predominating are coagulase-negative staphylococci (e.g., Staphylococccus epidermidis and Staphylococccus aureus). The aim of this study was to investigate modulation of CFTR, ENaC, mucins, proinflammatory cytokines, and inducible nitric oxide synthase (iNOS) in respiratory epithelial cells after S. epidermidis 94B080 and S. aureus 90B083 exposure. Bronchial epithelial cells were incubated with S. epidermidis 94B080 and S. aureus 90B083 (neonatal blood isolates) for 1-36 h. Expression of CFTR, ENaC, iNOS, and mucins was analyzed by real-time PCR and Western blotting. Release of cytokines was analyzed by ELISA, and production of NO by the Griess assay. Expression of CFTR significantly decreased after 36 h incubation with S. epidermidis and more prominently with S. aureus, whereas S. epidermidis caused a significant increase in the expression of ß- and γ-ENaC. Expression of iNOS increased, but NO was not detected. Both staphylococci caused a decrease in the intracellular Ca(2+) concentration. S. aureus induced increased secretion of IL-6, IL-8, and transforming nuclear factor (TNF)-α in a time-dependent manner as compared with S. epidermidis. In conclusion, expression of ENaC, CFTR, and iNOS is modulated by exposure to S. aureus 90B083 and S. epidermidis 94B080. S. aureus is more potent in causing release of IL-6, IL-8, and TNF-α by bronchial epithelial cells as compared with S. epidermidis. The mRNA expression for the mucus proteins MUC2, MUC5AC, and MUC5B could not be measured, neither in the presence nor in the absence of bacteria.
Asunto(s)
Bronquios/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/inmunología , Canales Epiteliales de Sodio/genética , Óxido Nítrico Sintasa de Tipo II/genética , Staphylococcus aureus/fisiología , Staphylococcus epidermidis/fisiología , Bronquios/microbiología , Bronquios/patología , Calcio/metabolismo , Línea Celular , Supervivencia Celular , Regulador de Conductancia de Transmembrana de Fibrosis Quística/inmunología , Citocinas/biosíntesis , Citocinas/inmunología , Células Epiteliales/microbiología , Células Epiteliales/patología , Canales Epiteliales de Sodio/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Recién Nacido , Mucinas/genética , Mucinas/inmunología , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/inmunología , Staphylococcus aureus/aislamiento & purificación , Staphylococcus epidermidis/aislamiento & purificaciónRESUMEN
Ambroxol, a mucokinetic anti-inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl(-) efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na(+) influx via the epithelial Na(+) channel (ENaC). In cells expressing wt-CFTR, ambroxol increased the Cl(-) conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real-time polymerase chain reaction (RT-PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl(-) transport was measured by Cl(-) efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl(-) efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α-ENaC, and of the CFTR protein. No significant difference was observed in ß-ENaC after exposure to ambroxol, whereas mRNA expression of γ-ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca(2+) concentration. Upregulation of CFTR and enhanced Cl(-) efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF-patients.
Asunto(s)
Ambroxol/farmacología , Bronquios/patología , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Western Blotting , Calcio/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio/genética , Colorantes Fluorescentes/metabolismo , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Since previous studies showed that the endogenous bronchodilator, S-nitrosglutathione (GSNO), caused a marked increase in CFTR-mediated chloride (Cl(-)) efflux and improved the trafficking of CFTR to the plasma membrane, and that also the nitric oxide (NO)-donor GEA3162 had a similar, but smaller, effect on Cl(-) efflux, it was investigated whether the NO-donor properties of GSNO were relevant for its effect on Cl(-) efflux from airway epithelial cells. Hence, the effect of a number of other NO-donors, sodium nitroprusside (SNP), S-nitroso-N-acetyl-DL-penicillamine (SNAP), diethylenetriamine/nitric oxide adduct (DETA-NO), and diethylenetriamine/nitric oxide adduct (DEA-NONOate) on Cl(-) efflux from CFBE (∆F508/∆F508-CFTR) airway epithelial cells was tested. Cl(-) efflux was determined using the fluorescent N-(ethoxycarbonylmethyl)-6-methoxyquinoliniu bromide (MQAE)-technique. Possible changes in the intracellular Ca(2+) concentration were tested by the fluorescent fluo-4 method in a confocal microscope system. Like previously with GSNO, after 4 h incubation with the NO-donor, an increased Cl(-) efflux was found (in the order SNAP>DETA-NO>SNP). The effect of DEA-NONOate on Cl(-) efflux was not significant, and the compound may have (unspecific) deleterious effects on the cells. Again, as with GSNO, after a short (5 min) incubation, SNP had no significant effect on Cl(-) efflux. None of the NO-donors that had a significant effect on Cl(-) efflux caused significant changes in the intracellular Ca(2+) concentration. After 4 h preincubation, SNP caused a significant increase in the mRNA expression of CFTR. SNAP and DEA-NONOate decreased the mRNA expression of all ENaC subunits significantly. DETA-NO caused a significant decrease only in α-ENaC expression. After a short preincubation, none of the NO-donors had a significant effect, neither on the expression of CFTR, nor on that of the ENaC subunits in the presence and absence of L-cysteine. It can be concluded that the effect of GSNO on Cl(-) efflux is, at least in part, due to its properties as an NO-donor, and the effect is likely to be mediated by CFTR, not by Ca(2+)-activated Cl(-) channels.
Asunto(s)
Calcio/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Células Epiteliales/metabolismo , Canales Epiteliales de Sodio/genética , Donantes de Óxido Nítrico/farmacología , Bronquios/patología , Línea Celular , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Células Epiteliales/patología , Canales Epiteliales de Sodio/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/metabolismo , Triazoles/farmacologíaRESUMEN
The lantibiotic duramycin (Moli1901, Lancovutide) has been suggested as a drug of choice in the treatment for cystic fibrosis (CF). It has been proposed that duramycin may stimulate chloride secretion through Ca²(+) -activated Clâ» channels (CaCC). We investigated whether duramycin exhibited any effect on Clâ» efflux and intracellular Ca²(+) concentration ([Ca²(+)](i)) in CF and non-CF epithelial cells. Duramycin did stimulate Clâ» efflux from CF bronchial epithelial cells (CFBE) in a narrow concentration range (around 1 µM). However, 100 and 250 µM of duramycin inhibited Clâ» efflux from CFBE cells. An inhibitor of the CF transmembrane conductance regulator (CFTR(inh)â172) and a blocker of the capacitative Ca²(+) entry, gadolinium chloride, inhibited the duramycin-induced Clâ» efflux. No effect on Clâ» efflux was observed in non-CF human bronchial epithelial cells (16HBE), human airway submucosal gland cell line, human pancreatic epithelial cells, CF airway submucosal gland epithelial cells, and CF pancreatic cells. The [Ca²(+)](i) was increased by 3 µM duramycin in 16HBE cells, but decreased after 1, and 3 µM of duramycin in CFBE cells. The results suggest that the mechanism responsible for the stimulation of Clâ» efflux by duramycin is mainly related to unspecific changes of the cell membrane or its components rather than to effects on CaCC.
Asunto(s)
Bacteriocinas/farmacología , Calcio/metabolismo , Cloruros/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Péptidos/farmacología , Línea Celular , Células Epiteliales , Humanos , Transporte Iónico/fisiología , Microscopía Confocal , Microscopía FluorescenteRESUMEN
Defective chloride transport in epithelial cells increases mucus viscosity and leads to recurrent infections with high oxidative stress in patients with CF (cystic fibrosis). NAC (N-acetylcysteine) is a well known mucolytic and antioxidant drug, and an indirect precursor of glutathione. Since GSNO (S-nitrosoglutathione) previously has been shown to be able to promote Cl- efflux from CF airway epithelial cells, it was investigated whether NAC also could stimulate Cl- efflux from CF and non-CF epithelial cells and through which mechanisms. CFBE (CF bronchial epithelial cells) and normal bronchial epithelial cells (16HBE) were treated with 1 mM, 5 mM, 10 mM or 15 mM NAC for 4 h at 37 degrees C. The effect of NAC on Cl- transport was measured by Cl- efflux measurements and by X-ray microanalysis. Cl- efflux from CFBE cells was stimulated by NAC in a dose-dependent manner, with 10 mM NAC causing a significant increase in Cl- efflux with nearly 80% in CFBE cells. The intracellular Cl- concentration in CFBE cells was significantly decreased up to 60% after 4 h treatment with 10 mM NAC. Moreover immunocytochemistry and Western blot experiments revealed expression of CFTR channel on CFBE cells after treatment with 10 mM NAC. The stimulation of Cl- efflux by NAC in CF airway epithelial cells may improve hydration of the mucus and thereby be beneficial for CF patients.
Asunto(s)
Acetilcisteína/farmacología , Antioxidantes/farmacología , Cloruros/metabolismo , Células Epiteliales/metabolismo , Mucosa Respiratoria/metabolismo , Línea Celular , Fibrosis Quística/metabolismo , Humanos , Inmunohistoquímica , Estrés Oxidativo , Mucosa Respiratoria/citologíaRESUMEN
It was investigated whether azithromycin (AZM) stimulates chloride (Cl-) efflux from cystic fibrosis (CF) and non-CF airway epithelial cells, possibly secondary to up-regulation of the multidrug resistance protein (MDR). CF and non-CF human airway epithelial cell lines (CFBE and 16HBE) were treated with 0.4, 4, and 40 microg/mL AZM for 4 days. Cl- efflux was explored in the presence or absence of specific inhibitors of CFTR and alternative Cl- channels. Six CF patients received AZM (500 mg daily) for 6 months. The percentage of predicted forced vital capacity (FVC%), forced expiratory volume (FEV1%), and the number of acute exacerbations were compared before and after treatment. Nasal biopsies were taken before and after treatment, and mRNA expression of MDR and CFTR was determined by in situ hybridization. A significant dose-dependent increase of Cl- efflux from CFBE cells (but not from 16HBE cells) was observed after AZM treatment. A CFTR inhibitor significantly reduced AZM-stimulated Cl- efflux from CFBE cells. A significant improvement in FEV1%, and fewer exacerbations were observed. AZM treatment did not affect mRNA expression of MDR and CFTR. The stimulation of Cl- efflux could be part of the explanation for the clinical improvement seen among the patients.