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1.
Physiol Rep ; 2(8)2014 Aug 01.
Article in English | MEDLINE | ID: mdl-25107986

ABSTRACT

In cystic fibrosis (CF), the airway surface liquid (ASL) is depleted. We previously demonstrated that lipoxin A4 (LXA4) can modulate ASL height (ASLh) through actions on Cl(-) transport. Here, we report novel effects of lipoxin on the epithelial Na(+) channel ENaC in this response. ASL dynamics and ion transport were studied using live-cell confocal microscopy and short-circuit current measurements in CF (CuFi-1) and non-CF (NuLi-1) cell cultures. Low physiological concentrations of LXA4 in the picomolar range produced an increase in ASLh which was dependent on inhibition of an amiloride-sensitive Na(+) current and stimulation of a bumetanide-sensitive Cl(-) current. These ion transport and ASLh responses to LXA4 were blocked by Boc-2 an inhibitor of the specific LXA4 receptor ALX/FPR2. LXA4 affected the subcellular localization of its receptor and enhanced the localization of ALX/FPR2 at the apical membrane of CF cells. Our results provide evidence for a novel effect of low physiological concentrations of LXA4 to inhibit airway epithelial Na(+) absorption that results in an ASL height increase in CF airway epithelia.

2.
Am J Respir Cell Mol Biol ; 51(2): 178-90, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24588705

ABSTRACT

In cystic fibrosis (CF), the airway surface liquid (ASL) height is reduced as a result of impaired ion transport, which favors bacterial colonization and inflammation of the airway and leads to progressive lung destruction. Lipoxin (LX)A4, which promotes resolution of inflammation, is inadequately produced in the airways of patients with CF. We previously demonstrated that LXA4 stimulates an ASL height increase and epithelial repair. Here we report the molecular mechanisms involved in these processes. We found that LXA4 (1 nM) induced an apical ATP release from non-CF (NuLi-1) and CF (CuFi-1) airway epithelial cell lines and CF primary cultures. The ATP release induced by LXA4 was completely inhibited by antagonists of the ALX/FPR2 receptor and Pannexin-1 channels. LXA4 induced an increase in intracellular cAMP and calcium, which were abolished by the selective inhibition of the P2RY11 purinoreceptor. Pannexin-1 and ATP hydrolysis inhibition and P2RY11 purinoreceptor knockdown all abolished the increase of ASL height induced by LXA4. Inhibition of the A2b adenosine receptor did not affect the ASL height increase induced by LXA4, whereas the PKA inhibitor partially inhibited this response. The stimulation of NuLi-1 and CuFi-1 cell proliferation, migration, and wound repair by LXA4 was inhibited by the antagonists of Pannexin-1 channel and P2RY11 purinoreceptor. Taken together, our results provide evidence for a novel role of LXA4 in stimulating apical ATP secretion via Pannexin-1 channels and P2RY11 purinoreceptors activation leading to an ASL height increase and epithelial repair.


Subject(s)
Adenosine Triphosphate/metabolism , Cystic Fibrosis/metabolism , Epithelial Cells/metabolism , Lipoxins/metabolism , Lung/metabolism , Receptors, Purinergic P2/metabolism , Regeneration , Respiratory Mucosa/metabolism , Signal Transduction , Adaptor Proteins, Signal Transducing/metabolism , Autocrine Communication , Calcium Signaling , Cell Line , Cell Movement , Cell Proliferation , Connexins/metabolism , Cyclic AMP/metabolism , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , Cystic Fibrosis/physiopathology , Epithelial Cells/drug effects , Epithelial Cells/pathology , Humans , Lung/drug effects , Lung/pathology , Lung/physiopathology , Nerve Tissue Proteins/metabolism , Primary Cell Culture , Purinergic P2 Receptor Antagonists/pharmacology , Receptors, Formyl Peptide/metabolism , Receptors, Lipoxin/metabolism , Regeneration/drug effects , Respiratory Mucosa/drug effects , Respiratory Mucosa/pathology , Respiratory Mucosa/physiopathology , Signal Transduction/drug effects , Time Factors
3.
PLoS One ; 7(5): e37746, 2012.
Article in English | MEDLINE | ID: mdl-22662206

ABSTRACT

Cystic Fibrosis (CF) is a genetic disease characterised by a deficit in epithelial Cl(-) secretion which in the lung leads to airway dehydration and a reduced Airway Surface Liquid (ASL) height. The endogenous lipoxin LXA(4) is a member of the newly identified eicosanoids playing a key role in ending the inflammatory process. Levels of LXA(4) are reported to be decreased in the airways of patients with CF. We have previously shown that in normal human bronchial epithelial cells, LXA(4) produced a rapid and transient increase in intracellular Ca(2+). We have investigated, the effect of LXA(4) on Cl(-) secretion and the functional consequences on ASL generation in bronchial epithelial cells obtained from CF and non-CF patient biopsies and in bronchial epithelial cell lines. We found that LXA(4) stimulated a rapid intracellular Ca(2+) increase in all of the different CF bronchial epithelial cells tested. In non-CF and CF bronchial epithelia, LXA(4) stimulated whole-cell Cl(-) currents which were inhibited by NPPB (calcium-activated Cl(-) channel inhibitor), BAPTA-AM (chelator of intracellular Ca(2+)) but not by CFTRinh-172 (CFTR inhibitor). We found, using confocal imaging, that LXA(4) increased the ASL height in non-CF and in CF airway bronchial epithelia. The LXA(4) effect on ASL height was sensitive to bumetanide, an inhibitor of transepithelial Cl(-) secretion. The LXA(4) stimulation of intracellular Ca(2+), whole-cell Cl(-) currents, conductances and ASL height were inhibited by Boc-2, a specific antagonist of the ALX/FPR2 receptor. Our results provide, for the first time, evidence for a novel role of LXA(4) in the stimulation of intracellular Ca(2+) signalling leading to Ca(2+)-activated Cl(-) secretion and enhanced ASL height in non-CF and CF bronchial epithelia.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Calcium/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Cystic Fibrosis/metabolism , Lipoxins/pharmacology , Respiratory Mucosa/drug effects , Respiratory Mucosa/metabolism , Child, Preschool , Chlorides/metabolism , Dose-Response Relationship, Drug , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Primary Cell Culture , Receptors, Formyl Peptide/metabolism , Receptors, Lipoxin/metabolism
4.
J Cyst Fibros ; 7(6): 463-8, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18541460

ABSTRACT

UNLABELLED: Antibiotics are largely prescribed for cystic fibrosis (CF) respiratory exacerbations. Effects of antibiotics on the inflammatory profile of the patients have been shown but remain controversial. Lipoxin A(4) (LXA(4)) is a lipid mediator, reported to play a central role in resolving airway inflammation. The aim of the study was to investigate the consequences of antibiotherapy on LXA(4) and IL-8 levels in CF patients' airways. METHODS: Eighteen CF patients (7 females, median age 20, range 8 to 47 years) consecutively admitted at the CF center of Montpellier for antibiotics during pulmonary exacerbation, were enrolled. Before and after antibiotics, all patients underwent spirometry (FEV(1) and FVC), bacterial cultures and cell counts in sputa. IL-8 and LXA(4) concentrations were determined in sputum samples by the median of immunometric assays. RESULTS: As previously reported, after antibiotics therapy, FEV(1) and FVC significantly improved. While neutrophil cell counts and IL-8 levels decreased, the LXA(4) levels significantly increased after antibiotics therapy and were inversely correlated with IL-8 levels. In conclusion, we reported a correlation between antibiotics treatments and inflammatory markers in CF sputum. Our data provide evidences for a novel effect of antibiotics increasing the concentration of the anti-inflammatory lipid mediator LXA(4).


Subject(s)
Anti-Bacterial Agents/therapeutic use , Cystic Fibrosis/drug therapy , Cystic Fibrosis/metabolism , Interleukin-8/metabolism , Lipoxins/metabolism , Sputum/chemistry , Adolescent , Adult , Child , Cystic Fibrosis/etiology , Female , Forced Expiratory Volume , Humans , Infusions, Intravenous , Male , Middle Aged , Prospective Studies , Sputum/cytology , Sputum/microbiology , Treatment Outcome , Vital Capacity , Young Adult
5.
J Biol Chem ; 280(43): 35807-14, 2005 Oct 28.
Article in English | MEDLINE | ID: mdl-16040608

ABSTRACT

Glucocorticoids have been shown to produce rapid nongenomic responses in airway epithelia. By using an intracellular pH (pH(i)) spectrofluorescence imaging system and the NH4Cl acid-loading technique, we have shown that the synthetic glucocorticoid,dexamethasone, accelerated intracellular pH recovery after an acid load in a human bronchial epithelial cell line (16HBE14o- cells). Exposure to NH4Cl (20 mm) elicited an intracellular acidification, followed by a pH(i) recovery. Inhibition of the Na+/H+ exchanger decreased the steady-state pH(i) and antagonized the dexamethasone stimulation of pH(i) regulation. The rapid effect of dexamethasone on pH(i) was neither affected by the inhibitor of transcription, cycloheximide, nor by the classical glucocorticoid and mineralocorticoid receptors antagonists, RU486 and spironolactone, respectively. The dexamethasone effect on pH(i) regulation was reduced by inhibitors of adenylate cyclase, cAMP-dependent protein kinase and mitogenactivated protein kinase (ERK1/2). By using a PepTag assay system and Western blotting, we have shown that dexamethasone stimulated cAMP-dependent protein kinase and mitogen-activated protein kinase activities. Taken together our results provide evidence for the rapid stimulation of Na+/H+ exchange activity by glucocorticoids in bronchial epithelial cells via a nongenomic mechanism involving cAMP-dependent protein kinase and mitogen-activated protein kinase ERK1/2 pathways.


Subject(s)
Bronchi/cytology , Dexamethasone/pharmacology , Epithelial Cells/cytology , Sodium-Hydrogen Exchangers/metabolism , Anti-Inflammatory Agents/pharmacology , Bronchi/pathology , Calcium/metabolism , Cell Line , Cells, Cultured , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/metabolism , Glucocorticoids/metabolism , Humans , Hydrogen-Ion Concentration , MAP Kinase Signaling System , Mifepristone/pharmacology , Mineralocorticoids/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Pertussis Toxin/pharmacology , Phosphorylation , Spectrometry, Fluorescence , Steroids/metabolism , Time Factors
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