Oxidant stress stimulates anion secretion from the human airway epithelial cell line calu-3: implications for cystic fibrosis lung disease (original) (raw)
2002, The Journal of Physiology
Exposure to reactive oxygen species (ROS) is associated with tissue damage in the lung and may be a common element in the pathogenesis of all inflammatory lung diseases. Exposure to the ROS hydrogen peroxide (H 2 O 2) evoked a rapid increase in transepithelial anion secretion across monolayers of the human submucosal gland serous cell line Calu-3. This increase was almost entirely abolished by the addition of diphenylamine-2-carboxylate (DPC), implicating the cystic fibrosis transmembrane conductance regulator (CFTR) Cl _ channel in the response. The response was also reduced by inhibitors of basolateral K + channels. Studies of electrically isolated apical and basolateral membranes revealed that H 2 O 2 stimulated both apical Cl _ and basolateral K + conductances (G Cl and G K). Apical G Cl was sensitive to DPC, but unaffected by 4,4‚-diisothiocyanatostilbene-2,2‚-disulfonic acid (DIDS), suggesting that CFTR is the major anion conduction pathway mediating the response to H 2 O 2. Additionally, H 2 O 2 had no effect on G Cl in the presence of the adenylate cyclase inhibitor SQ22536 or following maximal stimulation of G Cl with forskolin, implicating the cAMP-dependent protein kinase pathway in the apical response to H 2 O 2. Basolateral G K was reduced by the K + channel inhibitors clotrimazole and clofilium, indicating roles for KCNN4 and KCNQ1 in the H 2 O 2-stimulated response. We propose that ROS-stimulated anion secretion from serous cells plays an important role in keeping the airways clear from damaging radicals that could potentially initiate tissue destruction. Our finding that this response is CFTR dependent suggests that an important host defence mechanism would be dysfunctional in the cystic fibrosis (CF) lung. Loss of this compensatory protective mechanism could expose the CF lung to ROS for extended periods, which could be important in the pathogenesis of CF lung disease.
Related papers
Regulation of Basolateral Cl− Channels in Airway Epithelial Cells: The Role of Nitric Oxide
Journal of Membrane Biology, 2006
The presence of basolateral Cl) channels in airway epithelium has been reported in several studies, but little is known about their role in the regulation of anion secretion. The purpose of this study was to characterize regulation of these channels by nitric oxide (NO) in Calu-3 cells. Transepithelial measurements revealed that NO donors activated a basolateral Cl) conductance sensitive to 4,4¢-diisothiocyanatostilbene-2,2¢-disulfonic acid (DIDS) and anthracene-9-carboxylic acid. Apical membrane permeabilization studies confirmed the basolateral localization of NO-activated Cl) channels. Experiments using 8-bromo cyclic guanosine monophosphate (8Br-cGMP) and selective inhibitors of soluble guanylyl cyclase and inducible NO synthase (1H-[1, 2, 4] oxadiazolol-[4, 3-a] quinoxalin-1-one [ODQ] and 1400W [N-(3-Aminomethyl)benzyl)acetamidine], respectively) demonstrated that NO activated Cl) channels via a cGMP-dependent pathway. Anion replacement and 36 Cl) flux studies showed that NO affected both Cl) and HCO 3) secretion. Two different types of Cl) channels are known to be present in the basolateral membrane of epithelial cells: Zn 2+-sensitive ClC-2 and DIDS-sensitive bestrophin channels. S-Nitrosoglutathione (GSNO) activated Cl) conductance in the presence of Zn 2+ ions, indicating that ClC-2 channel function was not affected by GSNO. In contrast, DIDS completely inhibited GSNO-activated Cl) conductance. Bestrophin immunoprecipitation studies showed that under control conditions bestrophin channels were not phosphorylated but became phosphorylated after GSNO treatment. The presence of bestrophin in airway epithelia was confirmed using immunohistochemistry. We conclude that basolateral Cl) channels play a major role in the NO-dependent regulation of anion secretion in Calu-3 cells.
New Insights Into the Pathogenesis of Cystic Fibrosis
Treatments in Respiratory Medicine, 2004
channel' but recognized as a channel that also controls the efflux of other physiologically important anions, such as glutathione (GSH) and bicarbonate. More effective approaches to cystic fibrosis treatment may result from this reconceptualization of the CFTR by researchers and clinicians. For example, oxidant damage in cystic fibrosis has been assumed to be a significant part of the pathophysiology of the disease. Generally speaking, antioxidant status in cystic fibrosis is compromised. However, until recently this was seen as secondary to the excessive chemoattraction of neutrophils in this disease caused by mutation of the CFTR protein, leading to a high oxidant burden. New findings suggest that the cystic fibrosis mutations in fact cause a primary dysfunction in the system of one of the body's most important antioxidant and immune-signaling substances: the reduced GSH system. Cystic fibrosis mutations significantly decrease GSH efflux from cells without redundant channels to the CFTR; this leads to deficiency of GSH in the epithelial lining fluid of the lung, as well as in other compartments, including immune system cells and the gastrointestinal tract. This deficiency is exaggerated over time as the higher-than-normal oxidant burden of cystic fibrosis leads to successively larger decrements in GSH without the normal opportunity to fully recover physiologic levels. This GSH system dysfunction may be the trigger for initial depletion of other antioxidants and may also play a role in initiating the over-inflammation characteristic of cystic fibrosis. Proper GSH system functioning also affects immune system competence and mucus viscosity, both of relevance to cystic fibrosis pathophysiology. In a way, cystic fibrosis may be thought of as the first identified disease with GSH system dysfunction.
Pfl�gers Archiv European Journal of Physiology, 1994
The cystic fibrosis gene product (CFTR) is a C1-channel that possesses specific binding sites for cytosolic adenosine triphosphate (ATP) and is activated by cyclic adenosine monophosphate (cAMP)-dependent protein kinases. We explored the possibility that CFTR shares a common pharmacology with another ATP-regulated channel protein, the ATP-sensitive K + channel that is blocked by sulphonylureas and activated by diazoxide. cAMP-stimulated C1-effiuxes were measured with 36C1-in the epithelial cell line T84 which stably expresses CFTR. Neither glibenclamide (30 ~tM), tolbutamide (1 raM) nor diazoxide (100 pM) significantly affected forskolin-activated 36C1-effluxes in T84 cells. In patch-clamp experiments, glibenclamide exerted only weak inhibitory effects on the whole-cell currents through CFTR with an ICso of around 0.1 raM. Tolbutamide at I raM, but not at 0.1 raM, blocked a current of small amplitude which reversed near the equilibrium potential for K + ions. We conclude that sulphonylureas and diazoxide are not effective antagonists of endogenous CFTR C1-channels.
The Journal of physiology, 2015
Hypercapnia is clinically defined as an arterial blood partial pressure of CO2 of above 40 mmHg and is a feature of chronic lung disease. In previous studies we have demonstrated that hypercapnia modulates agonist-stimulated cAMP levels through effects on transmembrane adenylyl cyclase activity. In the airways, cAMP is known to regulate cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion and fluid secretion, which contributes to airway surface liquid homeostasis. The aim of the current work was to investigate if hypercapnia could modulate cAMP-regulated ion and fluid transport in human airway epithelial cells. We found that acute exposure to hypercapnia significantly reduced forskolin-stimulated elevations in intracellular cAMP as well as both adenosine and forskolin-stimulated increases in CFTR-dependent transepithelial short-circuit current, in polarised cultures of Calu-3 human airway cells. This CO2 -induced reduction in anion secretion was not due to a dec...
Physiological genomics, 2014
Although cystic fibrosis (CF) pathophysiology is explained by a defect in CF transmembrane conductance regulator (CFTR) protein, the broad spectrum of disease severity is the consequence of environmental and genetic factors. Among them, oxidative stress has been demonstrated to play an important role in the evolution of this disease, with susceptibility to oxidative damage, decline of pulmonary function, and impaired lung antioxidant defense. Although oxidative stress has been implicated in the regulation of inflammation, its molecular outcomes in CF cells remain to be evaluated. To address the question, we compared the gene expression profile in NuLi-1 cells with wild-type CFTR and CuFi-1 cells homozygous for ΔF508 mutation cultured at air-liquid interface. We analyzed the transcriptomic response of these cell lines with microarray technology, under basal culture conditions and after 24 h oxidative stress induced by 15 μM 2,3-dimethoxy-1,4-naphtoquinone. In the absence of oxidative...
Loading Preview
Sorry, preview is currently unavailable. You can download the paper by clicking the button above.