scholarly journals Naringenin Regulates CFTR Activation and Expression in Airway Epithelial Cells

2017 ◽  
Vol 44 (3) ◽  
pp. 1146-1160 ◽  
Author(s):  
Rui Shi ◽  
Zi-Ting Xiao ◽  
Yi-Jun Zheng ◽  
Yi-Lin Zhang ◽  
Jia-Wen Xu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
The Elderly ◽  
Airway Epithelial Cells ◽  
Intracellular Camp ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Camp Content ◽  
Basolateral Side

Background/Aims: Sputum symptoms are commonly seen in the elderly. This study aimed to identify an efficacious expectorant treatment stratagem through evaluating the secretion-promoting activation and cystic fibrosis transmembrane conductance regulator (CFTR) expression of the bioactive herbal monomer naringenin. Methods: Vectorial Cl- transport was determined by measuring short-circuit current (ISC) in rat airway epithelium. cAMP content was measured by ELISA in primary cultured epithelial cells and Calu-3 cells. CFTR expression in Calu-3 cells was determined by qPCR. Results: Addition of naringenin to the basolateral side of the rat airway led to a concentration-dependent sustained increase in ISC. The current was suppressed when exposed to Cl–-free solution or by bumetanide, BaCl2, and DPC but not by DIDS and IBMX. Forskolin-induced ISC increase and CFTRinh-172/MDL-12330A-induced ISC inhibition were not altered by naringenin. Intracellular cAMP content was significantly increased by naringenin. With lipopolysaccharide stimulation, CFTR expression was significantly reduced, and naringenin dose-dependently enhanced CFTR mRNA expression. Conclusion: These results demonstrate that naringenin has the ability to stimulate Cl- secretion, which is mediated by CFTR through a signaling pathway by increasing cAMP content. Moreover, naringenin can increase CFTR expression when organism CFTR expression is seriously hampered. Our data suggest a potentially effective treatment strategy for sputum.

scholarly journals Apical adenosine regulates basolateral Ca2+-activated potassium channels in human airway Calu-3 epithelial cells

2008 ◽  
Vol 294 (6) ◽  
pp. C1443-C1453 ◽  
Author(s):  
Dong Wang ◽  
Ying Sun ◽  
Wei Zhang ◽  
Pingbo Huang
Keyword(s):  
Epithelial Cells ◽  
Adenosine Receptors ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Specific Inhibitor ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Intracellular Camp ◽  
Anion Secretion ◽  
Human Airway

In airway epithelial cells, apical adenosine regulates transepithelial anion secretion by activation of apical cystic fibrosis transmembrane conductance regulator (CFTR) via adenosine receptors and cAMP/PKA signaling. However, the potent stimulation of anion secretion by adenosine is not correlated with its modest intracellular cAMP elevation, and these uncorrelated efficacies have led to the speculation that additional signaling pathways may be involved. Here, we showed that mucosal adenosine-induced anion secretion, measured by short-circuit current ( Isc), was inhibited by the PLC-specific inhibitor U-73122 in the human airway submucosal cell line Calu-3. In addition, the Isc was suppressed by BAPTA-AM (a Ca2+ chelator) and 2-aminoethoxydiphenyl borate (2-APB; an inositol 1,4,5-trisphosphate receptor blocker), but not by PKC inhibitors, suggesting the involvement of PKC-independent PLC/Ca2+ signaling. Ussing chamber and patch-clamp studies indicated that the adenosine-induced PLC/Ca2+ signaling stimulated basolateral Ca2+-activated potassium (KCa) channels predominantly via A2B adenosine receptors and contributed substantially to the anion secretion. Thus, our data suggest that apical adenosine activates contralateral K+ channels via PLC/Ca2+ and thereby increases the driving force for transepithelial anion secretion, synergizing with its modulation of ipsilateral CFTR via cAMP/PKA. Furthermore, the dual activation of CFTR and KCa channels by apical adenosine resulted in a mixed secretion of chloride and bicarbonate, which may alter the anion composition in the secretion induced by secretagogues that elicit extracellular ATP/adenosine release. Our findings provide novel mechanistic insights into the regulation of anion section by adenosine, a key player in the airway surface liquid homeostasis and mucociliary clearance.

Intracellular Ca2+ and regulation of ion transport across rabbit Clara cells

1992 ◽  
Vol 263 (1) ◽  
pp. L122-L127
Author(s):  
M. R. Van Scott ◽  
A. M. Paradiso
Keyword(s):  
Epithelial Cells ◽  
Ion Transport ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Bathing Solution ◽  
Airway Epithelial ◽  
Clara Cells ◽  
Tracheal Cell

We investigated whether Ca2+ was involved in regulation of ion transport across rabbit distal airway epithelial cells by studying the effects that elevation of intracellular Ca2+ (Cai) had on the bioelectric properties of nonciliated bronchiolar (Clara) cell epithelia in culture. Exposure of Clara cells to 5 x 10(-7) M ionomycin increased Cai concentration and transepithelial short-circuit current (Isc). Changing extracellular Ca2+ concentration in the presence of ionomycin demonstrated that changes in Isc paralleled changes in Cai. Another ionophore, 4-bromo-A23187, also increased Cai and Isc. Ionomycin-induced changes in Isc were insensitive to amiloride and were inhibited greater than 50% by pretreating the cells with bumetanide or substituting gluconate for Cl- in the bathing solution. Bradykinin and carbachol, which increased Cai and caused an increase in Isc across tracheal cell cultures, had no effect on Cai or Isc in Clara cell preparations. These results support the hypothesis that changes in Cai are linked to regulation of Cl- secretion across bronchiolar epithelial cells, but physiological regulators of Cai in Clara cells remain to be defined.

The rescue of F508del-CFTR by elexacaftor/tezacaftor/ivacaftor (Trikafta) in human airway epithelial cells is underestimated due to the presence of ivacaftor

2021 ◽  
pp. 2100671
Author(s):  
Frédéric Becq ◽  
Sandra Mirval ◽  
Thomas Carrez ◽  
Manuella Lévêque ◽  
Arnaud Billet ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Triple Combination Therapy ◽  
Whole Cell Patch Clamp ◽  
Membrane Location ◽  
And Function

Trikafta, currently the leading therapeutic in Cystic Fibrosis (CF), has demonstrated a real clinical benefit. This treatment is the triple combination therapy of two folding correctors elexacaftor/tezacaftor (VX445/VX661) plus the gating potentiator ivacaftor (VX770). In this study, our aim was to compare the properties of F508del-CFTR in cells treated with either lumacaftor (VX809), tezacaftor, elexacaftor, elexacaftor/tezacaftor with or without ivacaftor. We studied F508del-CFTR function, maturation and membrane localisation by Ussing chamber and whole-cell patch clamp recordings, Western blot and immunolocalization experiments. With human primary airway epithelial cells and the cell lines CFBE and BHK expressing F508del, we found that, whereas the combination elexacaftor/tezacaftor/ivacaftor was efficient in rescuing F508del-CFTR abnormal maturation, apical membrane location and function, the presence of ivacaftor limits these effects. The basal F508del-CFTR short-circuit current was significantly increased by elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor compared to other correctors and non-treated cells, an effect dependent on ivacaftor and cAMP. These results suggest that the level of the basal F508del-CFTR current might be a marker for correction efficacy in CF cells. When cells were treated with ivacaftor combined to any correctors, the F508del-CFTR current was unresponsive to the subsequently acute addition of ivacaftor unlike the CFTR potentiators genistein and Cact-A1 which increased elexacaftor/tezacaftor/ivacaftor and elexacaftor/tezacaftor-corrected F508del-CFTR currents. These findings show that ivacaftor reduces the correction efficacy of Trikafta. Thus, combining elexacaftor/tezacaftor with a different potentiator might improve the therapeutic efficacy for treating CF patients.

scholarly journals Carvedilol binding to β2-adrenergic receptors inhibits CFTR-dependent anion secretion in airway epithelial cells

2016 ◽  
Vol 310 (1) ◽  
pp. L50-L58 ◽  
Author(s):  
Elizabeth R. Peitzman ◽  
Nathan A. Zaidman ◽  
Peter J. Maniak ◽  
Scott M. O'Grady
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Protein Activation ◽  
Anion Secretion ◽  
G Protein Activation ◽  
Channel Expression

Carvedilol functions as a nonselective β-adrenergic receptor (AR)/α1-AR antagonist that is used for treatment of hypertension and heart failure. Carvedilol has been shown to function as an inverse agonist, inhibiting G protein activation while stimulating β-arrestin-dependent signaling and inducing receptor desensitization. In the present study, short-circuit current ( Isc) measurements using human airway epithelial cells revealed that, unlike β-AR agonists, which increase Isc, carvedilol decreases basal and 8-(4-chlorophenylthio)adenosine 3′,5′-cyclic monophosphate-stimulated current. The decrease in Isc resulted from inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR). The carvedilol effect was abolished by pretreatment with the β2-AR antagonist ICI-118551, but not the β1-AR antagonist atenolol or the α1-AR antagonist prazosin, indicating that its inhibitory effect on Isc was mediated through interactions with apical β2-ARs. However, the carvedilol effect was blocked by pretreatment with the microtubule-disrupting compound nocodazole. Furthermore, immunocytochemistry experiments and measurements of apical CFTR expression by Western blot analysis of biotinylated membranes revealed a decrease in the level of CFTR protein in monolayers treated with carvedilol but no significant change in monolayers treated with epinephrine. These results demonstrate that carvedilol binding to apical β2-ARs inhibited CFTR current and transepithelial anion secretion by a mechanism involving a decrease in channel expression in the apical membrane.

Partial correction of defective Cl− secretion in cystic fibrosis epithelial cells by an analog of squalamine

2001 ◽  
Vol 281 (5) ◽  
pp. L1164-L1172 ◽  
Author(s):  
Canwen Jiang ◽  
Edward R. Lee ◽  
Mathieu B. Lane ◽  
Yong-Fu Xiao ◽  
David J. Harris ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Synthetic Analog ◽  
Airway Epithelial ◽  
Thyroid Cells ◽  
Rat Thyroid ◽  
Partial Correction

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl− transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl− transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl− gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ∼30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl− currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl− solution, indicating restoration of Cl− secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl− secretion.

scholarly journals Chlorzoxazone or 1-EBIO increases Na+absorption across cystic fibrosis airway epithelial cells

2001 ◽  
Vol 281 (5) ◽  
pp. L1123-L1129 ◽  
Author(s):  
Lin Gao ◽  
James R. Yankaskas ◽  
Catherine M. Fuller ◽  
Eric J. Sorscher ◽  
Sadis Matalon ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Short Circuit ◽  
Fold Increase ◽  
Short Circuit Current ◽  
Airway Disease ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Cystic Fibrosis Airway ◽  
Cf Transmembrane Conductance Regulator

Previous studies demonstrated that chlorzoxazone or 1-ethyl-2-benzimidazolinone (1-EBIO) enhances transepithelial Cl− secretion by increasing basolateral K+ conductance ( G K) (Singh AK, Devor DC, Gerlach AC, Gondor M, Pilewski JM, and Bridges RJ. J Pharmacol Exp Ther 292: 778–787, 2000). Hence these compounds may be useful to treat cystic fibrosis (CF) airway disease. The goal of the present study was to determine whether chlorzoxazone or 1-EBIO altered ion transport across ΔF508-CF transmembrane conductance regulator homozygous CFT1 airway cells. CFT1 monolayers exhibited a basal short-circuit current that was abolished by apical amiloride (inhibition constant 320 nM) as expected for Na+ absorption. The addition of chlorzoxazone (400 μM) or 1-EBIO (2 mM) increased the amiloride-sensitive I sc ∼2.5-fold. This overlapping specificity may preclude use of these compounds as CF therapeutics. Assaying for changes in the basolateral G K with a K+ gradient plus the pore-forming antibiotic amphotericin B revealed that chlorzoxazone or 1-EBIO evoked an ∼10-fold increase in clotrimazole-sensitive G K. In contrast, chlorzoxazone did not alter epithelial Na+ channel-mediated currents across basolateral-permeabilized monolayers or in Xenopus oocytes. These data further suggest that alterations in basolateral G K alone can modulate epithelial Na+ transport.

A regulated apical Na+ conductance in dexamethasone-treated H441 airway epithelial cells

2004 ◽  
Vol 287 (2) ◽  
pp. L411-L419 ◽  
Author(s):  
S. J. Ramminger ◽  
K. Richard ◽  
S. K. Inglis ◽  
S. C. Land ◽  
R. E. Olver ◽  
...  
Keyword(s):  
Ion Transport ◽  
Rank Order ◽  
Short Circuit ◽  
Brefeldin A ◽  
Short Circuit Current ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Ionic Selectivity ◽  
Camp Content ◽  
Permeabilized Cells

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na+ channel α- and β-subunits and increased transepithelial ion transport (measured as short-circuit current, Isc) from <4 μA·cm−2 to 10–20 μA·cm−2. This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil ≥ amiloride > EIPA and can thus be attributed to active Na+ absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na+ pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na+ conductance ( GNa) from a negligible value to 100–200 μS·cm−2. Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na+ and Li+ and that the permeability to these cations was approximately fourfold greater than to K+. There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an ∼60% increase in Isc, and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in GNa. This cAMP-dependent control over GNa was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na+ transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.

scholarly journals A Ba2+-resistant, acid-sensitive K+ conductance in Na+-absorbing H441 human airway epithelial cells

2007 ◽  
Vol 292 (5) ◽  
pp. L1304-L1312 ◽  
Author(s):  
Sarah K. Inglis ◽  
Sean G. Brown ◽  
Maree J. Constable ◽  
Niall McTavish ◽  
Richard E. Olver ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Airway Epithelial Cells ◽  
Airway Epithelial ◽  
Human Airway ◽  
K2p Channels ◽  
Human Airway Epithelial Cells

By analysis of whole cell membrane currents in Na+-absorbing H441 human airway epithelial cells, we have identified a K+ conductance ( GK) resistant to Ba2+ but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K+ current ( IBl) whereas Ba2+ has only a weak inhibitory effect. IBl was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in IBl, acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K+ channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current ( ISC), and basolateral bupivacaine, lidocaine, quinidine, and Ba2+ inhibited ISC at concentrations similar to those needed to inhibit IBl, suggesting that the K+ channels underlying IBl are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K+ (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie GK in unstimulated cells and so maintain the driving force for Na+ absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology.

scholarly journals Deleterious impact of Pseudomonas aeruginosa on cystic fibrosis transmembrane conductance regulator function and rescue in airway epithelial cells

2015 ◽  
Vol 45 (6) ◽  
pp. 1590-1602 ◽  
Author(s):  
Nguyen Thu Ngan Trinh ◽  
Claudia Bilodeau ◽  
Émilie Maillé ◽  
Manon Ruffin ◽  
Marie-Claude Quintal ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Epithelial Cells ◽  
Short Circuit ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Transmembrane Conductance ◽  
Δf508 Cftr ◽  
Cystic Fibrosis Transmembrane

The epithelial response to bacterial airway infection, a common feature of lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis, has been extensively studied. However, its impact on cystic fibrosis transmembrane conductance regulator (CFTR) channel function is not clearly defined. Our aims were, therefore, to evaluate the effect of Pseudomonas aeruginosa on CFTR function and expression in non-cystic fibrosis airway epithelial cells, and to investigate its impact on ΔF508-CFTR rescue by the VRT-325 corrector in cystic fibrosis cells.CFTR expression/maturation was evaluated by immunoblotting and its function by short-circuit current measurements.A 24-h exposure to P. aeruginosa diffusible material (PsaDM) reduced CFTR currents as well as total and membrane protein expression of the wildtype (wt) CFTR protein in CFBE-wt cells. In CFBE-ΔF508 cells, PsaDM severely reduced CFTR maturation and current rescue induced by VRT-325. We also confirmed a deleterious impact of PsaDM on wt-CFTR currents in non-cystic fibrosis primary airway cells as well as on the rescue of ΔF508-CFTR function induced by VRT-325 in primary cystic fibrosis cells.These findings show that CFTR function could be impaired in non-cystic fibrosis patients infected by P. aeruginosa. Our data also suggest that CFTR corrector efficiency may be affected by infectious components, which should be taken into account in screening assays of correctors.

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