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.

scholarly journals Subtype-specific expression patterns of inositol 1,4,5-trisphosphate receptors in rat airway epithelial cells.

1996 ◽  
Vol 44 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
T Sugiyama ◽  
M Yamamoto-Hino ◽  
K Wasano ◽  
K Mikoshiba ◽  
M Hasegawa
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Specific Cell ◽  
Airway Epithelial ◽  
Clara Cells ◽  
Ciliated Cells ◽  
Inositol 1,4,5 Trisphosphate

We investigated the immunohistochemical localization of inositol 1,4,5-trisphosphate receptor (IP3R) Types 1, 2, and 3 in rat airway epithelium using the monoclonal antibodies KM1112, KM1083, and KM1082 specific for each type of IP3R. The epithelium from trachea to distal intrapulmonary airways (bronchioles) showed positive immunoreactivity for all types of IP3R. However, cell type as well as subcellular site immunoreactivity for each type of IP3R varied. IP3R Type 1 was found only in the apical thin cytoplasmic area of ciliated cells throughout all airway levels. IP3R Type 2 was exclusively localized to the entire cytoplasm of ciliated cells from the trachea to bronchioles. IP3R Type 3 was expressed mainly in the supranuclear cytoplasm not only of ciliated cells at all airway levels but also in Clara cells of the bronchiolar epithelium. Double fluorescent staining using combinations of KM1083 and Wisteria floribunda lectin or anti-rat 10-KD Clara cell-specific protein antibody confirmed that the IP3R Type 2-positive cells were neither seromucous cells nor Clara cells. These results indicate that the expression of three types of IP3Rs in different cell types and subcellular sites may reflect diverse physiological functions of IP3Rs within airway epithelial cells. The double staining studies suggested that the anti-IP3R Type 2 monoclonal antibody KM1083 would be a specific cell marker for ciliated cells of the airway epithelium.

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 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 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.

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.

Plasticity of airway cell proliferation and gene expression after acute naphthalene injury

1995 ◽  
Vol 269 (6) ◽  
pp. L791-L799 ◽  
Author(s):  
B. R. Stripp ◽  
K. Maxson ◽  
R. Mera ◽  
G. Singh
Keyword(s):  
Epithelial Cells ◽  
Cellular Proliferation ◽  
Airway Epithelial Cells ◽  
Clara Cell ◽  
Acute Injury ◽  
Alveolar Type ◽  
Airway Epithelial ◽  
Clara Cells ◽  
Proliferating Cells ◽  
Sequence Of Events

The goal of this study was to determine the temporal and spatial sequence of events that accompany lung injury and repair after parenteral administration of the Clara cell-specific cytotoxicant, naphthalene. Changes in airway epithelial cells were evaluated by measuring alterations in the expression of markers for differentiated Clara cells (CYPIIF and Clara cell 10-kDa secretory protein, CC10), distal airway/alveolar type II cells (surfactant protein B; SP-B) and for cycling/proliferating cells (cyclin dependent kinase 1;CDK1). Naphthalene-induced Clara cell cytotoxicity resulted in the exfoliation of epithelial cells containing CC10 protein. This was accompanied by a dramatic reduction in the abundance of mRNA for CC10 and CYPIIF. Large numbers of CDK1 mRNA-positive cells were identified in and around bronchioles and terminal bronchioles 48 h after treatment. This cellular proliferation resulted in the population of airways by immature epithelial cells lacking normal levels of CC10 mRNA but overexpressing SP-B mRNA. Seventy-two hours after naphthalene treatment a reduction in CDK1 mRNA-positive cells was noted within bronchioles and terminal bronchioles at all locations, with the exception of airway bifurcations. At airway bifurcations CDK1 mRNA appeared to be more abundant at the 72-h time point than at 48 h. Comparison of these sections with serial sections probed for CC10 mRNA demonstrated a correlation between the expression of CDK1 and CC10 mRNA at bifurcations. Temporal increases in the abundance of CC10 mRNA observed at later time points were largely accounted for by the processive maturation of newly repopulated cells neighboring bifurcations in bronchioles. These studies identify spatially distinct populations of cells that act in concert to repopulate naphthalene-injured airways and support the notion that branch point cells play an important role in the maturation of newly regenerated airway epithelial cells after acute injury.

Transgenic overexpression of β2-adrenergic receptors in airway epithelial cells decreases bronchoconstriction

2000 ◽  
Vol 279 (2) ◽  
pp. L379-L389 ◽  
Author(s):  
Dennis W. McGraw ◽  
Susan L. Forbes ◽  
Judith C. W. Mak ◽  
David P. Witte ◽  
Patricia E. Carrigan ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Airway Epithelium ◽  
Adrenergic Receptors ◽  
Airway Epithelial Cells ◽  
Airway Responsiveness ◽  
Ozone Exposure ◽  
Clara Cell ◽  
Whole Body ◽  
Airway Epithelial

Airway epithelial cells express β2-adrenergic receptors (β2-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of β2-ARs to airway epithelium of transgenic (CCSP-β2-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that β2-AR density in CCSP-β2-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED200) was greater for CCSP-β2-AR than for NTG mice (345 ± 34 vs. 157 ± 14 mg/ml; P < 0.01). CCSP-β2-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline ( P < 0.05) but remained unchanged in the CCSP-β2-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED200for ozone-exposed CCSP-β2-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell β2-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of β-agonists results from activation of receptors on both epithelial and smooth muscle cells.

Na+ and Cl- transport across rabbit nonciliated bronchiolar epithelial (Clara) cells

1989 ◽  
Vol 256 (4) ◽  
pp. C893-C901 ◽  
Author(s):  
M. R. Van Scott ◽  
C. W. Davis ◽  
R. C. Boucher
Keyword(s):  
Ion Transport ◽  
Short Circuit ◽  
Flux Ratio ◽  
Open Circuit ◽  
Clara Cell ◽  
Clara Cells ◽  
Transport Pathway ◽  
Transport Pathways ◽  
Serum Free ◽  
Significant Difference

Radioisotopic flux measurements were performed on rabbit Clara cell epithelium cultured in serum-free hormone-supplemented medium to identify the major ion transport pathways in the cell type. Clara cells cultured in serum-free hormone-supplemented medium exhibit a large short-circuit current compared with cells maintained in serum-containing medium (45 microA/cm2 vs. 15 microA/cm2). The responses to amiloride and isoproterenol, however, are similar for cells grown in the two media. A net amiloride-sensitive movement of Na+ in the mucosal (M)-to-serosal (S) direction undershort- and open-circuit conditions is detected (1.48 and 0.67 mueq.h-1.cm-2, respectively). No statistically significant difference in the unidirectional fluxes of Cl- is apparent in the basal state, but a net flux of Cl- in the S-to-M direction is observed after exposure of the apical membrane to amiloride (0.93 mueq.h-1.cm-2). The partial ionic conductances for Na+ and Cl- estimated from the fluxes measured in the passive directions (JNaS----M, JClM----S) exceed the total tissue conductance by 20%. Ussing flux ratio analyses of Cl- movements at clamped potentials between -60 and +20 mV show that Cl- movements are not strictly through passive conductive pathways at negative potentials. The movement of Cl- can be modeled by passive diffusion combined with Cl- -Cl- exchange equal to 20% of total passive fluxes of Na+ and Cl-. These observations indicate that 1) Na+ absorption is the major active ion transport pathway across cultured Clara cells, 2) active Cl- secretion is minimal in the basal state, and 3) approximately 20% of the unidirectional Cl- fluxes occur via nonconductive pathways.

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