Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl<sup>−</sup>
 conductance

Korbmacher, Jonas Philipp; Michel, C; Neubauer, Daniel; Thompson, Kristin; Mizaikoff, Boris; Frick, Manfred; Dietl, Paul; Wittekindt, Oliver H.

doi:10.1002/phy2.201

Cited by 14 publications

(13 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Our measurements cannot distinguish between these possibilities. These findings are broadly consistent with published results: in two studies CFTR inhibition caused a small decrease (NCI-H441 cells [39]) or no change (primary human type II cells [59]) in fluid absorption implying that CFTR-mediated Cl - absorption is not of primary importance while another study [11] found a small degree of CFTR-mediated Cl- secretion at baseline (~ 5% increase in TEPD following CFTR inhibition). In order to investigate the role of Cl - channels under stimulated conditions, NCI-H441 cells were treated with the cAMP agonist forskolin that can activate both ENaC and CFTR.…”

Section: Discussionsupporting

confidence: 90%

“…Some studies have used charcoal stripped serum instead of FBS which could offer better reproducibility [28, 39]. In our experiments there was little variation in the model characteristics with different batches of FBS.…”

Section: Discussionmentioning

confidence: 79%

“…While primary cell cultures are the gold standard for such models, difficulties in obtaining and long term passaging of primary cells have limited their use. We therefore focussed on the human cell lines NCI-H441 and A549 that have been widely used as models of the alveolar epithelium [18–23, 26–30, 33, 39, 51, 52]. Our study further evaluated these two cell models and focused on several important areas not covered by these studies: comparison with primary cell culture models, effect of culture conditions on model phenotype and function and the individual contribution of transcellular and paracellular pathways to ion transport.…”

Section: Discussionmentioning

confidence: 99%

“…A number of studies have identified ENaC and Na + -K + -ATPase expression in these cell lines as the primary pathway for sodium transport and characterised their function and regulation [26–32]. However, chloride transport is poorly understood: there are conflicting reports on the expression of CFTR in both NCI-H441 cells [30, 33, 34] and A549 cells [34–38], and its role in ion/fluid homeostasis is debated [29, 39]. Furthermore, none of these studies have examined sodium and chloride transport in conjunction and there is no information about the paracellular and transcellular components of net transport.…”

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

2016

View full text Add to dashboard Cite

Robust and reproducible in vitro models are required for investigating the pathways involved in fluid homeostasis in the human alveolar epithelium. We performed functional and phenotypic characterisation of ion transport in the human pulmonary epithelial cell lines NCI-H441 and A549 to determine their similarity to primary human alveolar type II cells. NCI-H441 cells exhibited high expression of junctional proteins ZO-1, and E-cadherin, seal-forming claudin-3, -4, -5 and Na+-K+-ATPase while A549 cells exhibited high expression of pore-forming claudin-2. Consistent with this phenotype NCI-H441, but not A549, cells formed a functional barrier with active ion transport characterised by higher electrical resistance (529 ± 178 Ω cm2 vs 28 ± 4 Ω cm2), lower paracellular permeability ((176 ± 42) ×10−8 cm/s vs (738 ± 190) ×10−8 cm/s) and higher transepithelial potential difference (11.9 ± 4 mV vs 0 mV). Phenotypic and functional properties of NCI-H441 cells were tuned by varying cell seeding density and supplement concentrations. The cells formed a polarised monolayer typical of in vivo epithelium at seeding densities of 100,000 cells per 12-well insert while higher densities resulted in multiple cell layers. Dexamethasone and insulin-transferrin-selenium supplements were required for the development of high levels of electrical resistance, potential difference and expression of claudin-3 and Na+-K+-ATPase. Treatment of NCI-H441 cells with inhibitors and agonists of sodium and chloride channels indicated sodium absorption through ENaC under baseline and forskolin-stimulated conditions. Chloride transport was not sensitive to inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) under either condition. Channels inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) contributed to chloride secretion following forskolin stimulation, but not at baseline. These data precisely define experimental conditions for the application of NCI-H441 cells as a model for investigating ion and water transport in the human alveolar epithelium and also identify the pathways of sodium and chloride transport.

show abstract

Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 79%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

2016

View full text Add to dashboard Cite

show abstract

“…The human lung adenocarcinoma cell line H441 is a well-established model for the investigation of pulmonary sodium transport processes (5,46,68,90). Cells were purchased from the American Type Culture Collection (distributed by LGC standards, Wesel, Germany) in the 65th passage and cultured exactly, as previously described (6).…”

Section: ϫ5mentioning

confidence: 99%

Hydrogen sulfide decreases β-adrenergic agonist-stimulated lung liquid clearance by inhibiting ENaC-mediated transepithelial sodium absorption

Agné

Baldin

Benjamin

et al. 2015

American Journal of Physiology-Regulatory, Integrative and Comp

View full text Add to dashboard Cite

-In pulmonary epithelia, ␤-adrenergic agonists regulate the membrane abundance of the epithelial sodium channel (ENaC) and, thereby, control the rate of transepithelial electrolyte absorption. This is a crucial regulatory mechanism for lung liquid clearance at birth and thereafter. This study investigated the influence of the gaseous signaling molecule hydrogen sulfide (H 2S) on ␤-adrenergic agonistregulated pulmonary sodium and liquid absorption. Application of the H2S-liberating molecule Na2S (50 M) to the alveolar compartment of rat lungs in situ decreased baseline liquid absorption and abrogated the stimulation of liquid absorption by the ␤-adrenergic agonist terbutaline. There was no additional effect of Na2S over that of the ENaC inhibitor amiloride. In electrophysiological Ussing chamber experiments with native lung epithelia (Xenopus laevis), Na 2S inhibited the stimulation of amiloride-sensitive current by terbutaline. ␤-adrenergic agonists generally increase ENaC abundance by cAMP formation and activation of PKA. Activation of this pathway by forskolin and 3-isobutyl-1-methylxanthine increased amiloride-sensitive currents in H441 pulmonary epithelial cells. This effect was inhibited by Na 2S in a dose-dependent manner (5-50 M). Na2S had no effect on cellular ATP concentration, cAMP formation, and activation of PKA. By contrast, Na 2S prevented the cAMP-induced increase in ENaC activity in the apical membrane of H441 cells. H441 cells expressed the H 2S-generating enzymes cystathionine-␤-synthase, cystathionine-␥-lyase, and 3-mercaptopyruvate sulfurtransferase, and they produced H 2S amounts within the employed concentration range. These data demonstrate that H 2S prevents the stimulation of ENaC by cAMP/PKA and, thereby, inhibits the proabsorptive effect of ␤-adrenergic agonists on lung liquid clearance.

show abstract

Role of CFTR in epithelial physiology

Saint‐Criq

Gray

2016

Cell. Mol. Life Sci.

319

291

View full text Add to dashboard Cite

Salt and fluid absorption and secretion are two processes that are fundamental to epithelial function and whole body fluid homeostasis, and as such are tightly regulated in epithelial tissues. The CFTR anion channel plays a major role in regulating both secretion and absorption in a diverse range of epithelial tissues, including the airways, the GI and reproductive tracts, sweat and salivary glands. It is not surprising then that defects in CFTR function are linked to disease, including life-threatening secretory diarrhoeas, such as cholera, as well as the inherited disease, cystic fibrosis (CF), one of the most common life-limiting genetic diseases in Caucasian populations. More recently, CFTR dysfunction has also been implicated in the pathogenesis of acute pancreatitis, chronic obstructive pulmonary disease (COPD), and the hyper-responsiveness in asthma, underscoring its fundamental role in whole body health and disease. CFTR regulates many mechanisms in epithelial physiology, such as maintaining epithelial surface hydration and regulating luminal pH. Indeed, recent studies have identified luminal pH as an important arbiter of epithelial barrier function and innate defence, particularly in the airways and GI tract. In this chapter, we will illustrate the different operational roles of CFTR in epithelial function by describing its characteristics in three different tissues: the airways, the pancreas, and the sweat gland.

show abstract

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl⁻ conductance

Cited by 14 publications

References 30 publications

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

Hydrogen sulfide decreases β-adrenergic agonist-stimulated lung liquid clearance by inhibiting ENaC-mediated transepithelial sodium absorption

Role of CFTR in epithelial physiology

Contact Info

Product

Resources

About

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl− conductance

Cited by 14 publications

References 30 publications

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

An Optimised Human Cell Culture Model for Alveolar Epithelial Transport

Hydrogen sulfide decreases β-adrenergic agonist-stimulated lung liquid clearance by inhibiting ENaC-mediated transepithelial sodium absorption

Role of CFTR in epithelial physiology

Contact Info

Product

Resources

About

Amiloride-sensitive fluid resorption in NCI-H441 lung epithelia depends on an apical Cl⁻ conductance