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Pflügers Archiv - European Journal of Physiology

, Volume 467, Issue 11, pp 2257–2273 | Cite as

Expression and function of the epithelial sodium channel δ-subunit in human respiratory epithelial cells in vitro

  • Elena Schwagerus
  • Svenja Sladek
  • Stephen T. Buckley
  • Natalia Armas-Capote
  • Diego Alvarez de la Rosa
  • Brian J. Harvey
  • Horst Fischer
  • Beate Illek
  • Hanno Huwer
  • Nicole Schneider-Daum
  • Claus-Michael Lehr
  • Carsten Ehrhardt
Ion channels, receptors and transporters

Abstract

Using human airway epithelial cell lines (i.e. NCI-H441 and Calu-3) as well as human alveolar epithelial type I-like (ATI) cells in primary culture, we studied the contribution of the epithelial sodium channel δ-subunit (δ-ENaC) to transepithelial sodium transport in human lung in vitro. Endogenous δ-ENaC protein was present in all three cell types tested; however, protein abundance was low, and no expression was detected in the apical cell membrane of these cells. Similarly, known modulators of δ-ENaC activity, such as capsazepine and icilin (activators) and Evans blue (inhibitor), did not show effects on short-circuit current (I SC), suggesting that δ-ENaC is not involved in the modulation of transcellular sodium absorption in NCI-H441 cell monolayers. Over-expression of δ-ENaC in NCI-H441 cells resulted in detectable protein expression in the apical cell membrane, as well as capsazepine and icilin-stimulated increases in I SC that were effectively blocked by Evans blue and that were consistent with δ-ENaC activation and inhibition, respectively. Consequently, these observations suggest that δ-ENaC expression is low in NCI-H441, Calu-3, and ATI cells and does not contribute to transepithelial sodium absorption.

Keywords

Airway epithelium Alveolar epithelium ENaC Ussing chamber Sodium absorption 

Notes

Acknowledgements

The authors thank M. Hittinger for his skilful technical support during the primary cell isolation. This work was supported by an IRCSET Government of Ireland Postgraduate Scholarship in Science, Engineering and Technology (ES, STB) and the COST Actions BM1201 (CE) and BM1301 (DA and BJH). HF and BI were supported by National Heart, Lung, and Blood Institute Grant HL86323, Cystic Fibrosis Research Inc. and Elizabeth Nash Foundation.

Supplementary material

424_2015_1693_MOESM1_ESM.docx (483 kb)
ESM 1 (DOCX 482 kb)

References

  1. 1.
    Tarran R (2004) Regulation of airway surface liquid volume and mucus transport by active ion transport. Proc Am Thorac Soc 1(1):42–6CrossRefPubMedGoogle Scholar
  2. 2.
    Bastacky J, Lee CY, Goerke J, Koushafar H, Yager D, Kenaga L, Speed TP, Chen Y, Clements JA (1995) Alveolar lining layer is thin and continuous: low-temperature scanning electron microscopy of rat lung. J Appl Physiol 79(5):1615–1628PubMedGoogle Scholar
  3. 3.
    Dobbs LG, Johnson MD (2007) Alveolar epithelial transport in the adult lung. Respir Physiol Neurobiol 159(3):283–300. doi: 10.1016/j.resp.2007.06.011 CrossRefPubMedGoogle Scholar
  4. 4.
    Brouard M, Casado M, Djelidi S, Barrandon Y, Farman N (1999) Epithelial sodium channel in human epidermal keratinocytes: expression of its subunits and relation to sodium transport and differentiation. J Cell Sci 112(19):3343–3352PubMedGoogle Scholar
  5. 5.
    Canessa CM, Horisberger JD, Rossier BC (1993) Epithelial sodium channel related to proteins involved in neurodegeneration. Nature 361(6411):467–470. doi: 10.1038/361467a0 CrossRefPubMedGoogle Scholar
  6. 6.
    Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367(6462):463–467. doi: 10.1038/367463a0 CrossRefPubMedGoogle Scholar
  7. 7.
    Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier BC (1994) Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol 127(6):1907–1921CrossRefPubMedGoogle Scholar
  8. 8.
    Johnson MD, Widdicombe JH, Allen L, Barbry P, Dobbs LG (2002) Alveolar epithelial type I cells contain transport proteins and transport sodium, supporting an active role for type I cells in regulation of lung liquid homeostasis. Proc Natl Acad Sci U S A 99(4):1966–1971. doi: 10.1073/pnas.042689399 PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Waldmann R, Champigny G, Bassilana F, Voilley N, Lazdunski M (1995) Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. J Biol Chem 270(46):27411–27414. doi: 10.1074/jbc.270.46.27411 CrossRefPubMedGoogle Scholar
  10. 10.
    Yamamura H, Ugawa S, Ueda T, Nagao M, Shimada S (2004) Protons activate the delta-subunit of the epithelial Na+ channel in humans. J Biol Chem 279(13):12529–12534. doi: 10.1074/jbc.M400274200 CrossRefPubMedGoogle Scholar
  11. 11.
    Giraldez T, Afonso-Oramas D, Cruz-Muros I, Garcia-Marin V, Pagel P, González-Hernández T, Alvarez de la Rosa D (2007) Cloning and functional expression of a new epithelial sodium channel delta subunit isoform differentially expressed in neurons of the human and monkey telencephalon. J Neurochem 102(4):1304–1315. doi: 10.1111/j.1471-4159.2007.04622.x CrossRefPubMedGoogle Scholar
  12. 12.
    Ji HL, Zhao RZ, Chen ZX, Shetty S, Idell S, Matalon S (2012) δ-ENaC: a novel divergent amiloride-inhibitable sodium channel. Am J Physiol Lung Cell Mol Physiol 303(12):L1013–L1026. doi: 10.1152/ajplung.00206.2012 PubMedCentralCrossRefPubMedGoogle Scholar
  13. 13.
    Hummler E, Barker P, Gatzy J, Beermann F, Verdumo C, Schmidt A, Boucher R, Rossier BC (1996) Early death due to defective neonatal lung liquid clearance in alpha-ENaC-deficient mice. Nat Genet 12(3):325–328. doi: 10.1038/ng0396-325 CrossRefPubMedGoogle Scholar
  14. 14.
    Li T, Folkesson HG (2006) RNA interference for alpha-ENaC inhibits rat lung fluid absorption in vivo. Am J Physiol Lung Cell Mol Physiol 290(4):L649–L660. doi: 10.1152/ajplung.00205.2005 CrossRefPubMedGoogle Scholar
  15. 15.
    Barker PM, Nguyen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, Boucher RC, Koller B (1998) Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism. J Clin Invest 102(8):1634–1640. doi: 10.1172/JCI3971 PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Elias N, Rafii B, Rahman M, Otulakowski G, Cutz E, O'Brodovich H (2007) The role of alpha-, beta-, and gamma-ENaC subunits in distal lung epithelial fluid absorption induced by pulmonary edema fluid. Am J Physiol Lung Cell Mol Physiol 293(3):L537–L545. doi: 10.1152/ajplung.00373.2006 CrossRefPubMedGoogle Scholar
  17. 17.
    McDonald FJ, Yang B, Hrstka RF, Drummond HA, Tarr DE, McCray PB Jr, Stokes JB, Welsh MJ, Williamson RA (1999) Disruption of the beta subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with a pseudohypoaldosteronism phenotype. Proc Natl Acad Sci U S A 96(4):1727–1731. doi: 10.1073/pnas.96.4.1727 PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Ji HL, Su XF, Kedar S, Li J, Barbry P, Smith PR, Matalon S, Benos DJ (2006) Delta-subunit confers novel biophysical features to alpha beta gamma-human epithelial sodium channel (ENaC) via a physical interaction. J Biol Chem 281(12):8233–8241. doi: 10.1074/jbc.M512293200 CrossRefPubMedGoogle Scholar
  19. 19.
    Nie HG, Chen L, Han DY, Li J, Song WF, Wei SP, Fang XH, Gu X, Matalon S, Ji HL (2009) Regulation of epithelial sodium channels by cGMP/PKGII. J Physiol 587(11):2663–2676. doi: 10.1113/jphysiol.2009.170324 PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Zhao RZ, Nie HG, Su XF, Han DY, Lee A, Huang Y, Chang Y, Matalon S, Ji HL (2012) Characterization of a novel splice variant of δ ENaC subunit in human lungs. Am J Physiol Lung Cell Mol Physiol 302(12):L1262–L1272. doi: 10.1152/ajplung.00331.2011 PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Bangel-Ruland N, Sobczak K, Christmann T, Kentrup D, Langhorst H, Kusche-Vihrog K, Weber WM (2010) Characterization of the epithelial sodium channel delta-subunit in human nasal epithelium. Am J Respir Cell Mol Biol 42(4):498–505. doi: 10.1165/rcmb.2009-0053OC CrossRefPubMedGoogle Scholar
  22. 22.
    Giraldez T, Rojas P, Jou J, Flores C, Alvarez de la Rosa D (2012) The epithelial sodium channel δ-subunit: new notes for an old song. Am J Physiol Renal Physiol 303(3):F328–F338. doi: 10.1152/ajprenal.00116.2012 CrossRefPubMedGoogle Scholar
  23. 23.
    Yamamura H, Ugawa S, Ueda T, Nagao M, Shimada S (2004) Capsazepine is a novel activator of the delta subunit of the human epithelial Na+ channel. J Biol Chem 279(43):44483–44489. doi: 10.1074/jbc.M408929200 CrossRefPubMedGoogle Scholar
  24. 24.
    Yamamura H, Ugawa S, Ueda T, Nagao M, Shimada S (2005) Icilin activates the delta-subunit of the human epithelial Na+ channel. Mol Pharmacol 68(4):1142–1147. doi: 10.1124/mol.104.010850 CrossRefPubMedGoogle Scholar
  25. 25.
    Yamamura H, Ugawa S, Ueda T, Shimada S (2005) Evans blue is a specific antagonist of the human epithelial Na+ channel delta-subunit. J Pharmacol Exp Ther 315(2):965–969. doi: 10.1124/jpet.105.092775 CrossRefPubMedGoogle Scholar
  26. 26.
    Salomon JJ, Muchitsch VE, Gausterer JC, Schwagerus E, Huwer H, Daum N, Lehr CM, Ehrhardt C (2014) The cell line NCl-H441 is a useful in vitro model for transport studies of human distal lung epithelial barrier. Mol Pharm 11(3):995–1006. doi: 10.1021/mp4006535 CrossRefPubMedGoogle Scholar
  27. 27.
    Ehrhardt C, Kim KJ, Lehr CM (2005) Isolation and culture of human alveolar epithelial cells. Methods Mol Med 107:207–216PubMedGoogle Scholar
  28. 28.
    Demling N, Ehrhardt C, Kasper M, Laue M, Knels L, Rieber EP (2006) Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells. Cell Tissue Res 323(3):475–488. doi: 10.1007/s00441-005-0069-0 CrossRefPubMedGoogle Scholar
  29. 29.
    Wesch D, Althaus M, Miranda P, Cruz-Muros I, Fronius M, González-Hernández T, Clauss WG, Alvarez de la Rosa D, Giraldez T (2012) Differential N termini in epithelial Na+ channel δ-subunit isoforms modulate channel trafficking to the membrane. Am J Physiol Cell Physiol 302(6):C868–C879. doi: 10.1152/ajpcell.00255.2011 CrossRefPubMedGoogle Scholar
  30. 30.
    Haerteis S, Krueger B, Korbmacher C, Rauh R (2009) The delta-subunit of the epithelial sodium channel (ENaC) enhances channel activity and alters proteolytic ENaC activation. J Biol Chem 284(42):29024–29040. doi: 10.1074/jbc.M109.018945 PubMedCentralCrossRefPubMedGoogle Scholar
  31. 31.
    Schönberger M, Althaus M, Fronius M, Clauss W, Trauner D (2014) Controlling epithelial sodium channels with light using photoswitchable amilorides. Nat Chem 6(8):712–719. doi: 10.1038/nchem.2004 PubMedGoogle Scholar
  32. 32.
    Yamamura H, Ugawa S, Ueda T, Nagao M, Shimada S (2008) Epithelial Na+ channel delta subunit mediates acid-induced ATP release in the human skin. Biochem Biophys Res Commun 373(1):155–158. doi: 10.1016/j.bbrc.2008.06.008 CrossRefPubMedGoogle Scholar
  33. 33.
    Hernández-González EO, Sosnik J, Edwards J, Acevedo JJ, Mendoza-Lujambio I, López-González I, Demarco I, Wertheimer E, Darszon A, Visconti PE (2006) Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm. J Biol Chem 281(9):5623–5633. doi: 10.1074/jbc.M508172200 CrossRefPubMedGoogle Scholar
  34. 34.
    Nie HG, Tucker T, Su XF, Na T, Peng JB, Smith PR, Idell S, Ji HL (2009) Expression and regulation of epithelial Na+ channels by nucleotides in pleural mesothelial cells. Am J Respir Cell Mol Biol 40(5):543–554. doi: 10.1165/rcmb.2008-0166OC PubMedCentralCrossRefPubMedGoogle Scholar
  35. 35.
    Kapoor N, Bartoszewski R, Qadri YJ, Bebok Z, Bubien JK, Fuller CM, Benos DJ (2009) Knockdown of ASIC1 and epithelial sodium channel subunits inhibits glioblastoma whole cell current and cell migration. J Biol Chem 284(36):24526–24541. doi: 10.1074/jbc.M109.037390 PubMedCentralCrossRefPubMedGoogle Scholar
  36. 36.
    Chang T, Ke Y, Ly K (2011) McDonald FJ (2011) COMMD1 regulates the delta epithelial sodium channel (δENaC) through trafficking and ubiquitination. Biochem Biophys Res Commun 411(3):506–511. doi: 10.1016/j.bbrc.2011.06.149 CrossRefPubMedGoogle Scholar
  37. 37.
    Behrendt HJ, Germann T, Gillen C, Hatt H, Jostock R (2004) Characterization of the mouse cold-menthol receptor TRPM8 and vanilloid receptor type-1 VR1 using a fluorometric imaging plate reader (FLIPR) assay. Br J Pharmacol 141(4):737–745. doi: 10.1038/sj.bjp.0705652 PubMedCentralCrossRefPubMedGoogle Scholar
  38. 38.
    Bevan S, Hothi S, Hughes G, James IF, Rang HP, Shah K, Walpole CS, Yeats JC (1992) Capsazepine: a competitive antagonist of the sensory neurone excitant capsaicin. Br J Pharmacol 107(2):544–552PubMedCentralCrossRefPubMedGoogle Scholar
  39. 39.
    Andersson DA, Chase HW, Bevan S (2004) TRPM8 activation by menthol, icilin and cold is differentially modulated by intracellular pH. J Neurosci 24(23):5364–5369. doi: 10.1523/jneurosci. 0890-04.2004 CrossRefPubMedGoogle Scholar
  40. 40.
    Kuenzi FM, Dale N (1996) Effect of capsaicin and analogues on potassium and calcium currents and vanilloid receptors in Xenopus embryo spinal neurones. Br J Pharmacol 119(1):81–90PubMedCentralCrossRefPubMedGoogle Scholar
  41. 41.
    Lee SY, Maniak PJ, Ingbar DH, O'Grady SM (2003) Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane. Am J Physiol Cell Physiol 284(6):C1614–C1624. doi: 10.1152/ajpcell.00429.2002 CrossRefPubMedGoogle Scholar
  42. 42.
    Bultmann R, Starke K (1993) Evans blue blocks P2X-purinoceptors in rat vas deferens. Naunyn-Schmiedeberg's Arch Pharmacol 348(6):684–687CrossRefGoogle Scholar
  43. 43.
    Hollywood MA, Cotton KD, McHale NG, Thornbury KD (1998) Enhancement of Ca2+-dependent outward current in sheep bladder myocytes by Evans blue dye. Pflugers Arch 435(5):631–636. doi: 10.1007/s004240050563 CrossRefPubMedGoogle Scholar
  44. 44.
    Manzanares D, Gonzalez C, Ivonnet P, Chen RS, Valencia-Gattas M, Conner GE, Larsson HP, Salathe M (2011) Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume. J Biol Chem 286(22):19830–19839. doi: 10.1074/jbc.M110.185074 PubMedCentralCrossRefPubMedGoogle Scholar
  45. 45.
    Gottardi CJ, Dunbar LA, Caplan MJ (1995) Biotinylation and assessment of membrane polarity: caveats and methodological concerns. Am J Physiol 268(2):F285–F295PubMedGoogle Scholar
  46. 46.
    Eaton DC, Chen J, Ramosevac S, Matalon S, Jain L (2004) Regulation of Na+ channels in lung alveolar type II epithelial cells. Proc Am Thorac Soc 1(1):10–16CrossRefPubMedGoogle Scholar
  47. 47.
    Albert AP, Woollhead AM, Mace OJ, Baines DL (2008) AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers. Am J Physiol Lung Cell Mol Physiol 295(5):L837–L848. doi: 10.1152/ajplung.90353.2008 PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Elena Schwagerus
    • 1
  • Svenja Sladek
    • 1
  • Stephen T. Buckley
    • 1
  • Natalia Armas-Capote
    • 2
  • Diego Alvarez de la Rosa
    • 2
  • Brian J. Harvey
    • 3
  • Horst Fischer
    • 4
  • Beate Illek
    • 4
  • Hanno Huwer
    • 5
  • Nicole Schneider-Daum
    • 6
  • Claus-Michael Lehr
    • 6
  • Carsten Ehrhardt
    • 1
  1. 1.School of Pharmacy and Pharmaceutical Sciences and Trinity Biomedical Sciences Institute, Trinity College DublinPanoz InstituteDublin 2Ireland
  2. 2.Departamento de Fisiología and Center for Biomedical Research of the Canary Islands (CIBICAN)Universidad de La LagunaLa LagunaSpain
  3. 3.Department of Molecular Medicine, Royal College of Surgeons in IrelandBeaumont HospitalDublin 9Ireland
  4. 4.Children’s Hospital Oakland Research Institute (CHORI)OaklandUSA
  5. 5.Department of Cardiothoracic SurgeryVölklingen Heart CentreVölklingenGermany
  6. 6.Helmholtz Institute for Pharmaceutical Research SaarlandSaarbrückenGermany

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