Cell and Tissue Research

, Volume 328, Issue 1, pp 77–84 | Cite as

P-glycoprotein (MDR1) functional activity in human alveolar epithelial cell monolayers

  • Sibylle Endter
  • Ulrich Becker
  • Nicole Daum
  • Hanno Huwer
  • Claus-Michael Lehr
  • Mark Gumbleton
  • Carsten EhrhardtEmail author
Regular Article


The distribution of the P-glycoprotein (P-gp/MDR1) efflux transporter at mucosal barriers has defined it as a functionally important element in limiting drug absorption into the systemic circulation. However, little is known about the distribution and functionality of P-gp/MDR1 in the human lung. Here, the presence of P-gp/MDR1 was investigated immunohistochemically in distal human lung tissue and at mRNA and protein levels in human alveolar epithelial cells (hAEpC) in primary culture. We studied the presence and activity of P-gp/MDR1 in hAEpC monolayers by Western blotting, by immunofluorescence microscopy and by conducting bi-directional transport studies employing a P-gp substrate (rhodamine 123) with and without a P-gp inhibitor (verapamil). The flux of fluorescein sodium was also examined as a paracellular transport marker. Alveolar tissue specimens showed P-gp localised at the luminal membranes of type I pneumocytes. Reverse transcription-polymerase chain reaction revealed the presence of mRNA encoding for P-gp/MDR1 in freshly isolated (i.e. type II) hAEpC and in monolayers of hAEpC cultured for 8 days (i.e. type I-like morphology). At the protein level, P-gp could be detected in hAEpC monolayers after 8 days in culture but not in freshly isolated type II pneumocytes. The flux of rhodamine 123 across hAEpC monolayers on day 8 in culture exhibited net secretion, which disappeared in the presence of verapamil. Fluorescein sodium fluxes showed no distinct directionality. Our findings indicate that P-gp is functionally active in the human alveolar airspace and that hAEpC monolayers might provide a suitable in vitro model for studying P-gp function mechanistically in the distal human lung.


ABC-transporters Cell culture Alveolar epithelium Pulmonary drug delivery Multi-drug resistance Alveolar epithelial cells Human 



The authors thank Ms. Susanne Kossek for her skilful technical assistance, Dr. Paul Buckland (Department of Psychological Medicine, Cardiff University) for his help in the PCR studies, and Prof. Kwang-Jin Kim (University of Southern California) for critical reading of the manuscript.


  1. Abulrob AG, Gumbleton M (1999) Transport of phosphatidylcholine in MDR3-negative epithelial cell lines via drug-induced MDR1 P-glycoprotein. Biochem Biophys Res Commun 262:121–126PubMedCrossRefGoogle Scholar
  2. Axiotis CA, Bani D, Bianchi S, Pioli P, Tanini A, Brandi ML (1995) P-glycoprotein is expressed in parathyroid epithelium and is regulated by calcium. Calcif Tissue Int 56:170–174PubMedCrossRefGoogle Scholar
  3. Bosch I, Dunussi-Joannopoulos K, Wu RL, Furlong ST, Croop J (1997) Phosphatidylcholine and phosphatidylethanolamine behave as substrates of the human MDR1 P-glycoprotein. Biochemistry 36:5685–5694PubMedCrossRefGoogle Scholar
  4. Campbell L, Abulrob AN, Kandalaft LE, Plummer S, Hollins AJ, Gibbs A, Gumbleton M (2003) Constitutive expression of P-glycoprotein in normal lung alveolar epithelium and functionality in primary alveolar epithelial cultures. J Pharmacol Exp Ther 304:441–452PubMedCrossRefGoogle Scholar
  5. Chen J, Chen Z, Narasaraju T, Jin N, Liu L (2004) Isolation of highly pure alveolar epithelial type I and type II cells from rat lungs. Lab Invest 84:727–735 (Erratum in: Lab Invest 85:1181)PubMedCrossRefGoogle Scholar
  6. Cordon-Cardo C, O’Brien LP, Boccia J, Casals D, Bertino JR, Melamed MR (1990) Expression of the multidrug resistance gene product (P-glycoprotein) in human normal and tumor tissues. J Histochem Cytochem 38:1277–1287PubMedGoogle Scholar
  7. Demeule M, Jodoin J, Gingras D, Beliveau R (2000) P-glycoprotein is localized in caveolae in resistant cells and in brain capillaries. FEBS Lett 466:219–224PubMedCrossRefGoogle Scholar
  8. Demeule M, Shedid D, Beaulieu E, Del Maestro RF, Moghrabi A, Ghosn PB, Moumdjian R, Berthelet F, Beliveau R (2001) Expression of multidrug-resistance P-glycoprotein (MDR1) in human brain tumors. Int J Cancer 93:62–66PubMedCrossRefGoogle Scholar
  9. 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 323:475–488PubMedCrossRefGoogle Scholar
  10. Dobbs LG, Gonzales RF (2002) Isolation and culture of pulmonary alveolar epithelial type II cells. In: Freshney RI, Freshney M (eds) Culture of epithelial cells, 2nd edn. Wiley, New York, pp 277–301Google Scholar
  11. Ehrhardt C, Kneuer C, Fiegel J, Hanes J, Schaefer UF, Kim KJ, Lehr CM (2002) Influence of apical fluid volume on the development of functional intercellular junctions in the human epithelial cell line 16HBE14o-: implications for the use of this cell line as an in vitro model for bronchial drug absorption studies. Cell Tissue Res 308:391–400PubMedCrossRefGoogle Scholar
  12. Ehrhardt C, Kim KJ, Lehr CM (2005) Isolation and culture of human alveolar epithelial cells. Methods Mol Med 107:207–216PubMedGoogle Scholar
  13. Ehrhardt C, Collnot EM, Baldes C, Becker U, Laue M, Kim KJ, Lehr CM (2006) Towards an in vitro model of cystic fibrosis small airway epithelium: characterisation of the human bronchial epithelial cell line CFBE41o-. Cell Tissue Res 323:405–415PubMedCrossRefGoogle Scholar
  14. Elbert KJ, Schafer UF, Schafers HJ, Kim KJ, Lee VH, Lehr CM (1999) Monolayers of human alveolar epithelial cells in primary culture for pulmonary absorption and transport studies. Pharm Res 16:601–608PubMedCrossRefGoogle Scholar
  15. Florea BI, Cassara ML, Junginger HE, Borchard G (2003) Drug transport and metabolism characteristics of the human airway epithelial cell line Calu-3. J Control Release 87:131–138PubMedCrossRefGoogle Scholar
  16. Forbes B, Ehrhardt C (2005) Human respiratory epithelial cell culture for drug delivery applications. Eur J Pharm Biopharm 60:193–205PubMedCrossRefGoogle Scholar
  17. Foster KA, Oster CG, Mayer MM, Avery ML, Audus KL (1998) Characterization of the A549 cell line as a type II pulmonary epithelial cell model for drug metabolism. Exp Cell Res 243:359–366PubMedCrossRefGoogle Scholar
  18. Fuchs S, Hollins AJ, Laue M, Schaefer UF, Roemer K, Gumbleton M, Lehr CM (2003) Differentiation of human alveolar epithelial cells in primary culture: morphological characterization and synthesis of caveolin-1 and surfactant protein-C. Cell Tissue Res 311:31–45PubMedCrossRefGoogle Scholar
  19. Greiner B, Eichelbaum M, Fritz P, Kreichgauer HP, Richter O von, Zundler J, Kroemer HK (1999) The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin. J Clin Invest 104:147–153PubMedCrossRefGoogle Scholar
  20. Gruenert DC, Finkbeiner WE, Widdicombe JH (1995) Culture and transformation of human airway epithelial cells. Am J Physiol 268:L347–L360PubMedGoogle Scholar
  21. Hamilton KO, Backstrom G, Yazdanian MA, Audus KL (2001) P-glycoprotein efflux pump expression and activity in Calu-3 cells. J Pharm Sci 90:647–658PubMedCrossRefGoogle Scholar
  22. Higgins CF (1995) Volume-activated chloride currents associated with the multidrug resistance P-glycoprotein. J Physiol (Lond) 482:31S–36SGoogle Scholar
  23. Kennedy BG, Mangini NJ (2002) P-glycoprotein expression in human retinal pigment epithelium. Mol Vis 8:422–430PubMedGoogle Scholar
  24. Kim KJ, Borok Z, Crandall ED (2001) A useful in vitro model for transport studies of alveolar epithelial barrier. Pharm Res 18:253–255PubMedCrossRefGoogle Scholar
  25. Lechapt-Zalcman E, Hurbain I, Lacave R, Commo F, Urban T, Antoine M, Milleron B, Bernaudin JF (1997) MDR1-Pgp 170 expression in human bronchus. Eur Respir J 10:1837–1843PubMedCrossRefGoogle Scholar
  26. Manford F, Tronde A, Jeppsson AB, Patel N, Johansson F, Forbes B (2005) Drug permeability in 16HBE14o- airway cell layers correlates with absorption from the isolated perfused rat lung. Eur J Pharm Sci 26:414–420PubMedCrossRefGoogle Scholar
  27. Mi Q, Cui B, Silva GL, Lantvit D, Lim E, Chai H, You M, Hollingshead MG, Mayo JG, Kinghorn AD, Pezzuto JM, Pervilleine A (2001) A novel tropane alkaloid that reverses the multidrug-resistance phenotype. Cancer Res 261:4030–4037Google Scholar
  28. Patton JS, Fishburn CS, Weers JG (2004) The lungs as a portal of entry for systemic drug delivery. Proc Am Thorac Soc 1:338–344PubMedCrossRefGoogle Scholar
  29. Steimer A, Haltner E, Lehr CM (2005) Cell culture models of the respiratory tract relevant to pulmonary drug delivery. J Aerosol Med 18:137–182PubMedCrossRefGoogle Scholar
  30. Stouch TR, Gudmundsson O (2002) Progress in understanding the structure-activity relationships of P-glycoprotein. Adv Drug Deliv Rev 54:315–328PubMedCrossRefGoogle Scholar
  31. Wichert P von, Seifart C (2005) The lung, an organ for absorption? Respiration 72:552–558CrossRefGoogle Scholar
  32. Wise J, Lechner JF (2002) Normal human bronchial epithelial cell culture. In: Freshney RI, Freshney M (eds) Culture of epithelial cells, 2nd edn. Wiley, New York, pp 257–276Google Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Sibylle Endter
    • 1
    • 2
  • Ulrich Becker
    • 3
  • Nicole Daum
    • 3
  • Hanno Huwer
    • 4
  • Claus-Michael Lehr
    • 3
  • Mark Gumbleton
    • 2
  • Carsten Ehrhardt
    • 1
    • 3
    Email author
  1. 1.School of Pharmacy and Pharmaceutical SciencesTrinity College DublinDublin 2Ireland
  2. 2.Welsh School of PharmacyCardiff UniversityCardiffUK
  3. 3.Biopharmaceutics and Pharmaceutical TechnologySaarland UniversitySaarbrückenGermany
  4. 4.Department of Cardiothoracic SurgeryVölklingen Heart CentreVölklingenGermany

Personalised recommendations