The Cell Line NCl-H441 Is A Useful In Vitro Model For Transport Studies ...

Full text links CiteDisplay options Display options Format AbstractPubMedPMID

Abstract

The lack of a well characterized, continuously growing in vitro model of human distal lung epithelial phenotype constitutes a serious limitation in the area of inhalation biopharmaceutics, particularly in the context of transepithelial transport studies. Here, we investigated if a human lung adenocarcinoma cell line, NCl-H441, has potential to serve as an in vitro model of human distal lung epithelium. The development of barrier properties was studied by immunocytochemistry (ICC) against the junction proteins zonula occludens protein 1 (ZO-1) and E-cadherin and measurement of transepithelial electrical resistance (TEER). Moreover, transport studies with the paracellular marker compounds fluorescein sodium and fluorescein isothiocyanate (FITC)-labeled dextrans of molecular weights ranging from 4 to 70 kDa were carried out. The expression of P-glycoprotein (P-gp; ABCB1) and organic cation transporters (OCT/Ns; SLC22A1-A5) was investigated by ICC and immunoblot. P-gp function was assessed by monolayer release and bidirectional transport studies using rhodamine 123 (Rh123) and the inhibitors verapamil and LY335979. Uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide (ASP(+)) was measured, in order to assess organic cation transporter function in vitro. Furthermore, the inhibitory potential of several organic cations on ASP(+) uptake was studied. NCl-H441 cells, when grown under liquid-covered conditions, formed confluent, electrically tight monolayers with peak TEER values of approximately 1000 Ω·cm(2), after 8-12 days in culture. These monolayers were able to differentiate paracellularly transported substrates according to their molecular weight. Presence of P-gp, OCT1, OCT2, OCT3, OCTN1, and OCTN2 was confirmed by Western blot and ICC and was similar to data from freshly isolated human alveolar epithelial cells in primary culture. Rh123 release from NCI-H441 monolayers was time-dependent and showed low, albeit significant attenuation by both inhibitors. In transport studies, Rh123 exhibited net secretion, which again was inhibitable by bona fide P-gp modulators. The uptake of ASP(+) was time- and temperature-dependent with Km = 881.2 ± 195.3 μM and Vmax = 2.07 ± 0.26 nmol/min/mg protein. TEA, amantadine, quinidine, and verapamil significantly inhibited ASP(+) uptake into NCl-H441 cells, whereas the effect of d- and l-carnitine and ergothioneine, two OCTN substrates, was less pronounced. NCl-H441 cells are the first cell line of human distal lung epithelial origin with the ability to form monolayers with appreciable barrier properties. Moreover, drug transporter expression and activity in NCl-H441 cells was consistent with what has been reported for human alveolar epithelial cells in primary culture.

Keywords: P-glycoprotein; absorption; drug transporters; inhalation biopharmaceutics; organic cation transporters; pulmonary drug disposition.

PubMed Disclaimer

Publication types

• Research Support, Non-U.S. Gov't Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search

MeSH terms

• ATP Binding Cassette Transporter, Subfamily B / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Adenocarcinoma / metabolism Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Adenocarcinoma / pathology Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Biological Transport Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Blotting, Western Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Cells, Cultured Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Epithelial Cells / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Humans Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Immunoenzyme Techniques Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• In Vitro Techniques Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Lung / cytology Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Lung / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Lung Neoplasms / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Lung Neoplasms / pathology Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Models, Biological* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Organic Cation Transport Proteins / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Pulmonary Alveoli / cytology Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Pulmonary Alveoli / metabolism* Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Rhodamine 123 / metabolism Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Tight Junctions / metabolism Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search

Substances

• ATP Binding Cassette Transporter, Subfamily B Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Organic Cation Transport Proteins Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search
• Rhodamine 123 Actions
  • Search in PubMed
  • Search in MeSH
  • Add to Search

LinkOut - more resources

• Full Text Sources

  • American Chemical Society

• Other Literature Sources

  • The Lens - Patent Citations Database
  • scite Smart Citations

• Medical

  • MedlinePlus Health Information

• Research Materials

  • NCI CPTC Antibody Characterization Program

• Miscellaneous

  • NCI CPTAC Assay Portal