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Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2

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ABSTRACT

Purpose

Studies were conducted in primary cultured rat alveolar epithelial cell monolayers to characterize peptide transporter expression and function.

Methods

Freshly isolated rat lung alveolar epithelial cells were purified and cultured on permeable support with and without keratinocyte growth factor (KGF). Messenger RNA and protein expression of Pept1 and Pept2 in alveolar epithelial type I- and type II-like cell monolayers (±KGF, resp.) were examined by RT-PCR and Western blotting. 3H–Glycyl-sarcosine (3H–gly-sar) transmonolayer flux and intracellular accumulation were evaluated in both cell types.

Results

RT-PCR showed expression of Pept2, but not Pept1, mRNA in both cell types. Western blot analysis revealed presence of Pept2 protein in type II-like cells, and less in type I-like cells. Bi-directional transmonolayer 3H–gly-sar flux lacked asymmetry in transport in both types of cells. Uptake of 3H–gly-sar from apical fluid of type II-like cells was 7-fold greater than that from basolateral fluid, while no significant differences were observed from apical vs. basolateral fluid of type I-like cells.

Conclusions

This study confirms the absence of Pept1 from rat lung alveolar epithelium in vitro. Functional Pept2 expression in type II-like cell monolayers suggests its involvement in oligopeptide lung disposition, and offers rationale for therapeutic development of di/tripeptides, peptidomimetics employing pulmonary drug delivery.

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Abbreviations

A549:

Human alveolar epithelial adenocarcinoma cell line

BEAS-2B or 16HBE14o-:

Immortalized human bronchial epithelial cell lines

BSA:

Bovine serum albumin

Caco-2:

Human colorectal adenocarcinoma cell line

Calu-3:

Human lung submucosal glandular epithelial adenocarcinoma cell line

DMEM/F12:

Dulbecco’s minimum essential and Ham’s F-12 media

HEPES:

Hydroxyethylpiperazine ethane sulfonate

KGF:

Keratinocyte growth factor

MES:

Morpholinoethanesulfonic acid

NCI-H441:

Human papillary adenocarcinoma cell line

PD:

Potential difference

Pept1/2:

Peptide transporter 1/2

rGAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

SDS:

Sodium dodecylsulfate

SDS-PAGE:

SDS polyacrylamide gel electrophoresis

SLC15 A1/A2:

Also known as solute carrier family 15 member

TEER:

Transepithelial electrical resistance

REFERENCES

  1. Siekmeier R, Scheuch G. Systemic treatment by inhalation of macromolecules--principles, problems, and examples. J Physiol Pharmacol. 2008;59(Suppl 6):53–79.

    PubMed  Google Scholar 

  2. Groneberg DA, Fischer A, Chung KF, Daniel H. Molecular mechanisms of pulmonary peptidomimetic drug and peptide transport. Am J Respir Cell Mol Biol. 2004;30(3):251–60.

    Article  CAS  Google Scholar 

  3. Bur M, Huwer H, Lehr CM, Hagen N, Guldbrandt M, Kim KJ, et al. Assessment of transport rates of proteins and peptides across primary human alveolar epithelial cell monolayers. Eur J Pharm Sci. 2006;28(3):196–203.

    Article  CAS  Google Scholar 

  4. Patton JS, Brain JD, Davies LA, Fiegel J, Gumbleton M, Kim KJ, et al. The particle has landed--characterizing the fate of inhaled pharmaceuticals. J Aerosol Med Pulm Drug Deliv. 2010;23(Suppl 2):S71–87.

    Article  CAS  Google Scholar 

  5. Baginski L, Tachon G, Falson F, Patton JS, Bakowsky U, Ehrhardt C. Reverse transcription polymerase chain reaction (RT-PCR) analysis of proteolytic enzymes in cultures of human respiratory epithelial cells. J Aerosol Med Pulm Drug Deliv. 2011;24(2):89–101.

    Article  CAS  Google Scholar 

  6. Morimoto K, Yamahara H, Lee VH, Kim KJ. Dipeptide transport across rat alveolar epithelial cell monolayers. Pharm Res. 1993;10(11):1668–74.

    Article  CAS  Google Scholar 

  7. Gumbleton M, Al-Jayyoussi G, Crandon-Lewis A, Francombe D, Kreitmeyr K, Morris CJ, et al. Spatial expression and functionality of drug transporters in the intact lung: objectives for further research. Adv Drug Deliv Rev. 2011;63(1–2):110–8.

    Article  CAS  Google Scholar 

  8. Nickel S, Clerkin CG, Selo MA, Ehrhardt C. Transport mechanisms at the pulmonary mucosa: implications for drug delivery. Expert Opin Drug Deliv. 2016;13(5):667–90.

    Article  CAS  Google Scholar 

  9. Boll M, Herget M, Wagener M, Weber WM, Markovich D, Biber J, et al. Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter. Proc Natl Acad Sci U S A. 1996;93(1):284–9.

    Article  CAS  Google Scholar 

  10. Doring F, Walter J, Will J, Focking M, Boll M, Amasheh S, et al. Delta-aminolevulinic acid transport by intestinal and renal peptide transporters and its physiological and clinical implications. J Clin Invest. 1998;101(12):2761–7.

    Article  CAS  Google Scholar 

  11. Groneberg DA, Eynott PR, Doring F, Dinh QT, Oates T, Barnes PJ, et al. Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung. Thorax. 2002;57(1):55–60.

    Article  CAS  Google Scholar 

  12. Groneberg DA, Nickolaus M, Springer J, Doring F, Daniel H, Fischer A. Localization of the peptide transporter PEPT2 in the lung: implications for pulmonary oligopeptide uptake. Am J Pathol. 2001;158(2):707–14.

    Article  CAS  Google Scholar 

  13. Lu H, Klaassen C. Tissue distribution and thyroid hormone regulation of Pept1 and Pept2 mRNA in rodents. Peptides. 2006;27(4):850–7.

    Article  CAS  Google Scholar 

  14. Saito H, Terada T, Okuda M, Sasaki S, Inui K. Molecular cloning and tissue distribution of rat peptide transporter PEPT2. Biochim Biophys Acta. 1996;1280(2):173–7.

    Article  Google Scholar 

  15. Rubio-Aliaga I, Daniel H. Peptide transporters and their roles in physiological processes and drug disposition. Xenobiotica. 2008;38(7–8):1022–42.

    Article  CAS  Google Scholar 

  16. Bleasby K, Castle JC, Roberts CJ, Cheng C, Bailey WJ, Sina JF, et al. Expression profiles of 50 xenobiotic transporter genes in humans and pre-clinical species: a resource for investigations into drug disposition. Xenobiotica. 2006;36(10–11):963–88.

    Article  CAS  Google Scholar 

  17. Endter S, Francombe D, Ehrhardt C, Gumbleton M. RT-PCR analysis of ABC, SLC and SLCO drug transporters in human lung epithelial cell models. J Pharm Pharmacol. 2009;61(5):583–91.

    Article  CAS  Google Scholar 

  18. Takano M, Horiuchi T, Sasaki Y, Kato Y, Nagai J, Yumoto R. Expression and function of PEPT2 during transdifferentiation of alveolar epithelial cells. Life Sci. 2013;93(17):630–6.

    Article  CAS  Google Scholar 

  19. Borok Z, Hami A, Danto SI, Lubman RL, Kim KJ, Crandall ED. Effects of EGF on alveolar epithelial junctional permeability and active sodium transport. Am J Phys. 1996;270(4 Pt 1):L559–65.

    CAS  Google Scholar 

  20. Kim KJ, Cheek JM, Crandall ED. Contribution of active Na+ and Cl- fluxes to net ion transport by alveolar epithelium. Respir Physiol. 1991;85(2):245–56.

    Article  CAS  Google Scholar 

  21. Borok Z, Hami A, Danto SI, Zabski SM, Crandall ED. Rat serum inhibits progression of alveolar epithelial cells toward the type I cell phenotype in vitro. Am J Respir Cell Mol Biol. 1995;12(1):50–5.

    Article  CAS  Google Scholar 

  22. Widera A, Beloussow K, Kim KJ, Crandall ED, Shen WC. Phenotype-dependent synthesis of transferrin receptor in rat alveolar epithelial cell monolayers. Cell Tissue Res. 2003;312(3):313–8.

    Article  CAS  Google Scholar 

  23. Cheek JM, Evans MJ, Crandall ED. Type I cell-like morphology in tight alveolar epithelial monolayers. Exp Cell Res. 1989;184(2):375–87.

    Article  CAS  Google Scholar 

  24. Okamoto T, Okabe S. Ultraviolet absorbance at 260 and 280 nm in RNA measurement is dependent on measurement solution. Int J Mol Med. 2000;5(6):657–9.

    CAS  PubMed  Google Scholar 

  25. Sodowich BI, Fadl I, Burns C. Method validation of in vitro RNA transcript analysis on the Agilent 2100 bioanalyzer. Electrophoresis. 2007;28(14):2368–78.

    Article  CAS  Google Scholar 

  26. Uchiyama T, Fujita T, Gukasyan HJ, Kim KJ, Borok Z, Crandall ED, et al. Functional characterization and cloning of amino acid transporter B(0,+) (ATB(0,+)) in primary cultured rat pneumocytes. J Cell Physiol. 2008;214(3):645–54.

    Article  CAS  Google Scholar 

  27. Takahashi K, Masuda S, Nakamura N, Saito H, Futami T, Doi T, et al. Upregulation of H(+)-peptide cotransporter PEPT2 in rat remnant kidney. Am J Physiol Ren Physiol. 2001;281(6):F1109–16.

    Article  CAS  Google Scholar 

  28. Takahashi K, Nakamura N, Terada T, Okano T, Futami T, Saito H, et al. Interaction of beta-lactam antibiotics with H+/peptide cotransporters in rat renal brush-border membranes. J Pharmacol Exp Ther. 1998;286(2):1037–42.

    CAS  PubMed  Google Scholar 

  29. Takano M, Sugimoto N, Ehrhardt C, Yumoto R. Functional expression of PEPT2 in the human distal lung epithelial cell line NCl-H441. Pharm Res. 2015;32(12):3916–26.

    Article  CAS  Google Scholar 

  30. Thwaites DT, Hirst BH, Simmons NL. Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco-2): identification of substrates that undergo H(+)-coupled absorption. Br J Pharmacol. 1994;113(3):1050–6.

    Article  CAS  Google Scholar 

  31. Thwaites DT, Brown CD, Hirst BH, Simmons NL. H(+)-coupled dipeptide (glycylsarcosine) transport across apical and basal borders of human intestinal Caco-2 cell monolayers display distinctive characteristics. Biochim Biophys Acta. 1993;1151(2):237–45.

    Article  CAS  Google Scholar 

  32. Thwaites DT, Hirst BH, Simmons NL. Direct assessment of dipeptide/H+ symport in intact human intestinal (Caco-2) epithelium: a novel method utilising continuous intracellular pH measurement. Biochem Biophys Res Commun. 1993;194(1):432–8.

    Article  CAS  Google Scholar 

  33. Thwaites DT, Kennedy DJ, Raldua D, Anderson CM, Mendoza ME, Bladen CL, et al. H/dipeptide absorption across the human intestinal epithelium is controlled indirectly via a functional Na/H exchanger. Gastroenterology. 2002;122(5):1322–33.

    Article  CAS  Google Scholar 

  34. Lubman RL, Crandall ED. Polarized distribution of Na(+)-H+ antiport activity in rat alveolar epithelial cells. Am J Phys. 1994;266(2 Pt 1):L138–47.

    CAS  Google Scholar 

  35. Lubman RL, Danto SI, Crandall ED. Evidence for active H+ secretion by rat alveolar epithelial cells. Am J Phys. 1989;257(6 Pt 1):L438–45.

    CAS  Google Scholar 

  36. Meredith D, Boyd CA. Dipeptide transport characteristics of the apical membrane of rat lung type II pneumocytes. Am J Phys. 1995;269(2 Pt 1):L137–43.

    CAS  Google Scholar 

  37. Sondergaard HB, Brodin B, Nielsen CU. hPEPT1 is responsible for uptake and transport of Gly-Sar in the human bronchial airway epithelial cell-line Calu-3. Pflugers Arch. 2008;456(3):611–22.

    Article  Google Scholar 

  38. Sporty JL, Horalkova L, Ehrhardt C. In vitro cell culture models for the assessment of pulmonary drug disposition. Expert Opin Drug Metab Toxicol. 2008;4(4):333–45.

    Article  CAS  Google Scholar 

  39. Irie M, Terada T, Sawada K, Saito H, Inui K. Recognition and transport characteristics of nonpeptidic compounds by basolateral peptide transporter in Caco-2 cells. J Pharmacol Exp Ther. 2001;298(2):711–7.

    CAS  PubMed  Google Scholar 

  40. Sawada K, Terada T, Saito H, Inui K. Distinct transport characteristics of basolateral peptide transporters between MDCK and Caco-2 cells. Pflugers Arch. 2001;443(1):31–7.

    Article  CAS  Google Scholar 

  41. Terada T, Sawada K, Saito H, Hashimoto Y, Inui K. Functional characteristics of basolateral peptide transporter in the human intestinal cell line Caco-2. Am J Phys. 1999;276(6 Pt 1):G1435–41.

    CAS  Google Scholar 

  42. Helliwell PA, Meredith D, Boyd CA, Bronk JR, Lister N, Bailey PD. Tripeptide transport in rat lung. Biochim Biophys Acta. 1994;1190(2):430–4.

    Article  CAS  Google Scholar 

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Author information

Author notes

  1. Hovhannes J. Gukasyan

    Present address: Allergan plc, Irvine, California, USA

  2. Tomomi Uchiyama

    Present address: Oozora Pharmacy, Hamamatsu, Shizuoka, Japan

  3. Sharon K. Wu

    Present address: Amgen, Inc., Thousand Oaks, California, USA

  4. Vincent H.L. Lee

    Present address: School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, 8/F, Lo Kwee-Seong Integrated Biomedical Sciences Building, Area 39, Shatin, N.T. Hong Kong SAR, China

  5. Hovhannes J. Gukasyan and Tomomi Uchiyama contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, California, USA

    Hovhannes J. Gukasyan, Tomomi Uchiyama, Kwang-Jin Kim, Sharon K. Wu & Vincent H.L. Lee

  2. Will Rogers Institute Pulmonary Research Center, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Kwang-Jin Kim, Zea Borok & Edward D. Crandall

  3. Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Kwang-Jin Kim, Zea Borok & Edward D. Crandall

  4. Department of Physiology and Biophysics, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Kwang-Jin Kim

  5. Department of Biomedical Engineering, Viterbi School of Engineering, University of Southern California, Los Angeles, California, USA

    Kwang-Jin Kim

  6. School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland

    Carsten Ehrhardt

  7. Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Zea Borok

  8. Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA

    Edward D. Crandall

  9. Mork Family Department of Chemical Engineering and Materials Science, Viterbi School of Engineering, University of Southern California, Los Angeles, California, USA

    Edward D. Crandall

Authors
  1. Hovhannes J. Gukasyan
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  2. Tomomi Uchiyama
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  8. Vincent H.L. Lee
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Corresponding author

Correspondence to Vincent H.L. Lee.

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Gukasyan, H.J., Uchiyama, T., Kim, KJ. et al. Oligopeptide Transport in Rat Lung Alveolar Epithelial Cells is Mediated by Pept2. Pharm Res 34, 2488–2497 (2017). https://doi.org/10.1007/s11095-017-2234-z

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  • DOI: https://doi.org/10.1007/s11095-017-2234-z

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