Skip to main content

Advertisement

SpringerLink
Log in

Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells

  • Published:
AAPS PharmSci Aims and scope Submit manuscript

Abstract

Purpose. To determine the localization of the human intestinal H+/peptide cotransporter (hPepT1) and its function in intestinal epithelial cells after adenoviral transduction. Methods. Caco-2 cells grown on Transwell membrane filters were transduced with a recombinant replication-deficient adenovirus carrying the hPepT1 gene. The transport of Gly-Sar across both apical and basolateral membranes was measured after adenoviral transduction as a function of pH, temperature, inhibitors, and substrate concentration. The localization of hPepT1 was examined by immunocytochemistry using confocal laser scanning microscopy. Results. The apical-to-basolateral and basolateral-to-apical transport of Gly-Sar in Caco-2 cells after viral transduction was increased 3.3 and 3.5-fold, respectively. The similar magnitude of Gly-Sar permeability from either direction indicates involvement of identical transport pathways in both membranes. This was further confirmed by immunocytochemistry showing that hPepT1 was localized in the apical and basolateral membrane of Caco-2 cells after adenoviral transduction. In both directions, Gly-Sar transport was enhanced in the presence of a pH gradient. In addition, the basolateral-to-apical Gly-Sar transport was dependent on temperature, multiplicity of infection (MOI), and Gly-Sar concentration. It was inhibited in the presence of excess Gly-Pro and cephalexin. Conclusions. Caco-2 cell monolayers represent an appropriate model to study gene expression in intestinal epithelial cells. Transport characteristics of Gly-Sar from the basolateral to the apical side in adenovirus-transduced Caco-2 cells are in agreement with those from the apical to the basolateral side, indicating that hPepT1 is also expressed in the basolateral membrane and displays a similar level of transport enhancement after adenovirus mediated hPepT1 gene expression.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Leibach FH, Ganapathy V. Peptide transporters in the intestine and the kidney. Ann Rev Nutr. 1996;16:99–119.

    Article  CAS  Google Scholar 

  2. Han H-K, Vrueh RLA, Rhie JK, Covitz KY, Smith PL, Lee C-P, Oh D-M, Sadee W, Amidon GL. 5′-Amino acid esters of antiviral nucleosides, acyclovir and AZT, are absorbed by the intestinal PEPT1 peptide transporter. Pharm Res. 1998;15:1154–1159.

    Article  CAS  PubMed  Google Scholar 

  3. Hu M, Subramanian P, Mosberg HI, Amidon GL. Use of the peptide carrier system to improve the intestinal absorption of L-a-methyldopa: carrier kinetics, intestinal permeabilities, and in vitro hydrolysis of dipeptidyl derivatives of L-a-methyldopa. Pharm Res. 1989;6:66–70.

    Article  CAS  PubMed  Google Scholar 

  4. Brody SL, Crystal RG. Adenovirus-mediated in vivo gene transfer. Ann. N Y Acad Sci. 1994;716:90–101.

    Article  CAS  PubMed  Google Scholar 

  5. Hsu C-P, Hilfinger JM, Walter E, Merkle HP, Roessler BJ, Amidon GL. Overexpression of human intestinal oligopeptide transporter in mammalian cells via adenoviral transduction. Pharm Res. 1998;15:1376–1381.

    Article  CAS  PubMed  Google Scholar 

  6. Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology 1989;96:736–749.

    CAS  PubMed  Google Scholar 

  7. Ogihara H, Saito H, Shin B-C, Terada T, Takenoshita S, Nagamachi Y, Inui K-I, Takata K. Immuno-localization of H+/peptide cotransporter in rat digestive tract. Biochem Biophy Res Comm. 1996;220:848–852.

    Article  CAS  Google Scholar 

  8. Sai Y, Tamai I, Sumikawa H, Hayashi K, Nakanishi T, Amano O, Numata M, Iseki S, Tsuji A. Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of b-lactam antibiotics. FEBS Letters. 1996;392:25–29.

    Article  CAS  PubMed  Google Scholar 

  9. Thwaites DT, Brown CD, Hirst BH, Simmons NL. Transepithelial glycylsarcosine transport in intestinal Caco-2 cells mediated by expression of H+-coupled carriers at both apical and basal membranes. J Biol Chem. 1993;268:7640–7642.

    CAS  PubMed  Google Scholar 

  10. Walker D, Thwaites DT, Simmons NL, Gilbert HJ, Hirst BH. Substrate upregulation of the human small intestinal peptide transporter, hPepT1. J Physiol. 1998;507:697–706.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  11. Gonzalez DE, Covitz KY, Sadée W, Mrsny RJ. An oligopeptide transporter is expressed at high levels in the pancreatic carcinoma cell lines AsPc-1 and Capan-2. Cancer Res. 1998;58:519–525.

    CAS  PubMed  Google Scholar 

  12. Bockman DE, Ganapathy V, Oblak TG, Leibach FH. Localization of peptide transporter in nuclei and lysosomes of the pancreas. Int J Pancreatology 1997;22:221–225.

    CAS  Google Scholar 

  13. Liang R, Fei YJ, Prasad PD, Ramamoorthy S, Han H, Yang-Feng TL, Hedinger MA, Ganapathy V, Leibach FH. Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J Biol Chem. 1995;270:6456–6463.

    Article  CAS  PubMed  Google Scholar 

  14. Basu SK, Shen J, Elbert KJ, Okamoto CT, Lee VHL, Grafenstein HV. Development and utility of anti-PepT1 anti-peptide polyclonal antibodies. Pharm Res. 1998;15:338–342.

    Article  CAS  PubMed  Google Scholar 

  15. Lozier JN, Yankaskas JR, Ramsey WJ, Chen L, Berschneider H, Morgan RA. Gut epithelial cells as targets for gene therapy of hemophilia. Human Gene Ther. 1997;8:1481–1490.

    Article  CAS  Google Scholar 

  16. Walter E, Croyle MA, Davidson BL, Roessler BJ, Hilfinger JM, Amidon GL. Adenovirus mediated gene transfer to intestinal epithelial cells as a potential approach for oral delivery of peptides and proteins. J Control Rel. 1997;46:75–87.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Johnson & Johnson, Drug Metabolism, Route 202, P.O. Box 300, 08869, Raritan, NJ

    Cheng-Pang Hsu, Elke Walter, Hans P. Merkle, Barbara Rothen-Rutishauser, Heidi Wunderli-Allenspach, John M. Hilfinger & Gordon L. Amidon

Authors
  1. Cheng-Pang Hsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elke Walter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hans P. Merkle
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Barbara Rothen-Rutishauser
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heidi Wunderli-Allenspach
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John M. Hilfinger
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gordon L. Amidon
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Cheng-Pang Hsu.

Additional information

Published: October 6, 1999.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hsu, CP., Walter, E., Merkle, H.P. et al. Function and immunolocalization of overexpressed human intestinal H+/peptide cotransporter in adenovirus-transduced Caco-2 cells. AAPS PharmSci 1, 12 (1999). https://doi.org/10.1208/ps010312

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1208/ps010312

Keywords

Search

Navigation