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A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.
Dea Herrera-Ruiz1, Teresa N Faria, Rajinder K Bhardwaj
1Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, The State University of New Jersey, 160 Frelinghuysen Road, Piscataway, New Jersey 08854-8022, USA.
Molecular Pharmaceutics
|April 19, 2005
Summary
Researchers developed a new cell model to study how human peptide transporter 1 (hPepT1) affects drug absorption. Higher hPepT1 expression directly correlated with increased peptide and peptide-like drug uptake and transport in vitro.
Area of Science:
- Pharmacology
- Molecular Biology
- Cell Biology
Background:
- Peptide transporters play a crucial role in the absorption of peptide-based drugs.
- Understanding the kinetics of peptide transporter function is essential for drug discovery.
- The human peptide transporter 1 (hPepT1) is a key transporter involved in peptide uptake.
Purpose of the Study:
- To establish and characterize stably transfected cell lines expressing varying levels of hPepT1.
- To quantify the relationship between hPepT1 expression levels and its functional kinetics in peptide and peptide-like drug uptake.
- To validate a novel in vitro model for screening drug transport mediated by hPepT1.
Main Methods:
- Amplification of hPepT1 sequence and insertion into pcDNA3.1-V5&His TOPO plasmid.
- Transfection of MDCK cells and selection of clonal cell lines with varying hPepT1 expression.
- Quantification of transgene protein levels using Western Blot analysis.
- Functional characterization of peptide uptake and transport using radiolabeled substrates ([14C]Gly-Sar and [3H]carnosine) at different pH levels.
Main Results:
- Established MDCK/hPepT1-V5&His clonal cell lines with low, medium, and high hPepT1 expression.
- Demonstrated sodium-independent and pH-dependent uptake of [14C]Gly-Sar, with increased rates at pH 6.0 in hPepT1-expressing cells.
- Observed a direct correlation between hPepT1 expression levels and the uptake/transport of [14C]Gly-Sar and [3H]carnosine.
- Confirmed a proportional relationship between Vmax and apparent permeability (Papp) and hPepT1 expression levels.
Conclusions:
- The developed MDCK/hPepT1-V5&His cell line accurately reflects hPepT1 function in vitro.
- hPepT1 expression levels directly influence the Vmax and Papp of peptide and peptide-like compounds.
- This cell line serves as a valuable tool for in vitro screening and quantification of drug transport mediated by hPepT1 in drug discovery.