TY - JOUR T1 - Delineation of Human Peptide Transporter 1 (hPepT1)-Mediated Uptake and Transport of Substrates with Varying Transporter Affinities Utilizing Stably Transfected hPepT1/Madin-Darby Canine Kidney Clones and Caco-2 Cells JF - Journal of Pharmacology and Experimental Therapeutics JO - J Pharmacol Exp Ther SP - 1093 LP - 1100 DO - 10.1124/jpet.105.087148 VL - 314 IS - 3 AU - Rajinder K. Bhardwaj AU - Dea Herrera-Ruiz AU - Patrick J. Sinko AU - Olafur S. Gudmundsson AU - Gregory Knipp Y1 - 2005/09/01 UR - http://jpet.aspetjournals.org/content/314/3/1093.abstract N2 - In the present investigation, the uptake and transport kinetics of valacyclovir (VACV), 5-aminolevulinic acid (5-ALA), and benzylpenicillin (BENZ) were studied in stably transfected Madin-Darby canine kidney (MDCK)/human peptide transporter 1 (hPepT1)-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein (low, medium, and high expression) and in Caco-2 cells to delineate hPepT1-mediated transport kinetics. These compounds were selected due to the fact that they are known PepT1 substrates, yet also have affinity for other transporters. Caco-2 cells, traditionally used for studying peptide-based drug transport, were included for comparison purposes. The time, pH, sodium, and concentration dependence of cellular uptake and permeability were measured using mock, clonal hPepT1-MDCK, and Caco-2 cells. A pH-dependent effect was observed in the hPepT1-expressing clones and Caco-2 cells, with an increase of 1.96-, 1.84-, and 2.05-fold for VACV, 5-ALA, and BENZ uptake, respectively, at pH 6 versus 7.4 in the high-expressing hPepT1 cells. BENZ uptake was significantly decreased in Caco-2 and MDCK cells in Na+-depleted buffer, whereas VACV uptake only decreased in Caco-2 cells. Concentration-dependent uptake studies in the mock-corrected hPepT1-MDCK and Caco-2 cells demonstrated hPepT1 affinity ranking of VACV > 5-ALA > BENZ. The apical-to-basal apparent permeability coefficient (Papp) values of VACV, 5-ALA, and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepT1-mediated transport in contrast to Caco-2 cells. Lower Km values and higher Papp in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells. Thus, hPepT1-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepT1. The American Society for Pharmacology and Experimental Therapeutics ER -