Delineation of Human Peptide Transporter 1(hPepTI)-Mediated Uptake and Transport of Substrates with Varying Transporter Affinities Utilizing Stably Transfected hPepTI/ Madin-Darby Canine Kidney Clones and Caco-2 Cells

摘要

In the present investigation,the uptake and transport kinetics of valacyclovir(VACV),5-aminolevulinic acid(5-ALA),and ben-zylpenicillin(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 hPepTI protein(low,medium,and high expression)and in Caco-2 cells to delineate hPepTI-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-depen-dent effect was observed in the hPepTI-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 hPepTI 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 hPepTI affinity ranking of VACV > 5-ALA > BENZ.The apical-to-basal apparent permeability coefficient(P_(app))values of VACV,5-ALA,and BENZ in mock-corrected hPepT1-MDCK cells showed solely hPepTI-mediated transport in contrast to Caco-2 cells.Lower K_m values and higher P_(app)in Caco-2 cells compared with hPepT1-MDCK cells suggested the involvement of multiple transporters in Caco-2 cells.Thus,hPepTI-MDCK cells corrected for endogenous transporter expression may be a more appropriate model for screening compounds for their affinity to hPepTI.