title: Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines.
abstract: The objective of this study is to delineate whether overexpression of human efflux transporters (P-gp, MRP2, and BCRP) in transfected MDCK cells affect the functional activities, and gene and protein expression of endogenous influx peptide transporter system (PepT). Real-time PCR, immunoblotting, uptake and permeability studies of [(3)H]Gly-Sar were conducted on transfected MDCKII and wild-type cells to investigate functional differences. Cellular [(3)H]Gly-Sar accumulation was significantly lower in transfected MDCKII cell lines compared to wild-type cells. Transport efficiency of apical peptide transporters was markedly reduced to around 25%, 30%, and 40% in P-gp-, MRP2-, and BCRP-overexpressed MDCK cell lines, respectively. With ascending cell-passage, transport efficiency was enhanced. A significantly higher Gly-Sar permeability was observed across parental cell-monolayers over transfected cells at all pHs. Levels of mRNA for both canine PepT1 and PepT2 were substantially reduced when efflux transporters overexpressed but enhanced when mRNA-levels of efflux genes diminished with ascending cell-passage of transfected cells. An inverse correlation was evident between endogenous PepT and exogenous efflux transporters in transfected MDCKII cells. Results of protein expression also supported these findings. Overexpression of MDR genes can affect endogenous PepT function which might be due to the phenomenon of transporter-compensation resulting in down-regulation of endogenous genes.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Health Sciences Building, 2464 Charlotte Street, Kansas City, MO 64108-2718, USA.
authors.forename: Zhiying
authors.initials: Z
authors.lastname: Wang
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashaben
authors.initials: A
authors.lastname: Patel
authors.forename: Deep
authors.initials: D
authors.lastname: Kwatra
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics*; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D004151:Dipeptides / Q000378:metabolism; D004285:Dogs; D015536:Down-Regulation; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D061985:Madin Darby Canine Kidney Cells; D026901:Membrane Transport Proteins / Q000235:genetics*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D009363:Neoplasm Proteins / Q000235:genetics*; D000074011:Peptide Transporter 1; D060888:Real-Time Polymerase Chain Reaction; D027981:Symporters / Q000235:genetics; D014162:Transfection
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000070997:ATP Binding Cassette Transporter, Subfamily G, Member 2; D018528:ATP-Binding Cassette Transporters; D004151:Dipeptides; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins; D000074011:Peptide Transporter 1; D027981:Symporters; C000715087:ABCC2 protein, human; C116614:ABCG2 protein, human; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine; C043912:peptide permease