title: Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2.
abstract: The purpose of this study was to construct stably transfected HeLa cells with human peptide transporters (hPEPT1/hPEPT2) and to identify the function of the transfected cells using the substrate JBP485 (a dipeptide) and a typical substrate for PEPTs, glycylsarcosine (Gly-Sar). An efficient and rapid method was established for the preparation and transformation of competent cells of Escherichia coli. After extraction and purification, hPEPT1/hPEPT2-pcDNA3 was transfected into HeLa cells by the liposome transfection method, respectively. HeLa-hPEPT1/hPEPT2 cells were selected by measuring the protein expression and the uptake activities of JBP485 and Gly-Sar. A simple and rapid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the simultaneous determination of JBP485 and Gly-Sar in biological samples. The Michaelis-Menten constant (K(m)) values of Gly-Sar uptake by the hPEPT1 and hPEPT2-expressing transfectants were 1.03 mM and 0.0965 mM, respectively, and the K(m) values of JBP485 uptake were 1.33 mM for PEPT1 and 0.144 mM for PEPT2. The uptake of Gly-Sar was significantly inhibited by JBP485 with a K(i) value of 8.11 mM (for PEPT1) and 1.05 mM (for PEPT2). Maximal uptake of Gly-Sar were detected at pH 5.8 (for PEPT1) and pH 6.5 (for PEPT2), suggesting that both HeLa-hPEPT1 and HeLa-hPEPT2 were H(+) dependent transporters. Stably transfected HeLa-hPEPT1/HeLa-hPEPT2 cells were constructed successfully, and the functions of hPEPT1/hPEPT2 were identified using their substrates, JBP485 and Gly-Sar. The transfected cells with transporters were used to investigate drug-drug interactions (DDIs) between JBP485 and other substrates (cephalexin or lisinopril) of PEPT1 and PEPT2.
authors.affiliation: Department of Clinical Pharmacology, College of Pharmacy, Dalian Medical University, China. guo.xinjin@163.com
authors.forename: Xinjin
authors.initials: X
authors.lastname: Guo
authors.forename: Qiang
authors.initials: Q
authors.lastname: Meng
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.forename: Changyuan
authors.initials: C
authors.lastname: Wang
authors.forename: Huijun
authors.initials: H
authors.lastname: Sun
authors.forename: Taiichi
authors.initials: T
authors.lastname: Kaku
authors.forename: Kexin
authors.initials: K
authors.lastname: Liu
mesh_terms: D001692:Biological Transport / Q000187:drug effects; D060846:Cell Engineering / Q000379:methods*; D002506:Cephalexin / Q000378:metabolism / Q000494:pharmacology; D002853:Chromatography, Liquid; D004151:Dipeptides / Q000378:metabolism / Q000494:pharmacology; D004347:Drug Interactions; D004926:Escherichia coli; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008081:Liposomes / Q000737:chemistry; D017706:Lisinopril / Q000378:metabolism / Q000494:pharmacology; D000074011:Peptide Transporter 1; D010456:Peptides, Cyclic / Q000378:metabolism / Q000494:pharmacology; D010957:Plasmids / Q000235:genetics; D027981:Symporters / Q000235:genetics / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D014162:Transfection
chemical_list: D004151:Dipeptides; C412308:JBP 485; D008081:Liposomes; D000074011:Peptide Transporter 1; D010456:Peptides, Cyclic; C082186:SLC15A1 protein, human; D027981:Symporters; C093694:hydrogen-coupled oligopeptide transporter PepT2; C004194:glycylsarcosine; D017706:Lisinopril; D002506:Cephalexin