Construction, identification and application of HeLa cells stably transfected with human PEPT1 and PEPT2

详细信息    查看全文

• 作者：Xinjin Guo^a ; ^{guo.xinjin@163.com} ; Qiang Meng^a ; ^c ; ^{mengq531@yahoo.cn} ; Qi Liu^a ; ^c ; ^{llaqii@yahoo.com.cn} ; Changyuan Wang^a ; ^c ; ^{wangcyuan@163.com} ; Huijun Sun^a ; ^c ; ^{sunhuijun@hotmail.com} ; Taiichi Kaku^b ; ^{tai_san@placenta-jbp.co.jp} ; Kexin Liu^a ; ^c ; ^{kexinliu@dlmedu.edu.cn}
• 关键词：JBP485 ; Gly-Sar ; Transfected cells ; PEPT1 ; PEPT2 ; Uptake
• 刊名：Peptides
• 出版年：2012
• 出版时间：April, 2012
• 年：2012
• 卷：34
• 期：2
• 页码：395-403
• 全文大小：1010 K

文摘

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.