Dipeptide transport: an active process with energy-and proton-dependence in the human intestinal cell line,Caco-2  

作　　者：SUN Bing-wei SHI Lei CHEN Xi CHEN Zhao-yong TAI Ning-zheng ZHAO Xiao-chen LI Ning 

机构地区：[1]Department of Bum and Plastic Surgery, Affiliated Hospital, Jiangsu University, Zhenjiang Jiangsu 212001 [2]Center of Drug Metabolism and Pharmacokinetics, China Pharmaceutical University, Nanjing Jiangsu 210009 [3]Research Institute of General Surgery, ChinesePLA General Hospital of Nanjing Military Area, Nanjing Jiangsu 210002, China

出　　处：《江苏大学学报（医学版）》2007年第1期9-14,18,共7页Journal of Jiangsu University：Medicine Edition

基　　金：The work was Supported by National Natural Science Foundation of China(30371392)

摘　　要：Objective: To determine the active process on dipeptide transport with proton-and energy-dependence in Caco-2 cells.Methods: A human intestinal cell monolayer(Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter(PepT1).Caco-2 cells grown on multiwell dishes(24 wells) and Transwell membrane filters were incubated in the culture medium. The transport and uptake experiments of cephalexin across apical membranes were then conducted with different temperature and different pH values.Uptake of cephalexin in Caco-2 cells gsown on multiple well dishes with addition of energy inhibitors(sodium azide,SA and 2,4-dinitrophenol,DNP) were then measured. Results: The accumulation of cephalexin into Caco-2 monolayers increased with the duration of culture.The uptake from the apical surface was markedly influenced by the pH of the apical medium,and the maximal uptake was achieved at pH 5.5;further acidification of the incubation medium may decrease transport of cephalexin despite an increase inward H+ gradient.Cephalexin uptake was linear over the concentration range when the cells were incubated at 4℃ while the uptake rate was enhanced and tended to be saturated as the cephalexin concentration increased when the cells were incubated at 37℃.The kinetic parameters for the cephalexin transport carrier were determined to be: Vmax of(22.173±1.9) nmol/min per mg protein,Km of(2.069±0.9)mol/L,the Kd was estimated to be(0.07±0.02) nmol/min per mg protein per mmol/L.Uptake of cephalexin was markedly inhibited by sodium azide(SA) and 2,4-dinitrophenol(DNP). Conclusion: Cephalexin was transported actively across Caco-2 cells,and the transport process was proton-and energy-dependent.In addition, Caco-2 cells taked up cephalexin by dipeptide transporters that closely resembled the transporters present in the intestine.Caco-2 cells represented an ideal cellular model for future studies of the dipeptide transporter.Objective： To determine the active process on dipeptide transport with proton- and energydependence in Caco-2 cells. Methods： A human intestinal cell monolayer （Caco-2） was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter （PepT1）. Caco-2 cells grown on multiwell dishes （24 wells） and Transwell membrane filters were incubated in the culture medium. The transport and uptake experiments of cephalexin across apical mem- branes were then conducted with different temperature and different pH values. Uptake of cephalexin in Caco-2 cells grown on multiple well dishes with addition of energy inhibitors（ sodium azide, SA and 2,4- dinitrophenol, DNP） were then measured. Results： The accumulation of cephalexin into Caco-2 monolayers increased with the duration of culture. The uptake from the apical surface was markedly influenced by the pH of the apical medium, and the maximal uptake was achieved at pH 5.5 ; further acidification of the incubation medium may decrease transport of cephalexin despite an increase inward H＋ gradient. Cephalexin uptake was linear over the concentration range when the cells were incubated at 4℃ while the uptake rate was enhanced and tended to be saturated as the cephalexin concentration increased when the cells were incubated at 37℃. The kinetic parameters for the cephalexin transport carrier were determined to be： Vmax of （22. 173 ±1.9） nmol/min per mg protein, Km of （2.069 ±0.9）mol/L, the Kd was estimated to be （0.07 ± 0.02） nmol/min per mg protein per mmol/L. Uptake of cephalexin was markedly inhibited by sodium azide （SA） and 2,4-dinitrophenol （DNP）. Conclusion： Cephalexin was transpor- ted actively across Caco-2 cells, and the transport process was proton- and energy-dependent. In addition, Caco-2 cells taked up cephalexin by dipeptide transporters that closely resembled the transporters present in the intestine. Gaco-2 cells represented an ideal cellular model for future

关 键 词：DIPEPTIDE transport UPTAKE active process ENERGY 

分 类 号：R[医药卫生]