Pflugers Arch - Eur J Physiol (2006) 452: 64–70 DOI 10.1007/s00424-005-0005-x EPITHELIAL TRANSPORT Megumi Irie . Tomohiro Terada . Masahiro Tsuda . Toshiya Katsura . Ken-ichi Inui Prediction of glycylsarcosine transport in Caco-2 cell lines expressing PEPT1 at different levels Received: 20 June 2005 / Accepted: 22 September 2005 / Published online: 10 November 2005 # Springer-Verlag 2005 Abstract H + -coupled peptide transporter 1 (PEPT1) and the basolateral peptide transporter mediate the absorption of small peptides and peptide-like drugs in the small in- testine. Recently, we constructed a mathematical model to simulate glycylsarcosine (Gly-Sar) transport in Caco-2 cells. In this study, we attempted to adjust our model to a change in the expression level of PEPT1. To obtain cell lines expressing PEPT1 at different levels, recloning of Caco-2 cells was performed, and nine clones were isolated. Compared with parental cells, clones 1 and 9 exhibited the lowest and the highest levels of [ 14 C]Gly-Sar uptake from the apical side, respectively, whereas activities of the baso- lateral peptide transporter were comparable. Kinetic anal- ysis demonstrated that the difference in the activity of PEPT1 was accounted by variations in V max . Moreover, PEPT1 mRNA level was positively related to the activity of [ 14 C]Gly-Sar uptake (r=0.55). Based on these findings, the V max value of PEPT1 was defined as a variable using the amount of PEPT1 mRNA as an index of the expression level. With this improved model, Gly-Sar transport in clones 1 and 9 was well-predicted, suggesting that our model can simulate Gly-Sar transport in cells expressing PEPT1 at different levels. Keywords Peptide transporter . Small intestine . Absorption . Simulation . Expression level Abbreviations PEPT1: H + -coupled peptide transporter 1 . Gly-Sar: glycylsarcosine Introduction H + -coupled peptide transporter 1 (PEPT1) expressed in brush-border membranes of intestinal epithelial cells trans- ports dipeptides and tripeptides from the lumen into cells by utilizing an inward H + gradient and mediates the ab- sorption of dipeptides and tripeptides [1, 6, 14, 23]. Because of its broad substrate specificity, PEPT1 can ac- cept various peptide-like drugs, such as oral β-lactam an- tibiotics and the anticancer agent bestatin; therefore, PEPT1 serves as a drug transporter [7, 23, 29]. On the other hand, it has been demonstrated that another peptide transporter is expressed in the basolateral membrane [10, 12, 19, 24, 26, 27]. The basolateral peptide transporter mediates the extrusion of substrates taken up by PEPT1 into the circulation and is involved in the absorption of peptide-like drugs. Recently, based on the influx and efflux properties of PEPT1 and the basolateral peptide transporter, we con- structed a computational model of glycylsarcosine (Gly-Sar) transport in Caco-2 cells [11]. This model was composed of three compartments (i.e., the apical, cellular, and basolat- eral compartments) and two functional factors (PEPT1 and the basolateral peptide transporter). To reproduce the sat- uration of both transporters, the rate constants of Gly-Sar transport by PEPT1 and the basolateral peptide transporter were defined as variables using the respective kinetic parameters K m and V max . With this model, the time course of Gly-Sar transport at various concentrations in the absorptive direction could be predicted well, indicating that the model could be used to underlie a simulator to forecast the absorption of peptide-like drugs in the small intestine. However, the expression level of PEPT1 was presumed to be constant and was not incorporated into this model as a variable factor, although the expression level of PEPT1 differed from the segment of the intestine [17] and the intestinal PEPT1 was regulated by various factors, such as food, hormones, drugs, and diurnal rhythm [2, 23]. It is, therefore, essential to enable the model to achieve a change in the expression level of PEPT1 for the development of a simulator of drug absorption in the small intestine. M. Irie . T. Terada . M. Tsuda . T. Katsura . K.-i. Inui (*) Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, 606-8507 Kyoto, Japan e-mail: inui@kuhp.kyoto-u.ac.jp Tel.: +81-75-7513577 Fax: +81-75-7514207