Vol. 281, Issue 3, 1415-1421, 1997

Characterization of Stably Transfected Kidney Epithelial Cell Line Expressing Rat H⁺/Peptide Cotransporter PEPT1: Localization of PEPT1 and Transport of -Lactam Antibiotics¹

Tomohiro Terada, Hideyuki Saito, Mayumi Mukai and Ken-Ichi Inui

Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Kyoto 606-01, Japan

We established stably transfected LLC-PK₁ cells expressing the rat H⁺/peptide cotransporter PEPT1 (designated LLC-rPEPT1) and examined membrane localization and uptake by rat PEPT1 of oral -lactam antibiotics. The LLC-rPEPT1 cells expressed a novel PEPT1 protein with an apparent molecular mass of 75 kdaltons, which was found in rat intestinal membranes. The cell surface biotinylation of LLC-rPEPT1 cell monolayers grown on membrane filters showed that PEPT1 was localized predominantly on the apical membranes and, to a lesser extent, on the basolateral membranes. The amount of [¹⁴C]glycylsarcosine uptake in LLC-rPEPT1 cell monolayers was 3-fold greater from the apical, than from the basolateral side, which suggested that rat PEPT1 expressed on both membranes was functionally active. LLC-rPEPT1 cells grown on plastic dishes transported differently charged oral cephalosporins such as ceftibuten (divalent anion lacking an -amino group) and cephradine (zwitterion with an -amino group) in the presence of an inward H⁺ gradient, whereas those transfected with the vector alone did not have transport activity. Kinetic analysis revealed that the LLC-rPEPT1 cells had much higher affinity for ceftibuten than for cephradine. Di- and tripeptides and bestatin, a dipeptide-like antineoplastic drug, potently inhibited the uptake of these cephalosporins. These results suggest that the LLC-rPEPT1 cells serve as a useful model with which to analyze the mechanisms involved in membrane targeting and substrate recognition by rat PEPT1.