Characterization of stably transfected kidney epithelial cell line expressing rat H+/peptide cotransporter PEPT1: localization of PEPT1 and transport of beta-lactam antibiotics.

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 beta-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 alpha-amino group) and cephradine (zwitterion with an alpha-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.