title: Characterization of stably transfected kidney epithelial cell line expressing rat H+/peptide cotransporter PEPT1: localization of PEPT1 and transport of beta-lactam antibiotics.
abstract: 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.
authors.affiliation: Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Terada
authors.forename: H
authors.initials: H
authors.lastname: Saito
authors.forename: M
authors.initials: M
authors.lastname: Mukai
authors.forename: K
authors.initials: K
authors.lastname: Inui
mesh_terms: D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D051381:Rats; D014162:Transfection; D047090:beta-Lactams / Q000378:metabolism*
chemical_list: D000900:Anti-Bacterial Agents; D010455:Peptides; D047090:beta-Lactams; D006859:Hydrogen