title: New insights into the carrier-mediated transport of estrone-3-sulfate in the Caco-2 cell model.
abstract: The current studies were undertaken to gain new insights into the interplay and mechanism of membrane transporters involved in the permeability of estrone-3-sulfate (E1S) in Caco-2 cells cultured either on the bottom of multiwell plastic dishes or on filter support. We demonstrate that Caco-2 cells from the "Deutsche sammlung von mikroorganismen und zellkulturen" (DSMZ) exhibit extensive and consistent carrier-mediated uptake of [(3)H]-E1S after a culture period of 11-13 days. The kinetic characterization, the inhibitory profile and the pH dependence for the initial linear uptake permeability (PUP) of [(3)H]-E1S suggest that the organic anion transporting polypeptide (OATP) 2B1 is the main transporter involved in the apical E1S PUP in Caco-2 cells from the DSMZ. Furthermore, our results indicate that the efflux transporter breast cancer resistance protein (BCRP) affects E1S PUP, even when uptake is measured at the initial linear uptake phase. Although almost identical results were obtained for cells cultured on plastic dishes and on filter supports, the OATP2B1 stimulator dexamethasone did not affect the PUP for cells grown on dishes but increased [(3)H]-E1S PUP by more than 2-fold for filter grown cells. The basolateral PUP of [(3)H]-E1S of filter grown cells was inhibited by several inhibitors of the bidirectional transporter organic solute transporter α/β (OSTα/β). Efflux studies were performed by loading the cells with either [(3)H]-E1S or [(3)H]-taurocholic acid (TCA) and subsequently measuring the efflux of radio labeled substance in the absence or presence of BCRP or OST α/β inhibitors. Similar effluxes of [(3)H]-E1S was observed across the apical and basolateral membrane, and the apical efflux was greatly decreased in the presence of the BCRP inhibitor fumitremorgin C. In contrast, efflux of [(3)H]-TCA to the basolateral compartment was clearly larger than to the apical compartment. Trans-stimulation of basolateral [(3)H]-E1S efflux was observed in the presence of taurolithocholic acid (TLC), although none of the applied OSTα/β inhibitors were able to confirm the existence of carrier-mediated efflux at the basolateral membrane, neither for [(3)H]-E1S nor for [(3)H]-TCA. These results highlight the importance of transporter interplay for E1S and drug compounds in Caco-2 cells and emphasize the importance of identifying the basolateral transporters in these cells.
authors.affiliation: Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen , Universitetsparken 2, 2100 Copenhagen, Denmark.
authors.forename: Anne Sophie
authors.initials: AS
authors.lastname: Grandvuinet
authors.forename: Lena
authors.initials: L
authors.lastname: Gustavsson
authors.forename: Bente
authors.initials: B
authors.lastname: Steffansen
mesh_terms: D001692:Biological Transport / Q000502:physiology*; D018938:Caco-2 Cells; D004347:Drug Interactions; D004970:Estrone / Q000031:analogs & derivatives* / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism; D013656:Taurocholic Acid / Q000378:metabolism
chemical_list: D026901:Membrane Transport Proteins; D004970:Estrone; D013656:Taurocholic Acid; C017296:estrone sulfate