Hepatobiliary elimination of many organic anions is initiated by OATP1B1 (OATP2, LST-1, OATP-C), OATP1B3 (OATP8), and OATP2B1 (OATP-B), which are the predominant uptake transporters of human hepatocytes. Thereafter, the unidirectional efflux pump ABCC2 (multidrug resistance protein 2) mediates the transport of organic anions, including glutathione conjugates and glucuronosides, into bile. In this study, we generated a Madin-Darby canine kidney (MDCKII) cell line stably expressing recombinant OATP1B1, OATP1B3, and OATP2B1 in the basolateral membrane and ABCC2 in the apical membrane. Double-transfected MDCKII cells stably expressing ABCC2 together with OATP1B1, OATP1B3, or OATP2B1 served as control cells. The quadruple-transfected cells exhibited high rates of vectorial transport of organic anions, including bromosulfophthalein, cholecystokinin peptide (CCK-8), and estrone 3-sulfate. The quadruple-transfected cells enabled the identification of substrates for uptake or vectorial transport that may be missed in studies with a double-transfected cell line, as exemplified by CCK-8, which is a substrate for OATP1B3 but not for OATP1B1 or OATP2B1. The broad substrate spectrum covered by the three hepatocellular OATP transporters enables representative analyses of the uptake of many organic anions into human hepatocytes. The broad spectrum of organic anions transported vectorially by the quadruple-transfected cells also provides valuable information on the substrate selectivity of ABCC2, without the need for studies in inside-out membrane vesicles containing the ABCC2 protein. The quadruple-transfected MDCKII-ABCC2/OATP1B1/1B3/2B1 cells may thus be useful for the identification of substrates and inhibitors, including drug candidates, undergoing uptake and secretion by human hepatocytes, under conditions that may be better defined than in primary cultures of human hepatocytes.

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In a search for sweet taste receptor interacting proteins, we have identified the calcium‐ and integrin‐binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα16gust44 and Gα15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5‐trisphosphate (InsP3)‐dependent Ca2+ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca2+ concentration, we used sweet and umami compounds as well as other InsP3‐generating ligands in FURA‐2‐based Ca2+ assays in wild‐type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short‐hairpin RNA increased the Ca2+ response of HEK293 cells to the InsP3‐generating ligands ATP, UTP and carbachol. Over‐expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin‐sensitive Ca2+ store of the endoplasmic reticulum (ER) and independent of extracellular Ca2+. The function of CIB1 on InsP3‐evoked Ca2+ release from the ER is most likely mediated by its interaction with the InsP3 receptor. Thus, CIB1 seems to be an inhibitor of InsP3‐dependent Ca2+ release in vivo.