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Cloning and molecular characterization of apical efflux transporters (ABCB1, ABCB11 and ABCC2) in rainbow trout (Oncorhynchus mykiss) hepatocytes

Fish probably poses similar mechanisms of billiary excretion of xeno(endo)biotics and their metabolites as found in higher vertebrates, with the network of various types of ABC efflux proteins expressed in apical membranes of polarized cells as key mediators of this vectorial transport. To test this hypothesis the main goals of this study were detection of the P-glycoprotein (Pgp), bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP) related genes and characterization of the related transport activities in primary cultured rainbow trout (Oncorhynchus mykiss) hepatocytes. We cloned three full gene sequences, which share a high degree of homology with mammalian Pgp1 (ABCB1), BSEP (ABCB11) and MRP2 (ABCC2) efflux transporters. Using quantitative real-time PCR (qRT-PCR) gene expression levels of these genes were determined in trout liver and primary hepatocytes. Same relative expression pattern of identified efflux transporters (BSEP>>MRP2>Pgp1) was found for both liver and primary hepatocytes but expressions of all three transporters were approximately 3- to 4- fold lower in primary hepatocytes in comparison to intact liver. The Pgp1-, BSEP- and MRP-like activities were detected and distinguished using specific fluorescent substrates (rhodamine 123, calcein-AM, bodipy-verapamil and dihydrofluorescein diacetat) and model inhibitors (verapamil, cyclosporine A, MK571, reversine 205, taurocholate and taurochenodeoxycholate). Furthermore, combinations of these inhibitors, using either calcein-AM or rhodamine 123 as substrates, showed that after complete inhibition of MRPs with MK571, a significant additional increase in substrates accumulation could be obtained through blockage of Pgp-like transport with either reversine 205 or verapamil (VER), and vice versa.

multidrug/multixenobiotic resistance (MDR/MXR); ABC transport proteins; gene identification; transport activity determination

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