Vectorial transport of the peptide CCK-8 by double-transfected MDCKII cells stably expressing the organic anion transporter OATP1B3 (OATP8) and the export pump ABCC2.
CCK-8 (L-aspartyl-L-tyrosyl-L-methionylglycyl-L-tryptophyl-L-methionyl-L-aspartyl-L-phenylalaninamide hydrogen sulfate ester), a derivative of the gastrointestinal peptide hormone cholecystokinin, is specifically taken up into human hepatocytes by the organic anion transporter OATP1B3 (OATP8). So far it was unknown which transporter mediates the excretion of CCK-8 into bile. Double-transfected Madin-Darby canine kidney strain II cells, expressing recombinant human OATP1B3 in the basolateral membrane together with human ABCC2 (multidrug resistance protein 2, MRP2) in the apical membrane, represent a valuable model system to study vectorial transport. The importance of an appropriate filter support for optimized protein localization and substrate transport was demonstrated by the comparison of filter pore densities of 2 x 10(6) and 1 x 10(8) per cm(2). At the high pore density, immunofluorescence microscopy showed an intense OATP1B3 signal in the basolateral membrane of all cells, and 82 +/- 8% of cells expressed ABCC2 in the apical membrane. Uptake and efflux of radiolabeled CCK-8 in the double-transfected cells grown at high pore density was enhanced 3.5- and 5.6-fold, respectively, compared with cells grown at lower pore density. Higher transport rates were also observed with [(3)H]bromosulfophthalein. The high-affinity ATP-dependent transport of CCK-8 by ABCC2 was directly demonstrated in ABCC2-containing membrane vesicles with a K(m) value of 8.1 microM. The uptake by OATP1B3 and hence the vectorial transport of CCK-8 was inhibited by cyclosporin A (K(i) 1.2 microM) and by MK571 [(3-(3-(2-(7-chloro-2-quinolinyl)ethenyl)phenyl) ((3-dimethylamino-3-oxopropyl)thio)methyl)thiopropanoic acid] (K(i) 0.6 microM); the respective K(i) values for the ABCC2-mediated transport were 24 and 8.5 microM. Thus, using an optimized filter support, we demonstrate vectorial transport of CCK-8 by OATP1B3 and by the apical export pump ABCC2.
Letschert
Katrin
K
Division of Tumor Biochemistry, German Cancer Research Center, Heidelberg. k.letschert@dkfz.de
Komatsu
Masaharu
M
Hummel-Eisenbeiss
Johanna
J
Keppler
Dietrich
D
D000818:Animals; D002460:Cell Line; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000096:biosynthesis* / Q000235:genetics; D029362:Organic Anion Transporters, Sodium-Independent / Q000096:biosynthesis* / Q000235:genetics; D011422:Propionates / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D011804:Quinolines / Q000494:pharmacology; D012844:Sincalide / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D014162:Transfection / Q000379:methods*
D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D029362:Organic Anion Transporters, Sodium-Independent; D011422:Propionates; D011804:Quinolines; D012844:Sincalide; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; C000715087:ABCC2 protein, human; C421209:SLCO1B3 protein, human; C059141:verlukast