TRANSIENT EXPRESSION OF OATP ORGANIC ANION TRANSPORTER IN MAMMALIAN-CELLS - IDENTIFICATION OF CANDIDATE SUBSTRATES

The cDNA for the rat liver organic anion-transporting polypeptide ''oa tp'' has been shown to encode transport of bromosulfophthalein (BSP) a nd bile salts in Xenopus oocytes (E. Jacquemin, B. Hagenbuch, B. Stieg er, A. W. Wolkoff, and P. J. Meier. Proc. Natl. Acad. Sci. USA 91: 133 -137, 1994). Because oatp mRNA is expressed strongly in the kidney, we sought to determine whether renal oatp might play a role in the known secretion of a large variety of organic anions by the kidney. We tran siently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa c ell monolayers using the recombinant vaccinia virus vtf7-3. We tested an array of organic anions as candidate substrates by determining thei r ability to compete with tracer BSP for transport. HeLa cell monolaye rs transfected with the oatp cDNA transported tracer BSP and taurochol ate at rates substantially higher than monolayers transfected with a c ontrol plasmid. Thus good expression can be obtained with the vaccinia -HeLa system using a standard plasmid cloning vector. BSP transport va ried as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions k nown to be secreted by the classic secretory pathway, including p-amin ohippurate (PAH), phenol red, and indigo carmine (10 mu M) failed to i nhibit oatp-mediated BSP transport. Direct testing using tracers revea led no oatp-mediated transport of sulfate, urate, PAH, several eicosan oids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by similar to 50% by 10 mu M corticosterone su lfate, spironolactone, and several other steroids. We conclude that th e functional properties of oatp expressed in the HeLa cell/vaccinia tr ansient expression system are comparable to those following expression in Xenopus oocytes and that steroids are likely to represent high-aff inity endogenous oatp substrates. The latter hypothesis is addressed i n a companion paper.