Transient expression of the organic anion transporter "oatp" in mammalian cells: identification of candidate substrates. Kanai, Naoaki, Run Lu, Yi Bao, Allan W. Wolkoff, Victor L. Schuster. Renal Division, Departments of Medicine, Physiology & Biophysics; Liver Research Center; Albert Einstein College of Medicine, Bronx, N.Y. 10461
APStracts 2:0165F, 1995.
The rat liver organic anion transporter cDNA "oatp" has been shown to encode transport of bromosulfophthalein (BSP) and bile salts in Xenopus oocytes (Jacquemin et al, Proc Nat'l Acad Sci 91: 133, 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 transiently expressed a full-length oatp cDNA, cloned in pSPORT, in HeLa cell monolayers using the recombinant vaccinia virus vtf 7-3. We tested an array of organic anions as candidate substrates by determining their ability to compete with tracer BSP for transport. HeLa cell monolayers transfected with the oatp cDNA transported tracer BSP and taurocholate at rates substantially higher than monolayers transfected with a control plasmid. Thus, good expression can be obtained with the vaccinia-HeLa system using a standard plasmid cloning vector. BSP transport varied as a function of the medium albumin, ionic conditions, and pH in a fashion similar to that in Xenopus oocytes. Several organic anions known to be secreted by the "classical" secretory pathway, including p-aminohippurate (PAH), phenol red, and indigo carmine (10 mM) failed to inhibit oatp -mediated BSP transport. Direct testing using tracers revealed no oatp-mediated transport of sulfate, urate, PAH, several eicosanoids, or unconjugated or conjugated bilirubin. On the other hand, BSP transport was inhibited by 50% by 10 mM corticosterone sulfate, spironolactone, and androsterone sulfate. We conclude that the functional properties of oatp expressed in the HeLa cell/vaccinia transient expression system are comparable to those following expression in Xenopus oocytes, and that steroids are likely to represent high-affinity endogenous oatp substrates. The latter hypothesis is addressed in greater detail in a companion paper. 

Received 22 February 1995; accepted in final form 21 August 1995.
APS Manuscript Number F61-5.
Article publication pending Am. J. Physiol. (Renal Fluid Electrolyte
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 31 October 95