Abstract
Organic cation transporters (OCT1–3) play an important role in renal elimination of many drugs. The goals of this study were to 1) identify a cell culture model which constitutively expressed OCT2 that could be used to study the characteristics and regulation of this transporter, and 2) to study the mechanisms by which xenobiotics and hormones regulate the activity of OCT2. We characterized the endogenous organic cation transporter (OCT) activity in Madin-Darby canine kidney (MDCK) cells. The activity was localized to the basolateral membrane and was pH and membrane potential-dependent. The uptake of the model organic cation, tetraethylammonium, was saturable (Km, 19.5 ± 4.6 μM;Vmax, 350 ± 19.4 pmol/mg of protein/10 min) and was inhibited by known OCT inhibitors (e.g., cimetidine and quinidine). A cDNA fragment (711 base pairs) isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) was greater than 83% identical to OCT2 cDNAs from mammalian species; no OCT1 or OCT3 was detected by RT-PCR, suggesting that OCT2 may be the primary basolateral OCT in MDCK. OCT2 mRNA levels were increased significantly following exposure of MDCK to the steroid hormones, dexamethasone (2.0-fold), hydrocortisone (2.4-fold), and testosterone (1.8-fold) with comparable increases in activity. Other compounds tested, including the cytochrome P450 inducers, rifampicin, phenobarbital, and phenytoin, and the OCT substrates, verapamil and metformin, had no inducing effects. Collectively, these data indicate that MDCK can serve as a useful and convenient tool in screening candidate drugs for interaction with OCT2 and for studying the regulation of this transporter. Furthermore, our data demonstrate that steroid hormones induce the transcription of OCT2 in the kidney.
Footnotes
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↵1 Current address: Sugen Inc., 230 East Grand Avenue, South San Francisco, CA 94080-4811.
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This work was supported by a Grant GM 36780 from the National Institutes of Health. L.M.M. was also supported by Pharmaceutical Chemistry, Pharmacology and Toxicology Training Grant GM 07175. Y.S. is a recipient of a Merck Sharp & Dohme International Fellowship in Clinical Pharmacology.
- Abbreviations:
- OCT
- organic cation transporter
- rOCT
- rat organic cation transporter
- CYP
- cytochrome P-450
- Pgp
- P-glycoprotein
- MDCK
- Madin-Darby canine kidney
- RT-PCR
- reverse transcriptase-polymerase chain reaction
- TEA
- tetraethylammonium
- GAPDH
- glyceraldehyde-3-phosphate dehydrogenase
- PAH
- p-aminohippuric acid
- PBS
- phosphate-buffered saline
- Received April 27, 2001.
- Accepted June 29, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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