Abstract
Recently, a polyspecific organic cation transporter, hOCT1, was cloned from human liver. To date, limited studies examining the functional characteristics of the transporter have been performed. The purpose of the present study was to develop a mammalian expression system for hOCT1 and to characterize the interactions of various compounds with the cloned transporter. Lipofection was used to transiently transfect the hOCT1 plasmid DNA in a human cell line, HeLa. We tested the interaction of an array of organic cations and other compounds with hOCT1 by determining Ki values in inhibiting14C-tetraethylammonium (TEA) transport in the transfected cells. Transient expression of hOCT1 activity was observed between 24 and 72 hr post-transfection, with maximal expression at approximately 40 hr. TEA transport was temperature dependent and saturable withVmax and Kmvalues of 2.89 ± 0.448 nmol/mg protein/30 min and 229 ± 78.4 μM, respectively. 14C-TEA uptake in hOCT1 plasmid DNA-transfected HeLa cells was trans-stimulated by unlabeled TEA and 1-methyl-4-phenyl-pyridinium. Organic cations, including clonidine, quinine, quinidine and verapamil (0.1 mM), significantly inhibited 14C-TEA uptake, whereas the organic anion, p-aminohippuric acid (5 mM), did not. The neutral compounds, corticosterone (Ki, 7.0 μM) and midazolam (Ki, 3.7 μM) potently inhibited14C-TEA uptake. The Ki values of several compounds in interacting with hOCT1 differed substantially from the corresponding values for the rat organic cation transporter, rOCT1, and the human kidney-specific organic cation transporter, hOCT2, determined in previous studies. Transiently transfected HeLa cells represent a useful tool in studying the interactions and kinetics of organic cations and other xenobiotics with hOCT1 and in understanding the molecular events involved in organic cation transport.
Footnotes
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Send reprint requests to: Kathleen M. Giacomini, Ph.D., Department of Biopharmaceutical Sciences, Box 0446, University of California, San Francisco, San Francisco, CA 94143-0446.
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↵1 This study was supported by grants from the National Institutes of Health (GM-36780 and GM-57656).
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↵2 Supported in part by the UCSF Chancellor’s Research Fellowship.
- Abbreviations:
- OCT
- organic cation transporter
- TEA
- tetraethylammonium
- MPP+
- 1-methyl-4-phenylpyridinium
- NMN
- N1-methylnicotinamide
- PAH
- p-aminohippuric acid
- dopa
- 3, 4-dihydroxyphenylalanine
- PBS
- phosphate-buffered saline
- Received November 20, 1997.
- Accepted March 6, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
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