Abstract
The organic anion-transporting polypeptide (OATP) 1B3 is a membrane transport protein that mediates hepatic uptake of many drugs and endogenous compounds. Currently, determination of OATP-mediated drug-drug interactions in vitro is focused primarily on direct substrate inhibition. Indirect inhibition of OATP1B3 activity is under-appreciated. OATP1B3 has putative protein kinase C (PKC) phosphorylation sites. Studies were designed to determine the effect of PKC activation on OATP1B3-mediated transport in human hepatocytes using cholecystokinin-8 (CCK-8), a specific OATP1B3 substrate, as the probe. A PKC activator, phorbol-12-myristate-13-acetate (PMA), did not directly inhibit [3H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [3H]CCK-8 accumulation. Treatment with a PKC inhibitor bisindolylmaleimide (BIM) I prior to PMA treatment blocked the inhibitory effect of PMA, indicating PKC activation is essential for downregulating OATP1B3 activity. PMA pretreatment did not affect OATP1B3 mRNA or total protein levels. To determine the mechanism(s) underlying the indirect inhibition of OATP1B3 activity upon PKC activation, adenoviral vectors expressing FLAG-Myc-tagged OATP1B3 (Ad-OATP1B3) were transduced into human hepatocytes; surface expression and phosphorylation of OATP1B3 were determined by biotinylation and by an anti–phosphor-Ser/Thr/Tyr antibody, respectively. PMA pretreatment markedly increased OATP1B3 phosphorylation without affecting surface or total OATP1B3 protein levels. In conclusion, PKC activation rapidly decreases OATP1B3 transport activity by post-translational regulation of OATP1B3. These studies elucidate a novel indirect inhibitory mechanism affecting hepatic uptake mediated by OATP1B3, and provide new insights into predicting OATP-mediated drug interactions between OATP substrates and kinase modulator drugs/endogenous compounds.
Footnotes
- Received January 22, 2014.
- Accepted September 8, 2014.
↵1 Current affiliation: Department of Pharmacokinetics and Drug Metabolism, Amgen Inc., Seattle, Washington.
K.K. and X.M. contributed equally to this work.
This research was supported by the National Institutes of Health National Institute of General Medical Sciences [Grants R01-GM094268 and R01-GM41935]; the University of North Carolina University Research Council Grant, the North Carolina Translational and Clinical Sciences [Grant 2KR90907]; College of Pharmacy University of Oklahoma Health Sciences Center start-up support; and Deutsche Forschungsgemeinschaft [Grant Ko4186/1-1]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Part of the work was presented as listed below: Yue W, Köck K, Brouwer KLR (2010) Regulation of organic anion transporting polypeptide (OATP) 1B3 function by protein kinase C, in 2010 FIP Pharmaceutical Sciences World Congress/AAPS Annual Meeting and Exposition; 2010 November 14-18; New Orleans, LA. Poster T3405. Meng X, Köck K, Li J, Brouwer KLR, Yue W (2013) Protein kinase C activation rapidly down-regulates OATP1B3 transport function in primary human hepatocytes, in 2013 AAPS Annual Meeting and Exposition; 2013 November 10-14; San Antonio, TX. Poster W4351.
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- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics
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