title: Novel mechanism of impaired function of organic anion-transporting polypeptide 1B3 in human hepatocytes: post-translational regulation of OATP1B3 by protein kinase C activation.
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 [(3)H]CCK-8 accumulation in human sandwich-cultured hepatocytes (SCH). However, pretreatment with PMA for as little as 10 minutes rapidly decreased [(3)H]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.
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: John
authors.initials: J
authors.lastname: Powell
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: Taleah
authors.initials: T
authors.lastname: Farasyn
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: Kathleen
authors.initials: K
authors.lastname: Köck
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Meng
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: Sonia
authors.initials: S
authors.lastname: Pahwa
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.).
authors.forename: Kim L R
authors.initials: KL
authors.lastname: Brouwer
authors.affiliation: Department of Pharmaceutical Sciences, College of Pharmacy, University of Oklahoma Health Sciences Center (J.P., T.F., X.M., S.P., W.Y.); Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.K., K.L.R.B.) wei-yue@ouhsc.edu.
authors.forename: Wei
authors.initials: W
authors.lastname: Yue
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D001483:Base Sequence; D002478:Cells, Cultured; D017931:DNA Primers; D015536:Down-Regulation; D004789:Enzyme Activation; D005260:Female; D022781:Hepatocytes / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008297:Male; D008875:Middle Aged; D029362:Organic Anion Transporters, Sodium-Independent / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D060888:Real-Time Polymerase Chain Reaction; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D055815:Young Adult
chemical_list: D017931:DNA Primers; D029362:Organic Anion Transporters, Sodium-Independent; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate