title: LC-MS/MS Based Quantitation of ABC and SLC Transporter Proteins in Plasma Membranes of Cultured Primary Human Retinal Pigment Epithelium Cells and Immortalized ARPE19 Cell Line.
abstract: The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier between neural retina and choroid. The RPE has several important vision supporting functions, such as transport mechanisms that may also modify pharmacokinetics in the posterior eye segment. Expression of plasma membrane transporters in the RPE cells has not been quantitated. The aim of this study was to characterize and compare transporter protein expression in the ARPE19 cell line and hfRPE (human fetal RPE) cells by using quantitative targeted absolute proteomics (QTAP). Among 41 studied transporters, 16 proteins were expressed in hfRPE and 13 in ARPE19 cells. MRP1, MRP5, GLUT1, 4F2hc, TAUT, CAT1, LAT1, and MATE1 proteins were detected in both cell lines within 4-fold differences. MPR7, OAT2 and RFC1 were detected in the hfRPE cells, but their expression levels were below the limit of quantification in ARPE19 cells. PCFT was detected in both studied cell lines, but the expression was over 4-fold higher in hfRPE cells. MCT1, MCT4, MRP4, and Na+/K+ATPase were upregulated in the ARPE19 cell line showing over 4-fold differences in the quantitative expression values. Expression levels of 25 transporters were below the limit of quantification in both cell models. In conclusion, we present the first systematic and quantitative study on transporter protein expression in the plasma membranes of ARPE19 and hfRPE cells. Overall, transporter expression in the ARPE19 and hfRPE cells correlated well and the absolute expression levels were similar, but not identical. The presented quantitative expression levels could be a useful basis for further studies on drug permeation in the outer blood-retinal barrier.
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland , 70210 Kuopio, Finland.
authors.forename: Laura
authors.initials: L
authors.lastname: Pelkonen
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Sendai, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Sato
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland , 70210 Kuopio, Finland.
authors.forename: Mika
authors.initials: M
authors.lastname: Reinisalo
authors.affiliation: Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , P.O. Box 56, FI-00014 Helsinki, Finland.
authors.forename: Heidi
authors.initials: H
authors.lastname: Kidron
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Sendai, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Tachikawa
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Sendai, Japan.
authors.forename: Michitoshi
authors.initials: M
authors.lastname: Watanabe
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Sendai, Japan.
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Uchida
authors.affiliation: School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland , 70210 Kuopio, Finland.|Centre for Drug Research, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki , P.O. Box 56, FI-00014 Helsinki, Finland.
authors.forename: Arto
authors.identifier: 0000-0001-6064-3102
authors.initials: A
authors.lastname: Urtti
authors.affiliation: Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University , Sendai, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Terasaki
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000378:metabolism*; D001813:Blood-Retinal Barrier / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D055213:Retinal Pigment Epithelium / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D000070590:Solute Carrier Proteins / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods; D015854:Up-Regulation / Q000502:physiology
chemical_list: D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D000070590:Solute Carrier Proteins; D000254:Sodium-Potassium-Exchanging ATPase
keywords: blood−retinal barrier; ocular pharmacokinetics; quantitative targeted absolute proteomics; retinal pigment epithelium; transporter