title: Functional activity of a monocarboxylate transporter, MCT1, in the human retinal pigmented epithelium cell line, ARPE-19.
abstract: The purpose of this study was to identify and characterize the functional activity of monocarboxylic acid transporter 1 (MCT1) on the human retinal pigmented epithelium (RPE) cell line, ARPE-19, and to evaluate whether the cell line can function as an in vitro screening tool for intravitreally administered drugs/prodrugs targeted to the MCT1 expressed in RPE. Uptake studies were carried out at 37 degrees C, for 30 s, with ARPE-19 cells. [(14)C]l-Lactic acid was selected as a substrate for this transporter. Uptake of [(14)C]L-lactic acid by ARPE-19 cells was found to exhibit saturable kinetics (K(m) = 3.1 +/- 0.6 mM and V(max) = 63.1 +/- 4.1 pmol/min/mg of protein). Monocarboxylic acids, such as benzoic acid, salicylic acid, and pyruvic acid, inhibited the uptake of [(14)C]L-lactic acid whereas di- and tricarboxylic acids, such as phthalic, succinic, and citric acids, did not demonstrate any inhibitory effect. Uptake was stereospecific where D-lactic acid was less effective in inhibiting [(14)C]L-lactic acid uptake than unlabeled L-lactic acid. ELISA indicated the expression of only MCT1, MCT4, and MCT8 isoforms by ARPE-19 cells. Increase in [(14)C]L-lactic acid uptake was observed as the uptake medium pH was lowered from 7.4 to 5.0. Moreover, inhibition of [(14)C]L-lactic acid uptake was observed in the presence of the protonophore 2,4-dinitrophenol. Uptake was significantly decreased in the presence of sodium azide, ouabain, p-chloromercuribenzoic acid (pCMBA), N-ethylmaleamide, dithiothreitol, and p-chloromercuribenzene sulfonate (pCMBS). However, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) and L-thyroxine did not inhibit [(14)C]L-lactic acid. RT-PCR studies and sequence analysis of the PCR product confirmed the expression of MCT1 by ARPE-19 cells. Our results indicate that MCT1 is functionally active and is the only MCT isoform involved in the apical uptake of monocarboxylates by ARPE-19 cells. This cell line may thus be used as an effective screening tool for intravitreally administered drugs/prodrugs targeted toward MCT1 expressed on the RPE.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, 5005 Rockhill Road, Kansas City, MO 64110, USA.
authors.forename: Soumyajit
authors.initials: S
authors.lastname: Majumdar
authors.forename: Sriram
authors.initials: S
authors.lastname: Gunda
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology; D000838:Anions; D019817:Benzoic Acid / Q000378:metabolism; D001667:Binding, Competitive; D018938:Caco-2 Cells; D002264:Carboxylic Acids / Q000737:chemistry; D002460:Cell Line; D045744:Cell Line, Tumor; D019343:Citric Acid / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D019344:Lactic Acid / Q000378:metabolism; D027501:Monocarboxylic Acid Transporters / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D010795:Phthalic Acids / Q000378:metabolism; D010857:Pigment Epithelium of Eye / Q000166:cytology*; D016133:Polymerase Chain Reaction; D020033:Protein Isoforms; D019289:Pyruvic Acid / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D020156:Salicylic Acid / Q000378:metabolism; D012984:Software; D019802:Succinic Acid / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000737:chemistry / Q000494:pharmacology; D027981:Symporters / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D013696:Temperature; D013974:Thyroxine / Q000737:chemistry / Q000378:metabolism; D013997:Time Factors; D014233:Tricarboxylic Acids / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid / Q000494:pharmacology; D000099233:Monocarboxylate Transport Protein 1
chemical_list: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002731:4-Chloromercuribenzenesulfonate; D000838:Anions; D019817:Benzoic Acid; D002264:Carboxylic Acids; D019343:Citric Acid; D018076:DNA, Complementary; D019344:Lactic Acid; D027501:Monocarboxylic Acid Transporters; D010795:Phthalic Acids; D020033:Protein Isoforms; D019289:Pyruvic Acid; D020156:Salicylic Acid; D019802:Succinic Acid; D013439:Sulfhydryl Reagents; D027981:Symporters; D013974:Thyroxine; D014233:Tricarboxylic Acids; D020245:p-Chloromercuribenzoic Acid; D000099233:Monocarboxylate Transport Protein 1; C032279:phthalic acid