title: P-Glycoprotein expression in human retinal pigment epithelium cell lines.
abstract: P-Glycoprotein (P-gp), an active efflux transporter encoded by the MDR1 gene, has recently been identified in the human and pig retinal pigment epithelium (RPE) in situ. Efflux pumps such as P-gp are major barriers to drug delivery in several tissues. We wished to establish whether human RPE cell lines express P-gp under the culture conditions recommended for each cell line so as to determine their suitability as in vitro models for predicting drug transport across the outer blood-retinal barrier. Three human RPE cell lines, ARPE19, D407 and h1RPE were investigated. Reverse transcriptase-polymerase chain reaction (RT-PCR) was carried out to determine the expression of MDR1 mRNA. Immunocytochemistry using the P-gp-specific antibody C219 was undertaken to investigate the presence of P-gp protein in each cell type. Uptake of rhodamine 123, a P-gp substrate, in the presence or absence of pre-treatment with a P-gp inhibitor, verapamil, was measured in each cell line to determine functional expression of P-gp. For all experiments, MDCK cells stably transfected with the human MDR1 gene (MDCK-MDR1) were used as a positive control. ARPE19 cells were consistently negative for P-gp as assessed by RT-PCR and immunocytochemistry. By contrast, RT-PCR of D407 and h1RPE samples yielded weak bands corresponding to MDR1; P-gp protein expression, as demonstrated by C219 immunoreactivity, was also present. Rhodamine uptake after treatment with verapamil was significantly greater in D407 and MDCK-MDR1, indicating functional expression of P-gp in these two cell lines. No evidence of functional P-gp was found in ARPE19 and h1RPE. In conclusion, D407 and h1RPE cells express P-gp, though functional activity was demonstrable only in D407 cells. ARPE19 cells do not express P-gp. Of these human RPE cells lines D407 could be considered as a suitable model for in vitro drug transport studies, particularly those involving P-gp substrates, without modification of their usual culture conditions.
authors.affiliation: Department of Optometry and Visual Science, Henry Wellcome Laboratories for Vision Sciences, Applied Vision Research Centre, City University, Northampton Square, London EC1V OHB, UK. p.a.constable@city.ac.uk
authors.forename: Paul A
authors.initials: PA
authors.lastname: Constable
authors.forename: John G
authors.initials: JG
authors.lastname: Lawrenson
authors.forename: Diana E M
authors.initials: DE
authors.lastname: Dolman
authors.forename: Geoffrey B
authors.initials: GB
authors.lastname: Arden
authors.forename: N Joan
authors.initials: NJ
authors.lastname: Abbott
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis*; D001692:Biological Transport / Q000502:physiology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005136:Eye Proteins / Q000032:analysis*; D019450:Genes, MDR / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D010857:Pigment Epithelium of Eye / Q000737:chemistry* / Q000187:drug effects; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012235:Rhodamines / Q000493:pharmacokinetics; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D005136:Eye Proteins; D012333:RNA, Messenger; D012235:Rhodamines; D014700:Verapamil