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  • Title: P-Glycoprotein expression in human retinal pigment epithelium cell lines.
    Author: Constable PA, Lawrenson JG, Dolman DE, Arden GB, Abbott NJ.
    Journal: Exp Eye Res; 2006 Jul; 83(1):24-30. PubMed ID: 16530756.
    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.
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