Preliminary Investigation into the Expression of Proton-Coupled Oligopeptide Transporters in Neural Retina and Retinal Pigment Epithelium (RPE): Lack of Functional Activity in RPE Plasma Membranes |
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Authors: | Ocheltree Scott M Keep Richard F Shen Hong Yang Dongli Hughes Bret A Smith David E |
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Institution: | (1) Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan, 48109-1065;(2) Departments of Neurosurgery and Physiology, The University of Michigan, Ann Arbor, Michigan, 48109;(3) Departments of Ophthalmology and Visual Sciences and Physiology, The University of Michigan, Ann Arbor, Michigan, 48109 |
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Abstract: | Purpose. To determine the expression and functional activity of proton-coupled oligopeptide transporters (POT) in retinal pigment epithelial (RPE) cells.
Methods. RT-PCR was used to probe the presence of POT mRNA in freshly isolated bovine RPE (BRPE) and human RPE (HRPE) cells, a human RPE cell line (ARPE-19), and human and bovine neural retina. 14C]GlySar uptake was used to characterize POT activity in cultured ARPE-19 cells and freshly isolated BRPE cell sheet suspensions.
Results. PHT1 mRNA was expressed in BRPE, HRPE, ARPE-19, and bovine and human neural retina. In contrast, PEPT2 and PHT2 were expressed only in bovine and human retina, and PEPT1 could not be detected. GlySar exhibited a linear uptake over 6 h at pH values of 6.0 and 7.4, with greater uptake at pH 7.4 (p < 0.01). GlySar uptake did not exhibit saturability (5-2000 M) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4°C or in the presence of endocytic inhibitors at 37°C (p < 0.01). Studies in BRPE cell sheet suspensions validated the results obtained in ARPE-19 cells and strongly suggested the absence of POT on the apical and basolateral membranes of RPE.
Conclusions. PHT1 mRNA is present in native bovine and human RPE and a human RPE cell line. However, the data argue against PHT1 being expressed on plasma membranes of RPE. Overall, GlySar appears to be taken up by RPE cells via a low-affinity, endocytic process. |
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Keywords: | PEPT1 PEPT2 PHT1 PHT2 RPE |
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