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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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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 μM) 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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Authors and Affiliations

  1. Department of Pharmaceutical Sciences, The University of Michigan, Ann Arbor, Michigan, 48109-1065

    Scott M. Ocheltree, Hong Shen & David E. Smith

  2. Departments of Neurosurgery and Physiology, The University of Michigan, Ann Arbor, Michigan, 48109

    Richard F. Keep

  3. Departments of Ophthalmology and Visual Sciences and Physiology, The University of Michigan, Ann Arbor, Michigan, 48109

    Dongli Yang & Bret A. Hughes

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  1. Scott M. Ocheltree
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Correspondence to David E. Smith.

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Ocheltree, S.M., Keep, R.F., Shen, H. et al. 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. Pharm Res 20, 1364–1372 (2003). https://doi.org/10.1023/A:1025741723724

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