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 microM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4 degrees C or in the presence of endocytic inhibitors at 37 degrees 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.
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 microM) and was unchanged when studied in the presence of 1 mM L-histidine. In contrast, GlySar uptake was significantly decreased when studied at 4 degrees C or in the presence of endocytic inhibitors at 37 degrees 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.