Functional Characterization of Large Neutral Amino Acid Transporter in Human Retinal Pigmented Epithelium(ARPE - 19) Cells

Abstract

Abstract: : Purpose: To identify and functionally characterize a large neutral amino acid transporter in human retinal pigmented epithelial (ARPE - 19) cells. Methods: ARPE-19 cells were used as in-vitro model for the experiments. Cells were cultured between passage 12-30 as confluent monolayers in DMEM/DME-F12. Uptake studies were performed using the appropriate substrates. Experiments were performed on confluent monolayers 37°C. Samples were analyzed using liquid Scintillation counter. The uptake of the radiolabeled large neutral amino acids like [³H] L-Phe was done using 12 /24 well cluster plates at 37°C. These studies were performed in triplicates within 17-27 days of plating cells. Inhibition studies were carried out in presence of other L- and D- amino acids and metabolic inhibitors like ouabain to delineate the mechanism of uptake. Results: Uptake of L-Phe, was found to be saturable with increasing concentrations. These results indicated the possible presence of a neutral amino acid transporter. The Km and Vmax for L-Phe on ARPE-19 cells were found to be 7.89(1.69) µM and 0.21(0.011) nmoles/min/mg protein respectively. The amino acid transport system was found to be sodium ( Na+ ) dependent . Metabolic inhibitors like ouabain and 2,4 DNP did not seem to have a significant effect on [³H] L-Phe uptake . Inhibition studies showed an increased specificity of this transporter for L - amino acids compared to the corresponding D isomers. L-Phenylalanine , L-Tryptophan, L- Tyrosine and Glycine were found to be substrates for this transporter. Conclusions: The amino acid transport system appears to be specific for aromatic amino acids. There seems to be no evidence of an energy dependent process involved. Some of the substrates studied undergo pH dependent uptake. Hence there may be one or more amino acid transporter(s) present on this cell line. These results strongly suggest the presence of an amino acid transporter belonging to the system B⁰. This system could be utilized for specific delivery using prodrug analogs of antiviral agents.

Keywords: ion transporters • retina • retinal pigment epithelium