Riboflavin transport mediated by riboflavin transporters (RFVTs/SLC52A) at the rat outer blood-retinal barrier

Abstract

The previous in vivo study revealed the carrier-mediated transport of riboflavin (vitamin B2) across the blood-retinal barrier (BRB). In the present study, the blood-to-retina supply of riboflavin across the outer BRB was assessed in RPE-J cells, a rat-derived in vitro cell model of the outer BRB that is formed by the retinal pigment epithelial cells. In the directional uptake analysis on collagen-coated Transwell® inserts, RPE-J cells showed higher basal-to-cell (B-to-C) uptake (22.8 μL/mg protein) of [3H]riboflavin than apical-to-cell (A-to-C) uptake (13.5 μL/mg protein). RPE-J cells showed concentration- and temperature-dependent uptake of [3H]riboflavin with a Km of 297 nM, suggesting the involvement of carrier-mediated process in the blood-to-retina transport of riboflavin across the outer BRB. In RPE-J cells, [3H]riboflavin uptake was affected under a K+-replacement condition while no effect was observed under a choline-replacement condition and at different pH values. Uptake of [3H]riboflavin by RPE-J cells was markedly reduced by riboflavin, flavin adenine dinucleotide (FAD), and lumichrome with no significant effect noted for other vitamins. The obtained results suggested the involvement of riboflavin transporters (SLC52A/RFVT) at the outer BRB, and this is supported by the expression and knockdown analyses of rRFVT2 (Slc52a2) and rRFVT3 (Slc52a3).