Regulation of reduced-folate transporter-1 (RFT-1) by homocysteine and identity of transport systems for homocysteine uptake in retinal pigment epithelial (RPE) cells

Reduced-folate transporter-1 (RFT-1) transports reduced-folates, such as N⁵-methyltetrahydrofolate (MTF), the predominant circulating form of folate. In RPE, RFT-1 is localized to the apical membrane and is thought to transport folate from RPE to photoreceptor cells. Folate is required for DNA, RNA, protein synthesis and the conversion of homocysteine (Hcy) to methionine. Decreased folate levels are associated with increased Hcy levels. In the present study, we asked whether RFT-1 activity in RPE is altered under high Hcy conditions and examined the transport mechanism for Hcy in RPE. Treatment of ARPE-19 cells, a human RPE cell line, with Hcy at concentrations higher than 50μM led to a significant decrease in RFT-1 activity. This effect increased as the treatment time increased. The inhibitory effect of Hcy on RFT-1 activity was not non-specific, as the activities of several other nutrient transporters were not affected under identical conditions. The effect of Hcy on RFT-1 was associated primarily with a decrease in the maximal velocity with no detectable change in substrate affinity. The decrease in RFT-1 activity was accompanied by parallel changes in RFT-1 mRNA and protein. Uptake of Hcy in ARPE-19 cells occurred via several transport systems, including Na⁺-independent systems L and b^0,+ and the Na⁺-dependent systems B ⁰, ATB^0,+ and A. Studies of the interaction of Hcy with one of the cloned transporters (ATB^0,+) provided direct evidence for the translocation of Hcy across the membrane via the transporter. We conclude that several transport systems operate in ARPE-19 cells for the entry of Hcy and that high levels of Hcy have deleterious effects on the expression and activity of RFT-1 in these cells.