| The purpose of these studies was to analyze the regulation of the folate transport protein, RFT-1 in RPE under conditions of hyperglycemia, hyperhomocysteinemia and increased endogenous levels of folate.; Human ARPE-19 cells were incubated for 4, 6, 9, 12 and 24 h in RPMI containing 45 mM glucose (high glucose), or 5 mM glucose + 40 mM mannitol (low glucose). The activity of RFT-1 was assessed by determining the ability of cells to take up [3H]-MTF, a reduced form of folate preferred by the transporter. Within 6 h of exposure to high glucose, MTF uptake was inhibited by 35% whereas the uptake of [3H]-glutamine and [14 C]-ascorbic acid was not inhibited. Dose-response experiments indicated that RFT-1 activity was attenuated by 37% following 6 h exposure to 35 mM glucose, but not to 15 mM or 25 mM glucose.; Decreased levels of folate are associated with increased levels of homocysteine and hyperhomocysteinemia is associated with diabetes. Therefore, the regulation of RFT-1 under hyperhomocysteinemic conditions was assessed. ARPE-19 cells were incubated in Na+ uptake buffer containing 1 mM D,L-homocysteine for varying time periods. Exposure of ARPE-19 cells for as short an incubation time as 4 h resulted in a 55% attenuation of [3H]-MTF uptake.; The transport of homocysteine into RPE cells has not been studied. Owing to a lack of available radiolabeled homocysteine, uptake by various systems was examined by competition studies using primary radiolabeled substrates ([3H]-leucine, [3H]-glutamate, [3H]-arginine, [3H]-alanine, [3H]-MeAIB) in the presence or absence of 5 mM D,L-homocysteine. Homocysteine transport studies showed the greatest inhibition in systems L, ATB0,+, A, b0,+ and B0 suggesting that homocysteine uses these transport systems to enter RPE cells.; These findings demonstrated for the first time that high levels of glucose, homocysteine and endogenous folate reduce the expression of RFT-1 and may have profound implications for the transport of folate by RPE in diabetes. The findings of these studies will form the basis of future experiments to understand alterations in the transport of folate in disease states, such as diabetic retinopathy. (Abstract shortened by UMI.)... |
