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摘要
PURPOSE. To analyze the mechanisms of folate uptake in retinal Muller cells. METHODS. RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Muller cells, and rMC-1 cells for the three known folate transport proteins folate receptor alpha (FR alpha), proton-coupled folate transporter (PCFT), and reduced folate carrier (RFC). Laser scanning confocal microscopy (LSCM) and immunoelectron microscopy were used to determine the subcellular location of FR alpha and PCFT in primary Muller cells. The pH dependence of the uptake of [(3)H]-methyltetrahydrofolate ([(3)H]-MTF) was assayed in Muller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC. RESULTS. FR alpha and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Muller cells and rMC-1 cells. LSCM localized these proteins to the plasma membrane, nuclear membrane, and perinuclear region. Immunoelectron microscopic studies revealed the colocalization of FR alpha and PCFT on the plasma membrane and nuclear membrane and within endosomal structures. Muller cell uptake of [(3)H]-MTF was robust at pH 5.0 to 6.0, consistent with PCFT activity, but also at neutral pH, reflecting RFC function. RFC was expressed in mouse Muller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells. CONCLUSIONS. FR alpha and PCFT are expressed in retinal Muller cells and colocalize in the endosomal compartment, suggesting that the two proteins may work coordinately to mediate folate uptake. The unexpected finding of RFC expression and activity in cultured Muller cells may reflect the upregulation of this protein under proliferative conditions. (Invest Ophthalmol Vis Sci. 2010; 51: 3226-3235) DOI:10.1167/iovs.09-4833
- 出版日期2010-6
