Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912-2000, USA.
Invest Ophthalmol Vis Sci. 2010 Jun;51(6):3226-35. doi: 10.1167/iovs.09-4833. Epub 2010 Jan 6.
To analyze the mechanisms of folate uptake in retinal Müller cells.
RT-PCR and Western blot analysis were performed in freshly isolated neural retina and RPE/eyecup, primary mouse Müller cells, and rMC-1 cells for the three known folate transport proteins folate receptor alpha (FRalpha), 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 FRalpha and PCFT in primary Müller cells. The pH dependence of the uptake of [(3)H]-methyltetrahydrofolate ([(3)H]-MTF) was assayed in Müller cells in the presence/absence of thiamine pyrophosphate, an inhibitor of RFC.
FRalpha and PCFT are expressed abundantly in the retina in several cell layers, including the inner nuclear layer; they are present in primary mouse Müller 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 FRalpha and PCFT on the plasma membrane and nuclear membrane and within endosomal structures. Müller 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 Müller cells that had been allowed to proliferate in culture, but not in freshly isolated primary cells.
FRalpha and PCFT are expressed in retinal Müller 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 Müller cells may reflect the upregulation of this protein under proliferative conditions.
分析视网膜 Müller 细胞中叶酸摄取的机制。
在新鲜分离的神经视网膜和 RPE/眼杯中、原代小鼠 Müller 细胞和 rMC-1 细胞中进行 RT-PCR 和 Western blot 分析，以检测三种已知的叶酸转运蛋白叶酸受体 α（FRα）、质子偶联叶酸转运蛋白（PCFT）和还原叶酸载体（RFC）。激光扫描共聚焦显微镜（LSCM）和免疫电子显微镜用于确定原代 Müller 细胞中 FRα 和 PCFT 的亚细胞定位。在存在/不存在硫胺素焦磷酸（RFC 的抑制剂）的情况下，测定 Müller 细胞中 [(3)H]-甲基四氢叶酸 ([(3)H]-MTF)摄取的 pH 依赖性。
FRα 和 PCFT 在视网膜的多个细胞层中大量表达，包括内核层；它们存在于原代小鼠 Müller 细胞和 rMC-1 细胞中。LSCM 将这些蛋白质定位到质膜、核膜和核周区域。免疫电子显微镜研究表明，FRα 和 PCFT 共定位于质膜和核膜以及内体结构内。Müller 细胞对 [(3)H]-MTF 的摄取在 pH 5.0 到 6.0 时非常活跃，与 PCFT 活性一致，但在中性 pH 时也很活跃，反映了 RFC 的功能。RFC 在已允许在培养中增殖的小鼠 Müller 细胞中表达，但在新鲜分离的原代细胞中不表达。
FRα 和 PCFT 在视网膜 Müller 细胞中表达，并在内体区室中共定位，表明这两种蛋白可能协同作用介导叶酸摄取。在培养的 Müller 细胞中发现 RFC 的表达和活性可能反映了该蛋白在增殖条件下的上调。
