Philp Nancy J, Wang Dian, Yoon Heeyong, Hjelmeland Leonard M
Department of Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
Invest Ophthalmol Vis Sci. 2003 Apr;44(4):1716-21. doi: 10.1167/iovs.02-0287.
To evaluate the expression and subcellular distribution of proton-coupled monocarboxylate transporters (MCTs) in human RPE in vivo and determine whether ARPE-19 cells retain the ability to express and differentially polarize these transporters.
Total RNA was prepared from human donor eyes and from ARPE-19 cell cultures. Expression of MCT transcripts was evaluated by RT-PCR amplification. Expression of MCT proteins in human RPE and ARPE-19 cells was evaluated by immunolocalization and Western blot analysis with isoform-specific anti-peptide antibodies.
The expression of MCTs in human RPE was investigated by immunofluorescence analysis on frozen sections of human donor eyes. MCT1 antibody labeled the apical membrane of the RPE intensely, whereas MCT3 labeling was restricted to the basolateral membrane. MCT4 was detected in the neural retina but not in the RPE. ARPE-19 cells constitutively expressed MCT1 and MCT4 mRNAs. Expression of MCT3 mRNA increased over time as ARPE-19 cells established a differentiated phenotype. Western blot analysis revealed that ARPE-19 cells expressed high levels of MCT1 and MCT4 but very little MCT3 protein. Sections of differentiated ARPE-19 cells were labeled with MCT1, MCT4, and glucose transporter-1 antibodies. MCT1 was polarized to the apical membrane and MCT4 to the basolateral membrane, whereas GLUT1 was expressed in both membrane domains. CD147, which is necessary for targeting MCTs to the plasma membrane, was detected in the apical and basolateral membranes of human RPE in situ and ARPE-19 cells.
These studies demonstrate for the first time that human RPE expresses two proton-coupled monocarboxylate transporters: MCT1 in the apical membrane and MCT3 in the basolateral membrane. The coordinated activities of these two transporters could facilitate the flux of lactate from the retina to the choroid. ARPE-19 cells express two MCT isoforms, polarized to different membrane domains: MCT1 to the apical membrane and MCT4 to the basolateral membrane. The polarized expression of MCTs in ARPE-19 demonstrates that these cells retain the cellular machinery necessary for transepithelial transport of lactate.
评估质子偶联单羧酸转运体（MCTs）在人视网膜色素上皮（RPE）中的体内表达及亚细胞分布，并确定ARPE - 19细胞是否保留表达这些转运体并使其差异化极化的能力。
从人供体眼和ARPE - 19细胞培养物中提取总RNA。通过逆转录聚合酶链反应（RT - PCR）扩增评估MCT转录本的表达。使用亚型特异性抗肽抗体，通过免疫定位和蛋白质印迹分析评估人RPE和ARPE - 19细胞中MCT蛋白的表达。
通过对人供体眼冰冻切片进行免疫荧光分析，研究了MCTs在人RPE中的表达。MCT1抗体强烈标记RPE的顶端膜，而MCT3标记仅限于基底外侧膜。在神经视网膜中检测到MCT4，但在RPE中未检测到。ARPE - 19细胞组成性表达MCT1和MCT4 mRNA。随着ARPE - 19细胞建立分化表型，MCT3 mRNA的表达随时间增加。蛋白质印迹分析显示，ARPE - 19细胞表达高水平的MCT1和MCT4，但MCT3蛋白含量很少。用MCT1、MCT4和葡萄糖转运体 - 1抗体标记分化的ARPE - 19细胞切片。MCT1极化至顶端膜，MCT4极化至基底外侧膜，而葡萄糖转运蛋白1在两个膜结构域均有表达。在人RPE原位和ARPE - 19细胞的顶端和基底外侧膜中检测到将MCTs靶向质膜所必需的CD147。
这些研究首次证明人RPE表达两种质子偶联单羧酸转运体：顶端膜中的MCT1和基底外侧膜中的MCT3。这两种转运体的协同活动可促进乳酸从视网膜向脉络膜的通量。ARPE - 19细胞表达两种MCT亚型，极化至不同的膜结构域：MCT1至顶端膜，MCT4至基底外侧膜。MCTs在ARPE - 19中的极化表达表明这些细胞保留了乳酸跨上皮运输所需的细胞机制。
