Department of Pharmaceutics, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.
Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Sendai, 980-8578, Japan.
Exp Eye Res. 2018 Mar;168:128-137. doi: 10.1016/j.exer.2018.01.016. Epub 2018 Jan 31.
The changes in the transport function of the outer blood-retinal barrier (BRB), formed by retinal pigment epithelial (RPE) cells, under pathological conditions need to be understood to normalize the retinal homeostasis in retinal diseases. Connexin 43 (Cx43) is known to be one of the basic units of gap junctions and hemichannels, which are opened by changes in extracellular conditions. The purpose of this study was to clarify the expression of Cx43 in RPE cells of the retina and Cx43 contribution to compound transport functions in RPE cells. Immunohistochemistry using guinea pig-derived polyclonal anti-Cx43 antibodies indicated that Cx43 is localized at the apical and intercellular membrane of mouse RPE cells. In addition, the immunoprecipitation study using the anti-Cx43 antibodies suggested that Cx43 at the intercellular membrane is associated with gap and adherent junctions in mouse RPE cells. The intercellular transfer after scrape loading of Lucifer Yellow (457 g/mol) among a human RPE cell line, ARPE-19 cells, was greater than that of fluorescein isothiocyanate-dextran (∼3000 g/mol). This Lucifer Yellow transfer was significantly inhibited by carbenoxolone, a connexin inhibitor, suggesting that connexins take part in compound transfer via gap junctions. In addition, Lucifer Yellow uptake by ARPE-19 cells in the absence of extracellular Ca, which is a condition of hemichannel opening, was increased compared with that under normal conditions. This uptake of Lucifer Yellow in the absence of extracellular Ca was significantly reduced in the presence of hemichannel inhibitors and Cx43-gene silencing conditions. This study suggests the involvement of Cx43 in dye transfer via gap junctions among RPE cells and hemichannel-mediated compound transport between the neural retina and RPE cells.
外血视网膜屏障（BRB）的转运功能发生变化，这需要在病理条件下了解视网膜色素上皮（RPE）细胞的转运功能，以使视网膜疾病中的视网膜内稳态正常化。连接蛋白 43（Cx43）已知是间隙连接和半通道的基本单位之一，它们由细胞外条件的变化打开。本研究的目的是阐明 Cx43 在视网膜 RPE 细胞中的表达以及 Cx43 对 RPE 细胞中化合物转运功能的贡献。使用豚鼠来源的多克隆抗 Cx43 抗体进行的免疫组织化学染色表明，Cx43 定位于小鼠 RPE 细胞的顶膜和细胞间膜。此外，使用抗 Cx43 抗体进行的免疫沉淀研究表明，细胞间膜上的 Cx43 与小鼠 RPE 细胞中的间隙和粘着连接有关。在人 RPE 细胞系 ARPE-19 细胞中，Lucifer Yellow（457g/mol）的划痕加载后细胞间转移大于荧光素异硫氰酸酯-葡聚糖（~3000g/mol）。这种 Lucifer Yellow 转移被间隙连接抑制剂 carbenoxolone 显著抑制，表明连接蛋白通过间隙连接参与化合物转移。此外，在不存在细胞外 Ca 的情况下，ARPE-19 细胞摄取 Lucifer Yellow，这是半通道打开的条件，与正常条件下相比增加。在存在半通道抑制剂和 Cx43 基因沉默条件下，这种缺乏细胞外 Ca 的 Lucifer Yellow 摄取显著减少。本研究表明 Cx43 参与了 RPE 细胞之间通过间隙连接的染料转移以及神经视网膜和 RPE 细胞之间的半通道介导的化合物转运。
