Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, Kyoto, Japan.
Invest Ophthalmol Vis Sci. 2012 May 22;53(6):3092-103. doi: 10.1167/iovs.11-8018.
To investigate the effect of a specific inhibitor of Rho-associated protein kinase, Y-27632, on monkey Schlemm's canal endothelial (SCE) cells.
SCE cells were isolated from cynomolgus monkey eyes. The effects of Y-27632 on aqueous outflow facility were evaluated using enucleated monkey eyes and a constant-pressure perfusion system. The effect of Y-27632 on the barrier function of the confluent SCE-cell monolayer was evaluated by measuring transendothelial electrical resistance (TEER) and fluorescein permeability. Y-27632-induced changes in the intracellular localization of ZO-1, claudin-5, β-catenin, pan-cadherin, and filamentous actin (F-actin) were examined by immunofluorescence. Gene-expression changes induced by Y-27632 were analyzed with microarray, and the functional categories of changed genes were identified by gene ontology analysis. The concentrations of intracellular calcium ions were estimated using Fluo-4/AM and a fluorescence microscope system.
Y-27632 significantly increased the outflow facility and the number of associated giant vacuoles, decreased TEER of the SCE-cell monolayer, and increased the transendothelial flux of fluorescein. Y-27632 disrupted ZO-1 and claudin-5 expression in a confluent SCE-cell monolayer. Among 12,544 genes, Y-27632 treatment increased the expression of 57 genes and decreased the expression of 15 genes. Gene ontology analysis revealed that changed genes were related to various cellular functions, including regulation of calcium ion transport into the cytosol. Y-27632 partially diminished the A23187-induced increase in intracellular calcium ions.
Y-27632 increased the permeability of the SCE-cell monolayer in association with disruption of the tight junction, F-actin depolymerization, and changes in various cell functions, including calcium transfer.
研究 Rho 相关蛋白激酶的特异性抑制剂 Y-27632 对猴氏施莱姆管内皮（SCE）细胞的影响。
从食蟹猴眼内分离 SCE 细胞。使用去眼猴和恒压灌注系统评估 Y-27632 对房水流出率的影响。通过测量跨内皮电阻（TEER）和荧光素通透性来评估 Y-27632 对 SCE 细胞单层屏障功能的影响。通过免疫荧光法观察 Y-27632 诱导的 ZO-1、claudin-5、β-连环蛋白、pan-钙粘蛋白和丝状肌动蛋白（F-actin）的细胞内定位变化。用微阵列分析 Y-27632 诱导的基因表达变化，并通过基因本体分析确定变化基因的功能类别。用 Fluo-4/AM 和荧光显微镜系统估计细胞内钙离子浓度。
Y-27632 显著增加了流出率和相关大空泡的数量，降低了 SCE 细胞单层的 TEER，并增加了荧光素的跨内皮通量。Y-27632 破坏了 SCE 细胞单层中的 ZO-1 和 claudin-5 的表达。在 12544 个基因中，Y-27632 处理增加了 57 个基因的表达，降低了 15 个基因的表达。基因本体分析显示，变化的基因与各种细胞功能有关，包括细胞质内钙离子转运的调节。Y-27632 部分减弱了 A23187 诱导的细胞内钙离子增加。
Y-27632 增加了 SCE 细胞单层的通透性，与紧密连接破坏、F-肌动蛋白解聚以及包括钙转移在内的各种细胞功能变化有关。
