Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.
Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.
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DOI：
10.1152/ajplung.00173.2005
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发表时间：
2006-05
期刊：
影响因子：
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通讯作者：
R. Yumoto;Hiromi Nishikawa;M. Okamoto;H. Katayama;J. Nagai;M. Takano
中科院分区：
文献类型：
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作者：
R. Yumoto;Hiromi Nishikawa;M. Okamoto;H. Katayama;J. Nagai;M. Takano
我们使用培养的 RLE-6TN 细胞检查了肺泡 II 型上皮细胞摄取 FITC-白蛋白的机制。在 RLE-6TN 细胞中检测到碱性磷酸酶活性和细胞角蛋白 19 mRNA 的表达，这是肺泡 II 型上皮细胞的特征。细胞对 FITC-白蛋白的摄取具有时间和温度依赖性，并显示出高亲和力和低亲和力运输系统的饱和动力学。 FITC-白蛋白的摄取受到天然白蛋白、化学修饰白蛋白、代谢抑制剂和巴弗洛霉素 A(1)（一种液泡 H(+)-ATP 酶抑制剂）的抑制。 FITC-白蛋白摄取后的共焦激光扫描显微镜分析显示细胞中荧光呈点状定位，部分定位于溶酶体中。 SDS-PAGE 后的荧光图像分析仪显示，细胞吸收的 FITC-白蛋白随着时间的推移逐渐降解。 RLE-6TN 细胞对 FITC-白蛋白的摄取不受制霉菌素、吲哚美辛或甲基-β-环糊精（小窝介导的内吞作用抑制剂）的抑制，但受到苯胂氧化物和氯丙嗪（网格蛋白介导的内吞作用抑制剂）的抑制，且呈浓度依赖性。钾消耗和高渗也会抑制摄取，这些条件已知会抑制网格蛋白介导的内吞作用。这些结果表明，培养的肺泡II型上皮细胞RLE-6TN中FITC-白蛋白的摄取是由网格蛋白介导的，而不是由小凹介导的内吞作用介导的，并且细胞内FITC-白蛋白在溶酶体中逐渐降解。讨论了参与该内吞系统的可能受体。
We examined mechanisms of FITC-albumin uptake by alveolar type II epithelial cells using cultured RLE-6TN cells. Alkaline phosphatase activity and the expression of cytokeratin 19 mRNA, which are characteristic features of alveolar type II epithelial cells, were detected in RLE-6TN cells. The uptake of FITC-albumin by the cells was time and temperature dependent and showed the saturation kinetics of high- and low-affinity transport systems. FITC-albumin uptake was inhibited by native albumin, by chemically modified albumin, and by metabolic inhibitors and bafilomycin A(1), an inhibitor of vacuolar H(+)-ATPase. Confocal laser scanning microscopic analysis after FITC-albumin uptake showed punctate localization of fluorescence in the cells, which was partly localized in lysosomes. FITC-albumin taken up by the cells gradually degraded over time, as shown by fluoroimage analyzer after SDS-PAGE. The uptake of FITC-albumin by RLE-6TN cells was not inhibited by nystatin, indomethacin, or methyl-beta-cyclodextrin (inhibitors of caveolae-mediated endocytosis) but was inhibited by phenylarsine oxide and chlorpromazine (inhibitors of clathrin-mediated endocytosis) in a concentration-dependent manner. Uptake was also inhibited by potassium depletion and hypertonicity, conditions known to inhibit clathrin-mediated endocytosis. These results indicate that the uptake of FITC-albumin in cultured alveolar type II epithelial cells, RLE-6TN, is mediated by clathrin-mediated but not by caveolae-mediated endocytosis, and intracellular FITC-albumin is gradually degraded in lysosomes. Possible receptors involved in this endocytic system are discussed.