Department of Anesthesiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America ; Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.
PLoS One. 2013 Nov 27;8(11):e81903. doi: 10.1371/journal.pone.0081903. eCollection 2013.
Although a critical role for caveolae-mediated albumin transcytosis in pulmonary endothelium is well established, considerably less is known about caveolae-independent pathways. In this current study, we confirmed that cultured rat pulmonary microvascular (RPMEC) and pulmonary artery (RPAEC) endothelium endocytosed Alexa488-labeled albumin in a saturable, temperature-sensitive mode and internalization resulted in co-localization by fluorescence microscopy with cholera B toxin and caveolin-1. Although siRNA to caveolin-1 (cav-1) in RPAEC significantly inhibited albumin uptake, a remnant portion of albumin uptake was cav-1-independent, suggesting alternative pathways for albumin uptake. Thus, we isolated and cultured mouse lung endothelial cells (MLEC) from wild type and cav-1(-/-) mice and noted that ~ 65% of albumin uptake, as determined by confocal imaging or live cell total internal reflectance fluorescence microscopy (TIRF), persisted in total absence of cav-1. Uptake of colloidal gold labeled albumin was evaluated by electron microscopy and demonstrated that albumin uptake in MLEC from cav-1(-/-) mice was through caveolae-independent pathway(s) including clathrin-coated pits that resulted in endosomal accumulation of albumin. Finally, we noted that albumin uptake in RPMEC was in part sensitive to pharmacological agents (amiloride [sodium transport inhibitor], Gö6976 [protein kinase C inhibitor], and cytochalasin D [inhibitor of actin polymerization]) consistent with a macropinocytosis-like process. The amiloride sensitivity accounting for macropinocytosis also exists in albumin uptake by both wild type and cav-1(-/-) MLEC. We conclude from these studies that in addition to the well described caveolar-dependent pulmonary endothelial cell endocytosis of albumin, a portion of overall uptake in pulmonary endothelial cells is cav-1 insensitive and appears to involve clathrin-mediated endocytosis and macropinocytosis-like process.
尽管小窝蛋白介导的白蛋白转胞作用在肺内皮细胞中起着至关重要的作用，但对于小窝蛋白非依赖性途径，人们了解得还相当少。在本研究中，我们证实，培养的大鼠肺微血管（RPMEC）和肺动脉内皮（RPAEC）以饱和、温度敏感的方式内吞荧光标记的白蛋白，荧光显微镜共定位显示内吞作用导致霍乱 B 毒素和小窝蛋白-1共定位。尽管 RPAEC 中的小窝蛋白-1（cav-1）siRNA 显著抑制白蛋白摄取，但白蛋白摄取的一小部分是 cav-1 非依赖性的，这表明了白蛋白摄取的替代途径。因此，我们从野生型和 cav-1（-/-）小鼠中分离和培养了小鼠肺内皮细胞（MLEC），并注意到，通过共聚焦成像或活细胞全内反射荧光显微镜（TIRF）测定，约 65%的白蛋白摄取在完全缺乏 cav-1 的情况下仍然存在。通过电子显微镜评估胶体金标记的白蛋白摄取，并证明 MLEC 从 cav-1（-/-）小鼠摄取白蛋白是通过小窝蛋白非依赖性途径（包括网格蛋白包被的凹陷），导致白蛋白在内体中的积累。最后，我们注意到，RPMEC 中的白蛋白摄取部分对药理学试剂（阿米洛利[钠转运抑制剂]、Gö6976[蛋白激酶 C 抑制剂]和细胞松弛素 D[肌动蛋白聚合抑制剂]）敏感，这与吞噬作用样过程一致。阿米洛利敏感性解释的吞噬作用也存在于野生型和 cav-1（-/-）MLEC 的白蛋白摄取中。我们从这些研究中得出结论，除了已描述的小窝蛋白依赖性肺内皮细胞对白蛋白的内吞作用外，肺内皮细胞中总摄取的一部分对小窝蛋白-1不敏感，似乎涉及网格蛋白介导的内吞作用和吞噬作用样过程。
