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Am J Physiol Lung Cell Mol Physiol 290: L946-L955, 2006. First published December 16, 2005; doi:10.1152/ajplung.00173.2005
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Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN

Ryoko Yumoto,1 Hiromi Nishikawa,1 Miho Okamoto,1 Hirokazu Katayama,2 Junya Nagai,1 and Mikihisa Takano1

1Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima; and 2Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Japan

Submitted 18 April 2005 ; accepted in final form 14 December 2005

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 A1, 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.

albumin clearance; caveolae-mediated endocytosis; endocytic receptor; modified albumin; protein transport


Address for reprint requests and other correspondence: M. Takano, Dept. of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima Univ., 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan (e-mail address: takanom{at}hiroshima-u.ac.jp)






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