Clathrin-mediated endocytosis of FITC-albumin in alveolar type II epithelial cell line RLE-6TN.
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.
Yumoto
Ryoko
R
Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Nishikawa
Hiromi
H
Okamoto
Miho
M
Katayama
Hirokazu
H
Nagai
Junya
J
Takano
Mikihisa
M
D000469:Alkaline Phosphatase / Q000378:metabolism; D001152:Arsenicals / Q000494:pharmacology; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D016650:Fluorescein-5-isothiocyanate / Q000493:pharmacokinetics*; D006801:Humans; D007213:Indomethacin / Q000494:pharmacology; D007633:Keratins / Q000235:genetics; D007700:Kinetics; D018613:Microscopy, Confocal; D009761:Nystatin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D020545:Respiratory Mucosa / Q000166:cytology / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D012710:Serum Albumin, Bovine / Q000493:pharmacokinetics*; D013816:Thermodynamics
D001152:Arsenicals; D002966:Clathrin; D012333:RNA, Messenger; C029341:oxophenylarsine; D009761:Nystatin; D012710:Serum Albumin, Bovine; D007633:Keratins; D000469:Alkaline Phosphatase; D016650:Fluorescein-5-isothiocyanate; D002746:Chlorpromazine; D007213:Indomethacin