title: Megalin/cubilin-mediated uptake of FITC-labeled IgG by OK kidney epithelial cells.
abstract: In this paper, we characterize the uptake mechanism of fluorescein isothiocyanate-labeled human immunoglobulin G (FITC-hIgG) in opossum kidney (OK) epithelial cells, which have been shown to express megalin and cubilin. Confocal immunofluorescence microscopy showed the punctate expression of the neonatal Fc receptor FcRn in the cytoplasm, but not on the cell surface membrane. Temperature- and energy-dependent uptake of FITC-hIgG was observed at pH 7.4 but not at pH 6.0, indicating that the internalization of FITC-hIgG might not be due to FcRn, which has a binding affinity for IgG under acidic conditions. Under physiological pH conditions, human and bovine serum γ-globulin decreased FITC-hIgG uptake in a concentration-dependent manner. In addition, FITC-hIgG uptake was inhibited by various megalin and/or cubilin ligands including albumin, cytochrome c, transferrin and gentamicin. Endosomal acidification inhibitors (bafilomycin A(1) and chloroquine) significantly decreased the uptake of FITC-hIgG. Clathrin-dependent endocytosis inhibitors (phenylarsine oxide and chlorpromazine) decreased FITC-hIgG uptake. Potassium depletion and hypertonicity, conditions known to inhibit clathrin-dependent endocytosis, also decreased FITC-hIgG uptake. In contrast, caveolin-dependent endocytosis inhibitors (nystatin and methyl-β-cyclodextrin) did not decrease, but rather increased the uptake of FITC-hIgG. These observations suggest that the internalization of FITC-hIgG in OK cells might be, at least in part, due to megalin/cubilin-mediated, clathrin-dependent endocytosis.
authors.affiliation: Department of Pharmaceutics and Therapeutics, Graduate School of Biomedical Sciences, Hiroshima University, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Nagai
authors.forename: Koya
authors.initials: K
authors.lastname: Sato
authors.forename: Ryoko
authors.initials: R
authors.lastname: Yumoto
authors.forename: Mikihisa
authors.initials: M
authors.lastname: Takano
mesh_terms: D000818:Animals; D001152:Arsenicals / Q000494:pharmacology; D002417:Cattle; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D004705:Endocytosis / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007074:Immunoglobulin G / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism*; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2 / Q000502:physiology*; D018613:Microscopy, Confocal; D009893:Opossums; D011956:Receptors, Cell Surface / Q000502:physiology*; D011961:Receptors, Fc / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000494:pharmacology; D005719:gamma-Globulins / Q000378:metabolism
chemical_list: D001152:Arsenicals; D002966:Clathrin; D015395:Histocompatibility Antigens Class I; D007074:Immunoglobulin G; D008024:Ligands; D026561:Low Density Lipoprotein Receptor-Related Protein-2; D011956:Receptors, Cell Surface; D011961:Receptors, Fc; D047392:beta-Cyclodextrins; D005719:gamma-Globulins; C029486:intrinsic factor-cobalamin receptor; C108732:methyl-beta-cyclodextrin; C029341:oxophenylarsine; D016650:Fluorescein-5-isothiocyanate; C100688:Fc receptor, neonatal; D002746:Chlorpromazine