title: Endosomal alkalinization reduces Jmax and Km of albumin receptor-mediated endocytosis in OK cells.
abstract: In this study, we investigated the effects of endosomal alkalinization on kinetics of endocytotic uptake in intact proximal tubule-derived opossum kidney cells. We used fluorescein isothiocyanate (FITC)-labeled albumin and FITC-dextran as endocytotic substrates for receptor-mediated endocytosis and fluid-phase endocytosis, respectively. The pH in endosomes labeled with either FITC-albumin or FITC-dextran rose in the presence of the vacuolar-type ATPase inhibitor, bafilomycin A1, and in the presence of NH4Cl. Cytoplasmic pH, decreased in the presence of bafilomycin A1, but was not significantly different from control during prolonged exposure of the cells to NH4Cl. Endocytotic uptake of FITC-dextran was not affected by endosomal pH changes. Endocytotic uptake of FITC-albumin was reduced markedly by bafilomycin A1 (decrease of maximum transport rate and apparent affinity). Selective alkalinization of endosomes using NH4Cl (i.e., with the cytoplasmic pH not different from control) reduced FITC-albumin uptake in a similar way but to a lesser extent than did bafilomycin A1. Intracellular albumin degradation was impaired by bafilomycin A1 and NH4Cl. Prevention of endosome-lysosome fusion (lowering the temperature to 20 degrees C) abolished the effects of endosomal alkalinization. Furthermore, specific binding of albumin to the plasma membrane was reduced after incubation with bafilomycin A1, indicating an impairment of receptor recycling. These data show that endosomal pH is an important determinant for the kinetics of receptor-mediated endocytotic uptake of albumin in the proximal tubule but not for fluid-phase endocytosis. Endosomal alkalinization disturbs intracellular ligand handling and receptor trafficking, leading to a reduction of endocytotic capacity and affinity.
authors.affiliation: Department of Physiology, University of Würzburg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Gekle
authors.forename: S
authors.initials: S
authors.lastname: Mildenberger
authors.forename: R
authors.initials: R
authors.lastname: Freudinger
authors.forename: S
authors.initials: S
authors.lastname: Silbernagl
mesh_terms: D000468:Alkalies / Q000378:metabolism*; D000643:Ammonium Chloride / Q000494:pharmacology; D000818:Animals; D003080:Cold Temperature; D003911:Dextrans; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009893:Opossums / Q000378:metabolism*; D017457:Receptors, Albumin / Q000502:physiology*; D012709:Serum Albumin
chemical_list: D000468:Alkalies; D003911:Dextrans; D017457:Receptors, Albumin; D012709:Serum Albumin; C015219:fluorescein isothiocyanate dextran; D000643:Ammonium Chloride; D016650:Fluorescein-5-isothiocyanate