ENDOSOMAL ALKALINIZATION REDUCES J(MAX) AND K-M OF ALBUMIN RECEPTOR-MEDIATED ENDOCYTOSIS IN OK CELLS

In this study, we investigated the effects of endosomal alkalinization on kinetics of endocytotic uptake in intact proximal tubule-derived o possum kidney cells. We used fluorescein isothiocyanate (FITC)-labeled albumin and FITC-dextran as endocytotic substrates for receptor-media ted endocytosis and fluid-phase endocytosis, respectively. The pH in e ndosomes labeled with either FITC-albumin or FITC-dextran rose in the presence of the vacuolar-type ATPase inhibitor, bafilomycin A(1), and in the presence of NH4Cl. Cytoplasmic pH, decreased in the presence of bafilomycin A(1), but was not significantly different from control du ring prolonged exposure of the cells to NH4Cl. Endocytotic uptake of F ITC-dextran was not affected by endosomal pH changes. Endocytotic upta ke of FITC-albumin was reduced markedly by bafilomycin A(1) (decrease of maximum transport rate and apparent affinity). Selective alkaliniza tion of endosomes using NH4Cl (i.e., with the cytoplasmic pH not diffe rent from control) reduced FITC-albumin uptake in a similar way but to a lesser extent than did bafilomycin A(1). Intracellular albumin degr adation was impaired by bafilomycin A(1) and NH4Cl. Prevention of endo some-lysosome fusion (lowering the temperature to 20 degrees C) abolis hed the effects of endosomal alkalinization. Furthermore, specific bin ding of albumin to the plasma membrane was reduced after incubation wi th bafilomycin A(1), indicating an impairment of receptor recycling. T hese data show that endosomal pH is an important determinant for the k inetics of receptor-mediated endocytotic uptake of albumin in the prox imal tubule but not for fluid-phase endocytosis. Endosomal alkalinizat ion disturbs intracellular ligand handling and receptor trafficking, l eading to a reduction of endocytotic capacity and affinity.