ENDOSOMAL ALKALINIZATION REDUCES J(MAX) AND K-M OF ALBUMIN RECEPTOR-MEDIATED ENDOCYTOSIS IN OK CELLS
ENDOSOMAL ALKALINIZATION REDUCES J(MAX) AND K-M OF ALBUMIN RECEPTOR-MEDIATED ENDOCYTOSIS IN OK CELLS
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DOI：
10.1152/ajprenal.1995.268.5.f899
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发表时间：
1995-05-01
期刊：
影响因子：
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通讯作者：
SILBERNAGL, S
中科院分区：
文献类型：
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作者：
GEKLE, M;MILDENBERGER, S;SILBERNAGL, S
在这项研究中，我们研究了内体碱化对完整近端小管来源的负鼠肾细胞内吞摄取动力学的影响。我们分别使用异硫氰酸荧光素 (FITC) 标记的白蛋白和 FITC-葡聚糖作为受体介导的内吞作用和液相内吞作用的内吞底物。在液泡型 ATP 酶抑制剂巴弗洛霉素 A(1) 存在和 NH4Cl 存在下，用 FITC-白蛋白或 FITC-葡聚糖标记的内体中的 pH 值升高。巴弗洛霉素 A(1) 存在时细胞质 pH 值降低，但在细胞长时间暴露于 NH4Cl 期间与对照没有显着差异。 FITC-葡聚糖的内吞摄取不受内体 pH 变化的影响。巴弗洛霉素 A(1) 显着降低 FITC-白蛋白的内吞摄取（最大转运速率和表观亲和力降低）。使用 NH4Cl 选择性碱化核内体（即细胞质 pH 值与对照没有不同）以类似的方式减少 FITC-白蛋白的摄取，但程度低于巴弗洛霉素 A(1)。巴弗洛霉素 A(1) 和 NH4Cl 会损害细胞内白蛋白的降解。防止内体-溶酶体融合（将温度降低至 20 摄氏度）消除了内体碱化的影响。此外，与巴弗洛霉素 A(1) 一起孵育后，白蛋白与质膜的特异性结合减少，表明受体再循环受损。这些数据表明，内体 pH 值是近端小管中受体介导的白蛋白内吞作用动力学的重要决定因素，但不是液相内吞作用的重要决定因素。内体碱化扰乱细胞内配体处理和受体运输，导致内吞能力和亲和力降低。
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 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 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 A(1) (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 A(1). Intracellular albumin degradation was impaired by bafilomycin A(1) 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 A(1), 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.