大鼠肾近球小管细胞胞饮体膜氢泵活性和水的渗透性转运的特征

本实验采用异硫氰酸-葡聚糖荧光素(fluorescein isothiocyanate-dextran,FITC-dextran)体内标记法,研究大鼠肾近球小管细胞胞饮体(endosome)膜上 H~+-ATP 酶的活性及水的渗透性转运。通过观察在胞饮体外加入一定量 ATP 后,胞饮体内 pH 值的时间反应曲线,从而测定 ATP-依赖的 H~+在胞饮体膜上的转运情况。胞饮体内的酸化速度及 pH 的最低值与加入的 ATP 浓度有关。在加入 ATP 前,胞饮体内的 pH 值为7.4,加入不同浓度的 ATP 后,即ATP]为0.005,0.05,0.5,5和10mmol/L,胞饮体内 pH 最低值分别为7.30,6.99,6.68,6.38和6.39。此种由 ATP 引起的酸化反应,被0.5mmol/L N-ethylmaleimide(NEM)抑制97%,但不被钒酸盐和 oligomycin 所抑制。实验还同时观察了此种胞饮体水的渗透性转运机制。通过在胞饮体膜内外建立一个蔗糖浓度梯度。观察 FITC-dextran 荧光信号的快速动力学变化过程,从而测定由于渗透压梯度引起的水在胞饮体膜上转运的特征。在230℃时,水的渗透性通透系数(osmotic water permeability coefficient,P_f)为0.03cm/s;加入0.5mmol/L HgCl_2后,水的转运被抑制70%。此抑制反应可被5mmol/L 巯基乙醇(β-Mcrcaptoethanol)完全逆转。上述结果提示:大鼠肾近球小管胞饮体膜含有H~+-ATP 酶和水的转运通道。胞饮

CHARACTERIZATION OF PROTON PUMP AND OSMOTIC WATER TRANSPORT IN ENDOCYTIC VESICLES FROM RAT KIDNEY PROXIMAL TUBULE

The characteristics of ATP-driven proton and osmotic water transport were studied in endocytic vesicles isolated from rat kidney proximal tubule labelled in vivo with fluorescein isothiocyanate-dextran (FITC-dextran). ATP-driven proton transport was measured from the time course of endosome pH following addition of external ATP. The rate of endosome acidification and the minimum pH were dependent on the ATP concentration. At an initial endosome pH of 7.4, the final pH values were 7.30, 6.99, 6.68, 6.38 and 6.39 at ATP] = 0.005, 0.05, 0.5, 5 and 10 mmol/L, respectively. The acidification was inhibited by 97% at 0.5 mmol/L N-ethylmaleimide but was not affected by vanadate and oligomycin. Osmotic water permeability was determined in the same endosomes from the rapid kinetics of FITC-dextran fluorescence following an inward sucrose gradient. The osmotic water permeability coefficient was 0.03 cm/s at 23 degrees C. Water permeability was inhibited by 70% with addition of 0.5 mmol/L mercuric chloride. The inhibition was reversed completely by adding 5 mmol/L mercaptoethanol. These data demonstrate that proximal tubule endosomes contain a proton ATPase and water channel. The endocytic process may be important for regulation of acidification and fluid resorption in the proximal tubule.