Acidification of vasopressin-induced endosomes in toad urinary bladder

It is well established that water channels (WC) are removed from the apical membrane of vasopressin-sensitive epithelia by endocytosis. The processing and the ultimate fate of endocytosed WC is, however, incompletely understood. In many cells, endosome acidification plays an important role in the processing and sorting of endocytosed proteins. Endosome acidification in the toad urinary bladder was therefore examined in vivo by fluorescence ratio video microscopy after induction of endocytosis by vasopressin removal and transepithelial water flow in the presence of the pH-sensitive fluid phase marker 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-dextran. Fifteen minutes after induction of endocytosis, the majority of endosomes had a neutral or slightly acidic pH. The number of acidic endosomes increased progressively with time. Two hours after endocytosis began, 98% of the endosomes had a pH < 6.0. Bafilomycin completely blocked endosome acidification, indicating that H+ transport is mediated by a vacuolar H(+)-adenosinetriphosphatase. Bafilomycin had no effect on transepithelial water flow in bladders repetitively stimulated by vasopressin. These findings, as well as the work of other investigators, suggest that if WC recycling occurs, it is not dependent on acidification of the endosomal compartment. Acidification of vasopressin-induced endosomes most likely represents a terminal event in the endocytic pathway.