Exocytosis regulates urinary acidification in turtle bladder by rapid insertion of H+ pumps into the luminal membrane

Urinary acidification by the turtle bladder is due to a H+-ATPase that is located in the luminal membrane. The rate of H+ transport is stimulated by an increase in the ambient CO2. Using the fluorescent dye acridine orange, we showed that the mitochondria-rich cell of this equilibrium contains vesicles whose internal pH is acidic. We measured the pH of these vesicles by using endocytosed fluorescein isothiocyanate-labeled dextran and found it to be near 5.0. The pH increased after treatment with protonophores or metabolic inhibitors, suggesting that it was due to a H+ pump rather than to a Donnan effect. In bladders preloaded with fluorescent dextran, CO2 stimulated exocytosis and H+ transport measured simultaneously in the same bladder. The increase in the H+ current correlated well with the extent of exocytosis, and both were inhibited by pretreatment with colchicine. We conclude that the turtle bladder contains an intracellular reserve of vesicles containing H+ pumps and CO2 stimulates rapid fusion of these vesicles with the luminal membrane with consequent insertion of H+ pumps, thereby stimulating H+ secretion across the whole epithelium.