Adaptation to metabolic acidosis by turtle urinary bladder.

We utilized the turtle urinary bladder to study the mechanisms responsible for adaptation to metabolic acidosis. Bladders removed from acidotic turtles had a higher rate of H+ secretion in vitro than bladders from control turtles, despite identical extracellular pH. HCO3 secretion, however, was not different between the two groups. The increase in H+ secretion could be mediated by a decrease in intracellular pH and/or by an increase in the number of cells thought to be responsible for H+ secretion. To study this issue, we measured intracellular pH with the fluorescent dye 6-carboxyfluorescein diacetate and quantified the number of cells by fluorescence microscopy utilizing acridine orange, rhodamine 123, and 6-carboxyfluorescein diacetate in turtles receiving different acid loads. Urinary acidification measured in vivo was increased in turtles fed a low-acid load for 48 h and in turtles fed a high-acid load for 24-48 h. Intracellular pH was lower in bladders from turtles fed a high-acid load for 48 h but it was not different from controls in the other groups, indicating that intracellular pH cannot account for the adaptive increase in H+ secretion. Bladders from all groups fed an acid load had a higher number of cells with positive staining for acridine orange compared with controls. Double labeling with acridine orange and the mitochondrial stain rhodamine 123 or 6-carboxyfluorescein showed a significant increase in the number of mitochondria-rich cells between control and bladders from turtles fed an acid load. The increase in the number of rhodamine 123- or 6-carboxyfluorescein-positive cells was lower than the increase in acridine orange-positive cells, suggesting that the apparent increase in the number of acridine orange-positive cells is due to an increase in the number of acidic vesicles in the mitochondria-rich cells and in the granular cells rather than solely to an increase in the number of mitochondria-rich cells. Plasma membrane fraction prepared from control and acidotic bladders failed to disclose an increase in the putative H+-ATPase as assessed by enzymatic activity and transport studies. In conclusion, the present study suggests that the adaptive increase in H+ secretion in metabolic acidosis is associated both with an increase in the number of mitochondria-rich cells as well as with an increase in the number of acidic vesicles in these cells.