Carbonic Anhydrase Function and the Epithelial Organization of H+ Secretion in Turtle Urinary Bladder

Abstract

The function of carbonic anhydrase in H⁺ secretion by the turtle bladder was studied in vitro. Dose response curves were obtained for the carbonic anhydrase inhibitors, acetazolamide and ethoxzolamide, with and without addition of CO₂ to the system. In addition, carbonic anhydrase was assayed in homogenates of mucosal cells. The activity in the homogenates was 155±16 U/g dry wt, of which only 11 U represented contamination from erythrocytes; after addition of 5 × 10⁻⁶m acetazolamide, no enzyme activity was detectable. In the intact preparation free of exogenous CO₂, the dose response curve for acetazolamide showed two plateaus of inhibition, one at 50% and one at more than 80% inhibition. At 50% inhibition (from 5 × 10⁻⁶ to 5 × 10⁻⁵m acetazolamide), H⁺ secretion was restored or enhanced by CO₂ addition to the same extent as bladders not exposed to acetazolamide. At concentrations of more than 1 × 10⁻⁴m, H⁺ secretion was no longer restorable by CO₂. Unlike acetazolamide, ethoxzolamide caused progressive inhibition of H⁺ secretion in the CO₂-free system. The maximal extent of inhibition with ethoxzolamide and the behavior of inhibition in the presence of 2.5% CO₂ were the same as for acetazolamide. Evidence is presented that all surface epithelial cells secrete H⁺ and generate OH⁻ within the cell interior. The capacity of cells to dispose of OH⁻ by CO₂ hydroxylation varies with the availability of cytoplasmic carbonic anhydrase. A small population of cells contains abundant carbonic anhydrase and secretes at high rates even when CO₂ is in short supply. On the basis of these results and histochemical data on the distribution of carbonic anhydrase within the mucosa, an analysis is presented of the epithelial organization of acidification by the turtle bladder.