Fluorescence identifies an alkaline cell in turtle urinary bladder.

Intracellular pH (pHi) of turtle bladder mucosal cells was studied by the trapped fluorescent indicator technique. Bladders efficiently accumulated and converted 4-methylumbelliferyl acetate to its pH-sensitive derivative 4-methylumbelliferone (4MU). Excited at the pH-indifferent wavelength 334 nm, bladders fluoresced a uniform blue. Using pH-sensitive 365-nm excitation, 10-20% of the mucosal cells fluoresced distinctly brighter, suggesting a more alkaline pHi. Using the 365/334 ratio to quantitate pHi, this difference averaged 0.1 pH units. Bright cells were more distinct after SITS or acetazolamide but disappeared after digitonin permeabilization, dinitrophenol, or treatment with propionate, DMO, and NH4Cl. Essentially the same population of bright cells was identified by carboxyfluorescein diacetate. The brighter cell corresponded exactly to a population of cells with distinctive acridine orange staining and bright costaining with the potential-sensing probes Di-O-C5, Di-S-C3, and 4-Di-5-Asp. Two extremes of bright cell shape were seen: an elongate cell, prevalent under conditions stimulating H+ secretion, and a more compact cell, when acidification was inhibited. These observations support the hypothesis that acidification represents H+ secretion via the luminal membrane and that a primary role of carbonic anhydrase in this process is to support the exit of base from the cell. The more alkaline cells appear to be the carbonic anhydrase-rich cells. These cells are chemically isolated from the surrounding granular cells and change their morphology in response to changes in acidification. These special properties indicate a unique role for the carbonic anhydrase cell in H+ secretion.