Permeabilizing the granular cell of toad and turtle bladder: lack of cell coupling

Mucosal cells of the toad and turtle urinary bladder have cell membranes that exclude charged fluorescent dyes. We describe a simple and effective method of transiently permeabilizing the apical membranes of mucosal cells in situ by applying mild mechanical stress to the bladder's mucosal face. The technique produces clusters of individually distinguishable granular cells, which are loaded with and retain membrane-impermeant fluoroprobes and macromolecules. Carbonic anhydrase-rich cells are resistant to the permeabilization. The apical membranes regain functional integrity after permeabilization, as indicated by a return of transepithelial electrical resistance, an inability to load cells except immediately after the stress, and the observation that loaded cells behave identically to normal cells in regulating cell pH. With the use of this technique, BCECF and fura-2 were loaded into granular cells and used successfully to follow cell pH and cell calcium. In granular cells loaded with these dyes or Lucifer Yellow, there was no detectable spread of dye into adjacent cells. This lack of dye coupling was confirmed by use of conventional iontophoresis of dye into normal granular cells. Electrical coupling was also undetectable between granular cells separated by distances less than 30 micron. We conclude that none of the mature cells of the bladder surface are directly coupled.