AJP - Renal Physiology, Vol 253, Issue 3 588-F594, Copyright © 1987 by American Physiological Society
Permeabilizing the granular cell of toad and turtle bladder: lack of cell coupling
M. Graber, P. R. Brink, D. DiLillo, P. Devine and E. Pastoriza-Munoz
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.
