AJP - Cell Physiology, Vol 262, Issue 5 C1109-C1118, Copyright © 1992 by American Physiological Society
Water and nonelectrolyte permeabilities of apical membranes of toad urinary bladder granular cells
E. B. Grossman, H. W. Harris Jr, R. A. Star and M. L. Zeidel
Medical Service, Brockton-West Roxbury, Department of Veterans Affairs Medical Center 02132.
Certain types of epithelial cells such as those lining the toad urinary
bladder have been classified as "tight" because their apical membranes
exhibit low permeabilities to water, ions, and small nonelectrolytes.
However, the permeability properties and structural features of these
specialized apical membranes remain unclear because these membranes have
never been purified. To isolate toad bladder granular cell apical
membranes, we derivatized the bladder apical surface with the
membrane-impermeant bifunctional reagent
N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin). After cell
disruption, these derivatized apical membranes were purified using
streptavidin-coated magnetic beads in a magnetic field. With the use of
lactoperoxidase-mediated radioiodination as a marker for apical membrane,
this preparative procedure purified apical membrane 48- or 72-fold as
compared with homogenate. Thin section electron microscopy revealed
unilamellar vesicles with some nonvesiculated membranes, while fragments of
organelles such as mitochondria were absent. Water and nonelectrolyte
permeabilities of purified apical membrane vesicles were similar to those
obtained in intact bladders in the absence of antidiuretic hormone
stimulation. The results demonstrate that isolated apical vesicles do not
contain water channels and confirm the applicability of Overton's rule to
the apical membrane of the toad urinary bladder. The technique has general
applicability to isolation of other plasma membranes, and the apical
membranes obtained are suitable for structural analysis.
