AJP - Renal Physiology, Vol 263, Issue 1 62-F76, Copyright © 1992 by American Physiological Society
Functional and structural characterization of endosomes from toad bladder epithelial cells
M. L. Zeidel, T. Hammond, B. Botelho and H. W. Harris Jr
Department of Veterans Affairs Medical Center, West Roxbury, Massachusetts 02132.
Previous functional studies of toad bladder endosomes have been complicated
by the presence of multiple endosome subpopulations each possessing
different permeability characteristics. To identify and characterize both
water channel-containing vesicles (WCV) and other endosome subpopulations,
we combined flow cytometry, electron microscopy, stop-flow fluorometry, and
sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Flow cytometry
of endosomes identified distinct populations of fluorescein-labeled
endosomes in bladders after removal of antidiuretic hormone (ADH)
stimulation (ADH withdrawal). Centrifugation separated the larger
fluorescein-labeled vesicles, sedimenting at lower speed (intermediate
pellet, IP), from the smaller fluorescein-labeled vesicles, sedimenting at
high speed (high-speed pellet, HSP). Permeability and structural studies of
these subpopulations revealed the following. 1) IP endosomes labeled 10 min
after ADH withdrawal (ADH IP) represented a highly purified population of
WCV with high water permeability (Pf) that exhibited a low-activation
energy and sensitivity to organic mercurials. 2) IP endosomes from
unstimulated bladders did not contain functional water channels. 3) HSP
from either ADH withdrawal or unstimulated bladders exhibited low Pf and
acidified after addition of extravesicular ATP; moreover, protein
compositions of purified HSP were distinct from those of purified IP. These
results suggest that HSPs represent constitutive and not ADH-sensitive
endosomes. 4) High permeability to protons (PH+) was seen in ADH IP
endosomes but not the other fractions, providing strong evidence that the
ADH water channel conducts protons. 5) Multivesicular bodies (MVB)
exhibited low Pf and PH+, indicating that they do not possess functional
water channels.(ABSTRACT TRUNCATED AT 250 WORDS)
