FUNCTIONAL AND STRUCTURAL CHARACTERIZATION OF ENDOSOMES FROM TOAD BLADDER EPITHELIAL-CELLS

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 sub-populations, 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 (P(f)) 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 P(f) 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 (P(H+)) 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 P(f) and P(H+), indicating that they do not possess functional water channels. 6) Because protein compositions of ADH IP and MVB were strikingly similar, it is possible that water channel proteins are transferred from the 10-min IP fraction to MVB and inactivated.