Ouabain-Induced Cytoplasmic Vesicles and Their Role in Cell Volume Maintenance

Figure 5

Aquaporin-8 localization in ouabain-induced vesicles and plasma membrane. Liver slices were incubated at 1°C for 90 min and then for 70 min at 38°C in the absence ((a), (b), and (c)) or presence ((d), (e), and (f)) of 2 mM ouabain. At the end of treatments, samples were slightly fixed (5 min in 4% paraformaldehyde) and cryofixed in liquid nitrogen. Ultrathin cryosections were obtained at the cryoultramicrotome, treated with a monoclonal antibody for AQ8 and then with protein A conjugated with 20 nm gold particles. Sections were postfixed and contrasted with osmium tetroxide vapors. (a) Control warm incubation. No gold particles are visible on the vesicle membranes. (b) Control warm incubation. Detail of (a). Few particles are visible on bile canalicular (BC) plasma membrane. (c) Control warm incubation. Occasional clusters of gold particles on the lateral plasma membrane. (d) Control warm incubation in the presence of 2 mM ouabain. A cluster of gold particles is present on the membranes where a typical electron-clear vesicle (V) is fusing with lateral plasma membrane (LP) (arrow). (e) Control warm incubation in the presence of 2 mM ouabain. A number of gold particles are visible on the membrane of an electron-clear vesicle. (f) Control warm incubation in the presence of 2 mM ouabain. Detail of (e) showing the close association between gold particles and vesicular membrane.