We and others recently have demonstrated adenosine triphosphate-dependent acidification in a variety of prelysosomal organelles isolated from liver including clathrin-coated vesicles, multivesicular bodies, and Golgi. Little is known, however, regarding the number or distribution of acidic compartments in intact hepatocytes. We therefore have utilized acridine orange, a fluorescent weak base, to study the number and distribution of acidic vesicles of rat hepatocytes in primary culture and compared these with the number and distribution of lysosomes and other storage vesicles. Hepatocytes were found to contain about 170 acidic compartments per cell by fluorescence microscopy. These vesicles were diffusely distributed throughout the cell cytoplasm, with about 50% in the perinuclear area by modified morphometry. The acridine orange staining of these vesicles was reversibly dissipated by monensin, NH4C1, chloroquine, and primaquine, indicating these vesicles exhibit an acidic interior established by active proton transport. In addition, the cholestatic agent chlorpromazine reversibly inhibited, in a dose-dependent fashion, the redevelopment of a pH gradient in the acidic vesicles after dissipation by monensin. The number and distribution of these acidic vesicles were not significantly different from the number and distribution of vesicles involved in the storage (up to 6 h after internalization) of the fluid phase marker fluorescein-dextran. By contrast, histochemically identifiable lysosomes were fewer in number and significantly more restricted in their distribution to the perinuclear area (89%) than either dextran-storing or acidic vesicles. Electron microscopic studies confirmed, that endocytosed dextran as well as another fluid phase marker, colloidal gold, were found predominantly in acid phosphatase- and arylsul f atase-negative vesicles for up to 6 h after internalization. These studies indicate that hepatocytes contain numerous intracellular vesicles acidi f ed by an active H+ transport mechanism. Based on their comparative number and distribution, acidic vesicles probably include vesicles involved in fluid-phase endocytosis but only a minority are lysosomes. The findings also indicate that fluid-phase markers are stored predominantly in vesicles other than histochemically identifiable 1ysosomes for up to 6 h after internalization. Finally, this technique also affords the opportunity for studying the movement of such vesicles in a vital preparation.
- fluorescein isothiocyanate dextran