Role of vacuolar pH on the receptor-mediated endocytosis of horseradish peroxidase in isolated rat sinusoidal endothelial cells

The role of vacuolar type H+-ATPases (v-ATPases) and vacuolar pH in the endocytosis of horseradish peroxidase, a mannose-terminated glycoprotein, was investigated in isolated rat sinusoidal endothelial cells. Toward this purpose, a specific inhibitor of v-ATPases, bafilomycin Al, was used to inhibit v-ATPases in the vacuolar system. Vacuolar pH was monitored using acridine orange with fluorescent microscopy. Uptake of the mannose-terminated glycoprotein was examined using horseradish peroxidase with electron microscopy. Fluorescent staining by acridine orange showed that bafilomycin Al rised the vacuolar pH of the endocytic compartments. Horseradish peroxidase was taken up via mannose receptors and was distributed in the tubulo-vesicular network in the isolated sinusoidal endothelial cells. Uptake of horseradish peroxidase was significantly inhibited by bafilomycin Al, and this findings were confirmed by the morphometrical analysis. Results suggest that: (a) v-ATPases are necessary for acidification of the endocytic compartments in the sinusoidal endothelial cells and (b) the vacuolar pH which is generated by v-ATPases is necessary for the receptor-mediated endocytosis of a mannose-terminated glycoprotein, horseradish peroxidase. And bafilomycin Al appears to be useful for studying various roles of v-ATPases in sinusoidal endothelial cells.