FLOW CYTOMETRIC ANALYSIS OF VESICULAR PH IN RAT HEPATOCYTES AFTER ETHANOL ADMINISTRATION

We examined the effect of ethanol administration on intravesicular pH in intact hepatocytes by applying a flow cytometric technique to detec t fluorescein-isothiocyanate-dextran (FITC-dextran) in acidic vesicles . Rats were pair-fed liquid diets containing either ethanol or isocalo ric carbohydrate for 1 to 5 weeks. Our study showed that ethanol admin istration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 pH units. This pH increase was sufficient to cause a significant redu ction in lysosomal protein degradation. Long-term ethanol administrati on also caused a significant alkalinization of hepatic endosomes, and this increased pH was sustained over the course of vesicular acidifica tion in hepatocytes incubated in vitro. Direct exposure of hepatocytes from rats fed control diet to either 25 mmol/L ethanol or 50 mu mol/L colchicine also brought about a rapid alkalinization of acidic vesicl es in a manner that resembled that seen in hepatocytes from ethanol-fe d rats. These same treatments augmented the vesicular alkalinization a lready present in cells from ethanol-fed animals. Although ethanol adm inistration had no effect on the content of the hepatic mannose-6-phos phate/IGFII receptor, the results indicate that sustained alkalinizati on of endosomes could have important functional consequences by impair ing M-6-P/IGFII receptor recycling, thereby disrupting the delivery of newly synthesized hydrolases to lysosomes. This decreased complement of hydrolases within lysosomes together with alkalinization of the int ralysosomal compartment would result in an overall decrease in lysosom al proteolysis.