Flow cytometric analysis of vesicular pH in rat hepatocytes after ethanol administration

Abstract

We examined the effect of ethanol administration on in-sponsible in part for a net hepatic protein gain in ethanolfed animals. 1,2 More recently, we showed that decreased degtravesicular pH in intact hepatocytes by applying a flow cytometric technique to detect fluorescein-isothiocyanate-dextran radation is associated with a deficiency in the content of cathepsins B and L in lysosomes of ethanol-fed rats. 3 This (FITC-dextran) in acidic vesicles. Rats were pair-fed liquid diets containing either ethanol or isocaloric carbohydrate for deficiency may arise from ethanol-induced alterations in the trafficking and processing of lysosomal hydrolases, 4 as indi-1 to 5 weeks. Our study showed that ethanol administration increased the in situ pH of hepatic lysosomes by 0.15 to 0.2 cated by our recent experiments that showed that ethanol consumption by rats delays the processing of procathepsin pH units. This pH increase was sufficient to cause a significant reduction in lysosomal protein degradation. Long-term etha-L to its mature form in hepatocytes from these animals. 4 A similar influence on the maturation of lysosomal b glucuronnol administration also caused a significant alkalinization of idase was reported after acute ethanol administration. 5 The hepatic endosomes, and this increased pH was sustained over mechanism(s) underlying these ethanol-elicited impairments the course of vesicular acidification in hepatocytes incubated is not clear, but altered vesicular acidification may play a in vitro. Direct exposure of hepatocytes from rats fed control role. For example, faulty acidification of intracellular vesicles diet to either 25 mmol/L ethanol or 50 mmol/L colchicine also may prevent the pH-dependent dissociation of procathepsins brought about a rapid alkalinization of acidic vesicles in a and other lysosomal precursors from the mannose-6-phosmanner that resembled that seen in hepatocytes from ethanolphate receptors, a class of proteins that targets these precurfed rats. These same treatments augmented the vesicular alkasors to lysosomes. [6][7][8] In addition, ethanol administration may linization already present in cells from ethanol-fed animals.affect the level or activity of the receptors themselves. PreviAlthough ethanol administration had no effect on the content ous reports of alcohol-induced changes in vesicular acidificaof the hepatic mannose-6-phosphate/IGFII receptor, the retion used isolated organelles to determine whether ethanol sults indicate that sustained alkalinization of endosomes consumption affected the acidification of vesicles along the could have important functional consequences by impairing endosomal-lysosomal pathway. Schneider and Manara 9 re-M-6-P/IGFII receptor recycling, thereby disrupting the delivported that alcohol ingestion decreases the H / -adenosine triery of newly synthesized hydrolases to lysosomes. This dephosphatase activity responsible for acidification of intracelcreased complement of hydrolases within lysosomes together lular vesicles and reduces adenosine triphosphate-...

Autophagy and ethanol-induced liver injury

“…Pulsechase analyses, using isolated hepatocytes demonstrated that the reduced lysosomal content of cathepsin L was the result of delayed trafficking of its nascent precursor and impaired processing to its mature catalytic form in cells from ethanol-fed rats [16,30,31] . Both flow cytometric and microscopy studies showed that vesicular movement and acidification of lysosomes in hepatocytes are both impeded after ethanol exposure [32,33] .…”

“…glycogen) to nucleic acids. Macromolecular catabolism takes place inside the lysosomal lumen at hydrogen ion concentrations that are 500-fold higher (pH about 4.7) than that in the cytoplasm (pH about 7.4) [1] . Maintenance of such an acidic lysosomal interior occurs through the continuous action of ATP-dependent proton transporters that reside on its membrane.…”

Multilevel regulation of autophagosome content by ethanol oxidation in HepG2 cells

“…[4][5][6] Lysosomes are crucial for the breakdown of all forms of cellular macromolecules. They participate in the catabolic phase of macroautophagy (autophagy), which begins with the formation of double-membrane-bound autophagic vacuoles (AVs or autophagosomes) that envelop and sequester soluble Acute and chronic ethanol administration increase autophagic vacuole (i.e., autophagosome; AV) content in liver cells.…”

Relevance of autophagy to fatty liver diseases and potential therapeutic applications

“…In mice fed with Lieber-DeCarli diet for 4 weeks, autophagy was elevated at a lower dose of ethanol, accounting for 29% of the caloric need, but was inhibited at a higher ethanol dose (accounting for 36% of the caloric need) (Lin et al 2013). Decrease in both the amount and the function of lysosomes was found in chronic alcohol treated rat livers, which may contribute to the autophagy suppression (Kharbanda et al 1996; Kharbanda et al 1997; Dolganiuc et al 2012). However, the liver injury is worse when autophagy is suppressed while enhancement of autophagy improves the condition (Lin et al 2013).…”