The hypotheses of this proposal are: 1) chronic ethanol consumption impairs lysosome biogenesis by preventing processing and trafficking of lysosomal hydrolases, causing their placement at other intracellular or extracellular sites; (2) Ethanol administration alters the ubiquitin- proteasome pathway by inactivating the proteasome which could lead to accumulation of modified proteins. Ethanol may differentially influence the proteasome in Kupffer cells. 2) Ethanol administration impairs the capacity of hepatocytes to degrade proteins modified by ethanol metabolism.
In Specific Aim 1 a, Lysosome biogenesis will be measured by examining the processing and compartmentalizatin of cathepsin L in hepatocytes isolated from control and ethanol-fed rats. This enzymes follows a specific pathway through vesicular compartments en route to the lysosome and our aim is to determine the step(s) at which ethanol impairs this process. Misrouting of cathepsin L and other hydrolases could potentially cause cell damage due to their potent hydrolytic capacities.
In Specific Aim 1 b we will use subcellular fractionation immunocytochemistry and functional assays to examine whether ethanol influences the distribution of the mannose-6-phosphate receptor. This receptor mediates lysosome assembly by targeting cathepsin L and other hydrolases to the lysosome. We postulate that ethanol may change the receptor's intracellular distribution.
In Specific Aim 2, the components of the ubiquitin-proteasome pathway will be examined in whole livers as well as parenchymal and Kupffer cells of control and ethanol- fed rats subjected to both ad lib and intragastric feeding specimens. This proteolytic pathway has a crucial role in 1) the degradation of altered proteins; and 2) the activation of the transcription factor NfkappaB which is involved in the expression of the inflammatory response. We postulate that while ethanol may down-regulate the proteasome in liver parenchymal cells, its activity may be regulated differentially in Kupffer cells, since the latter cells play a paracrine role in the pathogenesis of alcoholic liver disease.
Specific Aims 3 and 4 will address third hypothesis by testing the capacity of hepatocytes and their extracts to degrade altered (i.e. aldehyde-modified) and native forms of lysozyme.
