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.
In Specific Aim 1, lysosome biogenesis will be measured by examining the processing, and compartmentalization of the protease, cathepsin L in hepatocytes isolated from control and ethanol-fed rats. This enzyme follows a specific pathway through vesicular compartments en route to the lysosome and the investigator's Aim 1 is to determine the step(s) at which this process is impaired by ethanol, because misrouting of cathepsin L and other hydrolases could potentially cause cell damage due to their potent hydrolytic capacities.
In Specific Aim 1 b they will examine whether ethanol influences the distribution of the mannose-6-phosphate receptor by subcellular fractionation immunocytochemistry and functional assay studies. This receptor mediates lysosome assembly by targeting cathepsin L and other hydrolases to the lysosome. The investigators postulate that ethanol may change its intracellular distribution as well as its ligand affinity for procathepsin L.
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 intra gastric feeding regimens. This nonlysosomal proteolytic pathway has a crucial role in: 1) the degradation of altered proteins; and 2) the activation of the transcription factor NFkappaB which initiates transcription of genes involved in the inflammatory and immune responses. The investigators postulate that while ethanol may down-regulate the proteasome in liver parenchymal cells, its activity may be regulated differentially in Kupffer cells, since they play a paracrine role in the pathogenesis of alcoholic liver disease.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
2R01AA009384-05A1
Application #
2000288
Study Section
Special Emphasis Panel (ZRG4-ALTX-4 (01))
Project Start
1992-08-01
Project End
2000-03-31
Budget Start
1997-04-01
Budget End
1998-03-31
Support Year
5
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Nebraska Medical Center
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Omaha
State
NE
Country
United States
Zip Code
68198
Curry-McCoy, Tiana V; Osna, Natalia A; Nanji, Amin A et al. (2010) Chronic ethanol consumption results in atypical liver injury in copper/zinc superoxide dismutase deficient mice. Alcohol Clin Exp Res 34:251-61
Curry-McCoy, Tiana V; Osna, Natalia A; Donohue Jr, Terrence M (2009) Modulation of lysozyme function and degradation after nitration with peroxynitrite. Biochim Biophys Acta 1790:778-86
Osna, Natalia A; White, Ronda L; Krutik, Viatcheslav M et al. (2008) Proteasome activation by hepatitis C core protein is reversed by ethanol-induced oxidative stress. Gastroenterology 134:2144-52
Donohue, Terrence M; Curry-McCoy, Tiana V; Nanji, Amin A et al. (2007) Lysosomal leakage and lack of adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanol-induced liver injury in female rats. Alcohol Clin Exp Res 31:1944-52
Donohue Jr, Terrence M; Curry-McCoy, Tiana V; Todero, Sandra L et al. (2007) L-Buthionine (S,R) sulfoximine depletes hepatic glutathione but protects against ethanol-induced liver injury. Alcohol Clin Exp Res 31:1053-60
Osna, Natalia A; Clemens, Dahn L; Donohue Jr, Terrence M (2005) Ethanol metabolism alters interferon gamma signaling in recombinant HepG2 cells. Hepatology 42:1109-17
Donohue Jr, Terrence M; Kharbanda, Kusum K; Casey, Carol A et al. (2004) Decreased proteasome activity is associated with increased severity of liver pathology and oxidative stress in experimental alcoholic liver disease. Alcohol Clin Exp Res 28:1257-63
Osna, Natalia A; Haorah, James; Krutik, Viatcheslav M et al. (2004) Peroxynitrite alters the catalytic activity of rodent liver proteasome in vitro and in vivo. Hepatology 40:574-82
Donohue Jr, Terrence M; Osna, Natalia A (2003) Intracellular proteolytic systems in alcohol-induced tissue injury. Alcohol Res Health 27:317-24
Haorah, James; MacDonald, Richard G; Stoner, Julie A et al. (2003) Ethanol consumption decreases the synthesis of the mannose 6-phosphate/insulin-like growth factor II receptor but does not decrease its messenger RNA. Biochem Pharmacol 65:637-48

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