The goal of this proposed study is to define the molecular mechanisms by which alcohol, and its oxidized metabolite, acetaldehyde, impairs secretory and endocytic vesicular protein transport in hepatocytes. Specifically, we will test the hypothesis that alcohol disrupts the microtubule cytoskeleton and associated ATPases, or motor enzymes, which play a major role in the organization, transport, and targeting of different vesicle populations within the hepatocyte. Numerous reports by others have suggested that alcohol and its oxidized metabolites inhibit protein trafficking indirectly through a disruption of the microtubule cytoskeleton upon which most of these transport events depend. Despite these extensive in vitro studies, it has not been determined if alcohol alters microtubule organization within the hepatocyte, how this disruption occurs, and if other microtubule associated enzymes (motors) are also perturbed. Our goal is to utilize state of the art cell biological techniques to conduct a definitive and novel study on the mechanism by which alcohol interferes with microtubule- based vesicular trafficking within the hepatocyte. The proposed study has three specific aims. First, we will examine and compare how microtubules are organized and polarized within healthy control vs. ethanol-treated hepatocytes by: a) confocal-immunofluorescent and electron microscopic examination in conjunction with computer-aided reconstruction morphometry; b) polarity assays of microtubules in cultured hepatocyte couplets. Second, through the application of cell microinjection with computer- enhanced, fluorescent, video microscopy we will determine if secretory and endocytotic vesicular transport along microtubules in cultured hepatocyte couplets is altered by alcohol treatment. Third, we will test for disruptive effects of alcohol on vesicle transport and fusion by measuring alterations in the activity of microtubule-associated motor enzymes (kinesin, dynein, dynamin) through morphological and biochemical manipulation of permeabilized and homogenized hepatocytes, and cell-free systems using purified vesicular and cytoskeletal components. The technology and experiments described in this proposal are novel to the study of alcohol-induced cell-damage and to vesicular transport in hepatocytes. They will not only greatly expand our understanding of how alcohol impairs hepatocyte function but lend insight into the basic mechanisms of secretion and endocytosis in these cells.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA009227-03
Application #
2045450
Study Section
Biochemistry, Physiology and Medicine Subcommittee (ALCB)
Project Start
1992-04-01
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Mayo Clinic, Rochester
Department
Type
DUNS #
City
Rochester
State
MN
Country
United States
Zip Code
55905
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Chen, X M; Levine, S A; Tietz, P et al. (1998) Cryptosporidium parvum is cytopathic for cultured human biliary epithelia via an apoptotic mechanism. Hepatology 28:906-13
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Torok, N; Marks, D; Hsiao, K et al. (1997) Vesicle movement in rat hepatocytes is reduced by ethanol exposure: alterations in microtubule-based motor enzymes. Gastroenterology 113:1938-48
Urrutia, R; Henley, J R; Cook, T et al. (1997) The dynamins: redundant or distinct functions for an expanding family of related GTPases? Proc Natl Acad Sci U S A 94:377-84
Torok, N; Urrutia, R; Nakamura, T et al. (1996) Upregulation of molecular motor-encoding genes during hepatocyte growth factor- and epidermal growth factor-induced cell motility. J Cell Physiol 167:422-33
Larkin, J M; Oswald, B; McNiven, M A (1996) Ethanol-induced retention of nascent proteins in rat hepatocytes is accompanied by altered distribution of the small GTP-binding protein rab2. J Clin Invest 98:2146-57
Marks, D L; LaRusso, N F; McNiven, M A (1995) Isolation of the microtubule-vesicle motor kinesin from rat liver: selective inhibition by cholestatic bile acids. Gastroenterology 108:824-33
Torre, E; McNiven, M A; Urrutia, R (1994) Dynamin 1 antisense oligonucleotide treatment prevents neurite formation in cultured hippocampal neurons. J Biol Chem 269:32411-7

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