ETHANOL EFFECT ON ENDOCYTOSIS IN LIVER

This proposal represents a request for an ADAMHA
Research Scientist Development Award (RSDA). The central hypothesis of the
proposed investigation is that faulty acidification of endosomes and other
acidic organelles after ethanol administration is a major mechanism by
which ethanol impairs RME and other protein trafficking pathways.
Acidification of endocytic vesicles is critical to efficient sorting and
delivery of ligands and/or receptors to various compartments in the cell.
We have used asialoglycoproteins as model ligands for studying RME and have
identified several steps of the multi-step pathway that are affected by
ethanol treatment, including impaired receptor recycling, altered
dissociation of intracellular receptor-ligand complexes, and impaired
internalization of the complexes into coated pits. Mechanisms which have
been proposed to explain defective RME include formation of acetaldehyde
adducts to tubulin resulting in impaired microtubule function, improper
acidification of endosomes and defective receptor clustering in coated
pits. The initial objective of our study is to demonstrate that ethanol
administration impairs ATP-dependent acidification of prelysosomal
endosomes after 1-5 weeks of ethanol feeding. In addition we plan to
determine the effect of ethanol administration on ATP-dependent
acidification in other subcellular organelles which are known to contain
acidic interiors, namely Golgi apparatus, endoplasmic reticulum and
lysosomes. A third objective is to examine potential impairment of
acidification which may be induced by acetaldehyde. In these studies, we
hope to establish whether the major metabolite of ethanol induces
alterations in acidification similar to previously reported alterations in
protein trafficking. Investigation of whether faulty acidification could
lead to inactivation of the asialoglycoprotein receptor and subsequently
alter synthesis and turnover of the receptor will be the goal of the fourth
specific aim. A final objective is to clarify the effects of ethanol
administration on the clathrin-coated vesicle population, since initial
internalization via coated pits is impaired by ethanol treatment.
Clathrin-coated vesicles are acidic organelles with an ATP dependent proton
pump and we will measure acidification in these vesicles as well as rates
of vesicle uncoating, a process which is necessary before vesicle fusion
and subsequent transport of material across the cell can occur. The
proposed studies should give valuable information concerning the basic
molecular mechanism(s) of alcohol-induced hepatotoxicity. In addition to the research plans outlined above, the principal
investigator will also maintain an active interest in teaching and training
students in alcohol-related research. These additional areas of
participation include lecturing in graduate level course as well as
actively participating in other educational activities at the University of
Nebraska Medical Center including journal clubs and problem solving days.