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.
