Research will focus on the bile canaliculus, a specialized domain of the hepatocyte plasma membrane. Constituents of the canaliculus include structural elements, a variety of transporters for solute flux in and out of the canalicular lumen, a protease that cleaves plgA before its excretion into bile, and a rich web of cytoskeleton that is necessary for active canalicular contraction. Our approach is to first perturb the canaliculus with a toxicant and then to identify impairments in function, alterations in structure, and changes in canalicular constituents. Our ultimate goal is to clarify the interrelationships among these perturbations. In the prior funding period, we found rats treated with 1,1-dichloroethylene (DCE) to be an excellent in vivo model of canalicular injury. Our working hypothesis is that this target site is a consequence of DCE metabolism to reactive GS-conjugates which are stable enough to congregate in the pericanalicular regions, enroute to excretion in bile. DCE-induced structural changes were confined to zone 3 (centrolobulas, canaliculi, and were accompanied by diminished staining for 4 canalicular components indicating their loss or alteration. Concurrent changes in biliary function were slower organic anion excretion, > 90% decrease in a lgA and a 40% total protein excretion, but no alterations in bile volume, bile salt excretion, or Pi exclusion. Proposed function studies will use freely moving rats with exteriorized cannulas and physiological replacement of bile salt.
AIM 1 will establish the utility of fluorescent markers that allow zonal localization of functional impairments.
AIM 2 will attempt to distinguish injury-associated functional alterations from less relevant alterations due to a secondary depletion of GSH.
AIM 3 will test our protein, by impairing the canalicular-localized, pre-excretion processing of plgA to its secreted form.
AIM 4 will characterize effects on DCE on the pericanalicular cytoskeleton and verify the apparent sparing of the tight junctions between hepatocytes.
AIM 5 will use canalicular membrane vesicles and hepatocyte couplets to address mechanistic questions about the postulated role of the reactive DCE-GS conjugates in the canaliculi injury. New coinvestigator will facilitate this AIM by synthesizing stable and reactive forms of DCE-GS conjugates, by microinjecting these compounds into hepatocyte couplets for toxicity studies, and by providing an antibody to the canalicular membrane GS-conjugate transporter that we predict is a particularly vulnerable target. Information obtained should clarify the pathogenesis of DCE injury to the canaliculus.
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