Redistribution of Ca2+, Mg2+-ATPase activity in relation to alterations of the cytoskeleton and tight junctions in hepatocytes of cholestatic rat liver.

Ca2+, Mg2+-ATPase is a membrane-bound enzyme localized at the bile canalicular membranes of hepatocytes. Cytoskeleton and tight junctions are important for maintenance of the polar distribution of plasma membrane proteins. In order to understand the mechanisms involved in the redistribution of Ca2+, Mg2+-ATPase due to cholestasis, the relationship between Ca2+, Mg2+-ATPase, microfilaments and tight junctions was examined. Cholestasis was induced in rat liver by common bile duct ligation (CBDL) for 2 weeks. Localization of Ca2+, Mg2+-ATPase activity was studied at the light and electron microscopic level. Double-staining of the enzyme and F-actin was performed using phase-contrast and fluorescence microscopy of 7-nitrobenzene-2-oxa-1,3-diazole phallacidin (NBD-ph), respectively. Immunofluorescence microscopy of ZO-1 was applied for the observation of tight junctions. Furthermore, cytoskeleton and junctional complexes were investigated electron microscopically in saponin-extracted tissues. The results showed that CBDL induced redistribution of Ca2+, Mg2+-ATPase activity from the apical to the entire plasma membrane of hepatocytes, which seemed to occur independently of F-actin. F-actin was present at all membrane domains of hepatocytes in control liver, whereas CBDL increased the amounts of F-actin mainly at the bile canalicular membranes. An inverse distribution pattern of Ca2+, Mg2+-ATPase activity and F-actin was found in epithelial cells of bile ducts in control and cholestatic livers. Marked alterations in microfilaments were observed at the bile canaliculi, which were defined as hypertrophy and atrophy and were in association with changes in tight junctions. Structural impairment of the tight junctions was proven by disordered immunofluorescence of ZO-1. It is concluded that changes in the distribution of Ca2+, Mg2+-ATPase and F-actin due to CBDL are independent of each other. CBDL-induced disorders of microfilaments are related to impairment of structural integrity of tight junctions that is suggested to be responsible for the redistribution of Ca2+, Mg2+-ATPase in hepatocytes.