bromochloroacetic-acid and diacetylfluorescein

The role of cytokeratin filaments in the function of hepatocytes was investigated using a nickel-treated hepatocyte in vitro model. Cytokeratin intermediate filaments were selectively dissociated from the cell cortex by nickel treatment. Cytokeratins and ubiquitin were observed using immunofluorescence and immunoelectron microscopy. Hepatocytic function was assessed by visualizing uptake, transhepatic transport and secretion of fluorescein diacetate and horseradish peroxidase into the bile canaliculi. In control primary cultures, most of the bile canaliculi were surrounded by an inner layer of actin filaments and an outer pericanalicular sheath of cytokeratin filaments and microtubules. The cytoplasmic distribution of ubiquitin was diffuse and particulate. After treatment with NiCl2 (150 micrograms/ml) for 24 hr, the cytokeratin filaments and desmoplakin became focally detached from the cell cortex and retracted to form an aggregate around the nucleus. These aggregates were associated with intense ubiquitin immunoreactivity. Only a few attachments of the cytokeratin filaments to the cell cortex remained. F-actin remained attached to the cell cortex in the areas where the cytokeratin filaments had become detached. The pericanalicular sheath of cytokeratin filaments and the bile canaliculi disappeared and actin was dispersed over the entire cell periphery. Fluorescein diacetate secretion and horseradish peroxidase uptake were almost completely absent in the hepatocytes treated with nickel. The effects of nickel persisted 24 hr after its removal from the medium. It is concluded that cytokeratin intermediate filaments play a critical role in the formation of the bile canaliculus, secretion of fluorescein diacetate and uptake of horseradish peroxidase. Further, our study indicates that cytokeratin ubiquitination occurs during collapse and aggregation of the cytokeratin filaments. The formation of cytokeratin-ubiquitin conjugates during aggregation suggests a role of ubiquitin in the control of cytokeratin organization in hepatocytes in the response to cell stress.

Topics: Animals; Animals, Suckling; Bile Canaliculi; Bile Ducts, Intrahepatic; Biological Transport; Cells, Cultured; Cytoskeleton; Fluoresceins; Fluorescent Antibody Technique; Horseradish Peroxidase; Immunohistochemistry; Intermediate Filaments; Keratins; Liver; Male; Microscopy, Electron; Nickel; Rats; Rats, Inbred Strains; Ubiquitins

We determined the role of cytokeratin (CK) intermediate filaments in the excretory function of hepatocytes in cultured hepatocytes containing Mallory bodies (MBs) from the livers of griseofulvin (GF)-fed mice. Hepatocytes for primary culture were obtained from GF-fed and control mice using the 0.1% collagenase perfusion method. Each component of the cytoskeleton in cultured hepatocytes and liver frozen sections was visualized by immunofluorescence. The whole mount extraction of hepatocytes was carried out using 0.5% Triton X-100. To examine the excretory function of the bile canaliculi (BC), fluorescein diacetate and horseradish peroxidase were used as visible excretory products. Thin sections of the cultured cells were made by the "pop-off" method for electron microscopic examination. Frozen sections of livers from the GF-fed mice showed that the MBs were stained with a rat monoclonal antibody to mouse CK, but the CK filaments in the cells containing MBs did not stain. The intercellular BC were reduced in number in the livers of the GF-fed mice compared with the controls. At 3 hours after seeding, hepatocytes with MBs were not stained, but by 24 hours the CK filament network stained normally in cells containing MBs. The loss of staining of the CK filaments was therefore rapidly reversible in the absence of GF in tissue culture. This reversion to normal was prevented by adding 2 x 10(-4) m GF to the culture medium. Thus, the loss of the CK filament antigenic determinants was directly maintained by GF in vitro. The extracted hepatocytes showed spherical canalicular sheaths formed by the CK filaments within the cytoplasm. This was confirmed in "pop-off" sections which revealed that the canaliculi were lined by microvilli and by the localization of actin around the canaliculi as visualized by immunofluorescence. Excretion of fluorescein diacetate into the intracytoplasmic BC was seen both in the cells from GF and control mice but uptake of horseradish peroxidase was markedly reduced by the hepatocytes from the GF-fed mice. The results show that the hepatocytes containing MBs do not form intercellular BC and excretion of fluorescein diacetate into intracytoplasmic BC is not impaired but the uptake of horseradish peroxidase is markedly reduced. The results imply that the rearrangement of the cytoskeleton induced by GF causes both structural and functional deficits in the affected hepatocytes.

Topics: Actins; Animals; Antibodies, Monoclonal; Cells, Cultured; Cytoskeleton; Endocytosis; Fluoresceins; Fluorescent Antibody Technique; Griseofulvin; Horseradish Peroxidase; Immunohistochemistry; Inclusion Bodies; Intermediate Filaments; Keratins; Liver; Male; Mice; Mice, Inbred C3H; Microscopy, Electron; Tubulin