phalloidine and Cholestasis--Intrahepatic

We investigated the therapeutic effect of tauroursodeoxycholate on phalloidin-induced cholestasis in rats. Intrahepatic cholestasis was induced by administration of phalloidin (500 microg/kg, i.p.) for 7 days. From the day of the last phalloidin injection, tauroursodeoxycholate (60-360 micromol/kg) was given intravenously twice a day for 4 days. On the next day after the last tauroursodeoxycholate administration, bile flow, serum biochemical parameters and biliary lipid excretion rates were determined. Tauroursodeoxycholate significantly suppressed the decrease in bile flow and increases in serum alkaline phosphatase, leucine aminopeptidase and glutamic pyruvic transaminase activities, cholesterol, phospholipid and bile acid concentrations observed in phalloidin-induced cholestasis in rats. Furthermore, tauroursodeoxycholate significantly improved the biliary cholesterol and phospholipid excretion rates in phalloidin-induced cholestasis in rats. These results demonstrate the usefulness of tauroursodeoxycholate as a therapeutic agent in intrahepatic cholestasis.

Topics: Alanine Transaminase; Alkaline Phosphatase; Animals; Aspartate Aminotransferases; Bile; Bile Acids and Salts; Biliary Tract; Bilirubin; Cholestasis, Intrahepatic; Cholesterol; Dose-Response Relationship, Drug; Leucyl Aminopeptidase; Male; Phalloidine; Phospholipids; Rats; Rats, Wistar; Taurochenodeoxycholic Acid

To study the relationship between the dynamic actin web and bile secretion, we developed an acute model of cholestasis, using phalloidin, and examined sequential morphologic and biochemical events in rat liver. Biliary function (bite flow, bile, and canalicular membrane components) and cellular integrity (release of hepatic enzymes in serum and bile, canalicular structure, and microfilaments distribution) in rats given a single iv dose of phalloidin (0.8 mg/kg body weight) were assessed at 15, 45, and 90 min, 24 hr, and 5 days postinjection. Bile flow decreased significantly at 45 and 90 min, but cholestasis was transient since bile secretion returned to control levels at 24 hr. The biliary bile acid secretion rate was not modified during the same time period, indicating that cholestasis may have been due to impairment of the bile acid independent component of bile flow. Serum alanine aminotransferase and lactate dehydrogenase as well as biliary alkaline phosphatase and alkaline phosphodiesterase-1 activities were not altered by phalloidin treatment. These data, coupled with morphologic studies, provide no evidence of cell damage. Electron microscopy revealed that the pericanalicular actin web in both centrilobular and periportal hepatocytes was increased at 90 min and further enlarged at 24 hr and 5 days after phalloidin injection. At all time periods, the canalicular structure was well preserved. Na+K+ -ATPase and Mg2+ -ATPase activities in membrane fractions enriched in bile canalicular complexes decreased significantly at 15 min and remained low up to Day 5. Mg2+ -ATPase activity returned to control levels by Day 5. The lipid constituents of liver cell membranes enriched in canalicular complexes showed no significant variations 90 min after toxin treatment but, at 24 hr, phospholipid content rose and membrane fluidity increased. These results clearly indicate that the bile flow variation after a single low dose of phalloidin can be dissociated from specific pericanalicular microfilament distribution, lending further support to the view that normal biliary function is not strictly dependent on the integrity of the actin filament network.

Topics: Actin Cytoskeleton; Alanine Transaminase; Alkaline Phosphatase; Animals; Bile; Bile Acids and Salts; Bile Canaliculi; Cell Membrane; Cholestasis, Intrahepatic; Cholesterol; Injections, Intravenous; L-Lactate Dehydrogenase; Leucyl Aminopeptidase; Liver; Male; Phalloidine; Phosphodiesterase I; Phospholipids; Phosphoric Diester Hydrolases; Rats; Rats, Sprague-Dawley

To investigate the effect of a thickened pericanalicular ectoplasm in tubulovesicular transport and biliary excretion, we examined the ultrastructure of the intracellular membranous system in rat hepatocytes with and without phalloidin treatment, by transmission electron microscopy and scanning electron microscopy combined with the Aldehyde prefix Osmium-Dimethyl Sulfoxide-Osmium method. Hepatocytes possessed elaborate networks of tubules around bile canaliculi, and some of them extended to the bile canaliculi in control rats. Vesicles were also present around the bile canaliculus. Treatment of rats with phalloidin produced a thick pericanalicular ectoplasm around the bile canaliculus visualized by transmission electron microscopy, and the density of vesicles (p < 0.001) and tubules (p < 0.001) within 0.5 microns around the bile canaliculus significantly decreased in phalloidin-treated rats. The number of lysosomes in hepatocytes apparently increased in phalloidin-treated rats; however, they were rarely observed around the bile canaliculus. The Aldehyde prefix Osmium-Dimethyl Sulfoxide-Osmium method produced an organelle-free space around the bile canaliculus by removing the thick pericanalicular ectoplasm in scanning electron microscopic examination, and the thickened pericanalicular ectoplasm inhibited the approach of intracellular membranes to the canalicular membrane in the transmission electron microscopic examination. In some pathological cholestatic conditions, the thickened pericanalicular ectoplasm may inhibit not only bile canalicular contraction but also biliary excretion of substances, which is mediated by the tubulovesicular transport system.

Topics: Animals; Bile Canaliculi; Biological Transport; Cholestasis, Intrahepatic; Intracellular Membranes; Liver; Male; Microscopy, Electron; Microscopy, Electron, Scanning; Phalloidine; Rats; Rats, Wistar

Morphological alterations in the pattern of liver cell gap junctions were examined in phalloidin-treated rats to assess the role of gap junctions in experimental intrahepatic cholestasis. Double-labelled fluorescent staining of gap junctions and F-actin were performed using a monoclonal antibody against rat hepatocyte connexin 32 and rhodamine-phalloidin. Immunoelectron microscopy, using the anti-connexin 32 antibody, freeze-fracture replica electron microscopy, and conventional electron microscopy were also performed. In phalloidin-treated rat livers, the specific immunofluorescent staining of connexin 32 was markedly decreased in the pericentral area after 1 day of phalloidin treatment and, after 5 days of phalloidin treatment, there was a decrease in connexin 32 staining in the entire hepatic lobule. On the other hand, F-actin staining at the cell periphery and at the bile canaliculi was markedly increased in the pericentral area of the hepatic lobule after 1 day of phalloidin treatment and in the entire lobule after 5 days of treatment. Immunoelectron microscopy showed that both sides of the cytoplasmic domains of gap junctions were stained with anti-connexin 32 antibody in controls, whereas, in cholestatic rats, only one side of the cytoplasmic domain of some gap junctions was stained with anti-connexin 32 antibody after 1 or 3 days of phalloidin treatment. No gap junctions were observed after 5 days of phalloidin treatment either by freeze-fracture replica electron microscopy or by conventional electron microscopy. These results indicate that with phalloidin treatment, hepatocyte gap junctions decrease, first in the pericentral area, and finally throughout the entire lobule.

Topics: Actins; Animals; Cholestasis, Intrahepatic; Connexins; Gap Junction beta-1 Protein; Gap Junctions; Liver; Male; Phalloidine; Rats; Rats, Wistar

Topics: Actins; Animals; Bile Canaliculi; Cholestasis, Intrahepatic; Cytoskeleton; Keratins; Liver; Male; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains

Small doses of manganese or bilirubin administered alone to rats produce no morphologic alterations in the hepatocytes. However, combination of both treatments produced a rapid and severe decrease in bile flow that was paralleled by the appearance and aggravation of the cholestatic lesion: bile canaliculi progressively lost their microvilli; vacuolization occurred in the pericanalicular area and subsequently in the cytoplasm. When bile flow returned to normal, or when cholestasis was prevented by an injection of sulfobromophthalein or a pretreatment with phalloidin, no cholestatic signs were observed. A close correlation appears to exist between the presence of morphologic alterations and the decrease of bile flow in manganese-bilirubin cholestasis.

Topics: Animals; Bile; Bilirubin; Cholestasis, Intrahepatic; Liver; Male; Manganese; Phalloidine; Rats; Rats, Inbred Strains

Topics: Animals; Cholestasis, Intrahepatic; Cholesterol; Liver; Microscopy, Electron; Phalloidine; Rats; Rats, Inbred Strains; Taurolithocholic Acid

Topics: Alkaline Phosphatase; Animals; Cholestasis, Intrahepatic; Disease Models, Animal; Female; Hyperbilirubinemia; Liver; Male; Mitosis; Oligopeptides; Phalloidine; Rats; Rats, Inbred Strains; Sex Factors