Effects of phalloidin and colchicine on diethylmaleate-induced choleresis and ultrastructural appearance of rat hepatocytes.

Diethylmaleate is used as a model compound whose glutathione conjugates are secreted into bile, and which induce choleresis and the formation of Golgi-derived vesicles in hepatocytes. This study was performed to test the assumption that these vesicles are involved in the bile canalicular secretion of diethylmaleate. We reasoned that phalloidin and colchicine, two drugs acting on microfilaments and microtubules, respectively, can modify the movements of diethylmaleate-induced vesicles towards the bile canaliculus. Phalloidin induced the formation of a thick microfilamentous network around the bile canalicular plasma membrane domain. A significant decrease in diethylmaleate-stimulated choleresis was observed, associated with a striking accumulation of pericanalicular vesicles, which were confirmed by morphometric analysis. In contrast, in rats pretreated with colchicine, after diethylmaleate administration, only a few vesicles were observed around the bile canaliculus, while diethylmaleate-induced choleresis also decreased. These results suggest that: a) the thick microfilament network induced by phalloidin prevents diethylmaleate-associated vesicles reaching the bile canalicular plasma membrane; and b) colchicine produces a dispersion of these vesicles in the cytoplasm of hepatocytes by inhibiting the polymerization of microtubules. These observations support a role of vesicles in the transport of diethylmaleate by hepatocyte into bile, and are consistent with the existence of a vesicular pathway for the biliary secretion of diethylmaleate and possibly other organic anions.

Glucagon administration in vivo stimulates hepatic RNA and protein breakdown in fed and fasted rats.

Liver RNA and protein breakdown rates were measured simultaneously in fed and in 24 h-fasted rats during a short-term cyclic perfusion, 1 h after an intraperitoneal injection of glucagon or of saline. RNA was labelled in vivo by an intraperitoneal injection of [6-14C]orotic acid, 60 h before the start of the perfusion. The accumulation of radioactive cytidine and valine in the perfusion medium for 15 min was used to determine RNA breakdown and proteolysis respectively. The portal glucagon/insulin ratio was significantly higher in the fasted glucagon-treated rats than in their fed counterparts. Although glucagon administration significantly increased RNA and protein degradation rates in the fasted and in the fed groups, the effect was greater after 24 h of starvation. The relationship between these biochemical changes and the alterations of the hepatocyte lysosomal system was investigated by determining the fractional cytoplasmic volume of lysosomal structures (autophagic vacuoles and dense bodies) by morphometry in the fasted glucagon-treated rats and in their controls. Hyperlucagonaemia significantly enhanced the relative volume of autophagic vacuoles without affecting that of dense bodies. The results showed that hyperglucagonaemia induced in vivo stimulated both liver RNA and protein breakdown and that this effect was modulated by the nutritional status of the rats.

Immunoperoxidase localization of ursodeoxycholic acid in rat biliary epithelial cells. Evidence for a cholehepatic circulation.

To explain the hypercholeretic effect of ursodeoxycholic acid, a cholehepatic shunt circulation has been postulated. This pathway includes secretion by the hepatocyte into bile and absorption by the biliary epithelial cells. To test this possibility, we have attempted to localize ursodeoxycholic acid in hepatocytes and portal bile duct cells by an indirect immunoperoxidase technique using specific polyclonal antibodies against ursodeoxycholic acid and cholic acid conjugates. Rat livers were fixed with paraformaldehyde, incubated with antibodies directed against bile acids and examined by electron microscopy. After infusion of ursodeoxycholic acid and incubation with antibodies against this bile acid, an electron-dense staining was observed on the apical (luminal) membrane of the portal bile ductal cells. In contrast, no staining was observed when no ursodeoxycholic acid was infused, or after infusion with taurocholate and incubation with antibodies against cholic acid conjugates. These observations are consistent with absorption of ursodeoxycholic acid by the portal bile ducts and a cholehepatic circulation of this bile acid.

Heterogeneous lobular distribution of hepatocytes expressing acute-phase genes during the acute inflammatory reaction.

Functional heterogeneity in the lobule with regard to plasma protein synthesis is still debated. Therefore, we have localized in liver sections from normal rats and from rats with turpentine-induced AIR the mRNA and protein products of three genes with different alterations in their hepatic expression during an AIR: alpha 2M and alpha 1PI, two positively reacting acute-phase genes, and alpha 1I3, a negative acute-phase reactant. In normal liver, all hepatocytes expressed alpha 2M and alpha 1I3 mRNA, but a preferential expression of alpha 2M and alpha 1I3 mRNA and protein in the PP and ML zones was observed. During an AIR, the level of alpha 2M mRNA increased fourfold in the cytoplasm of PP and ML hepatocytes, while the level of cytoplasmic alpha 1I3 mRNA was decreased about fourfold in the same zones, with parallel variations in the expression of the corresponding proteins. In contrast, no significant modulation of the RNA and protein concentrations of both genes was detected in PV areas. alpha 1PI mRNA was expressed at the same levels in the three lobular zones in normal liver, but staining for the alpha 1PI protein was more intense in the PV zones. During the acute-phase response alpha 1PI mRNA levels were increased twofold in all three lobular zones, and alpha 1PI staining became homogeneous within the lobule. These results demonstrate that the location of a hepatocyte with the liver lobule can influence the expression of the three genes under study both at pre- and post-translational levels, in basal conditions, as well as during modulation of their expression during the inflammatory reaction.

Immunoperoxidase localization of bile salts in rat liver cells. Evidence for a role of the Golgi apparatus in bile salt transport.

The mechanisms of intracellular transport of bile acids from the sinusoidal pole to the canalicular pole of the hepatocyte are poorly understood. There is physiological and autoradiographic evidence for a vesicular pathway. The purpose of this study was to determine the localization of natural bile acids in the liver using antibodies against cholic acid conjugates and ursodeoxycholic acid. An indirect immunoperoxidase technique was used on rat liver sections fixed either with paraformaldehyde (PF) and saponin, a membrane-permeabilizing agent that allows penetration of antibodies into the cell, or with PF alone. Retention of taurocholate in the liver after tissue processing was 26 +/- SD 15% of the bile acid initially present. When sections fixed with PF and saponin were incubated with the antibody against cholic acid conjugates, a granular cytoplasmic staining was observed by light microscopy in all hepatocytes. By electron microscopy, strong electron-dense deposits were observed mostly on vesicles of the Golgi apparatus (GA) and, sometimes, in the smooth endoplasmic reticulum (SER). After taurocholate infusion, the intensity of the reaction increased. When the liver was fixed with PF alone, almost no reaction was visible on light microscopy, but on electron microscopy the label was localized on the hepatocyte plasma membrane, mainly on the bile canalicular domain and to a lesser extent on the sinusoidal domain. With the antibody against ursodeoxycholic acid, no staining was observed in three of four livers, and a slight staining was observed in one. However, after infusion of ursodeoxycholic acid, staining of GA and SER vesicles was observed when the liver was fixed with PF and saponin. With PF alone, the reaction was intense on the canalicular membrane. These results support the view that, within the limits of the method, vesicles from the GA and possibly vesicles of the SER are involved in the intracellular transport of bile acids before canalicular secretion.

Biosynthesis and regulation of rat alpha 1-inhibitor3, a negative acute-phase reactant of the macroglobulin family.

The biosynthesis of rat alpha 1-inhibitor3, a negative acute-phase reactant specifically found in rodents, was studied in vitro in a cell-free translation system from rabbit reticulocytes, in rat hepatocyte primary cultures and in vivo by immunocytochemistry using normal and turpentine-injected rats. By sucrose-gradient centrifugation and subsequent translation of the fractionated RNA in vitro it was found that the mRNA coding for alpha 1-inhibitor3 exhibited a size of about 28S. For the alpha 1-inhibitor3 translated in vitro an apparent Mr of 155,000 was determined. A continuous decrease in the level of alpha 1-inhibitor3 in serum during experimental inflammation induced by turpentine injection was demonstrated by means of quantitative 'rocket' immunoelectrophoresis. This result agrees with the observation by immunocytochemistry of a drastic decrease in alpha 1-inhibitor3 levels in hepatocytes 24 h after turpentine injection. At that time alpha 1-inhibitor3 is mainly located in the Golgi apparatus, whereas it is also present in the membranes of the rough and smooth endoplasmic reticulum when normal liver is used. All hepatocytes, but no other hepatic cells, contain alpha 1-inhibitor3. When hepatocyte primary cultures were labelled with [35S]methionine and alpha 1-inhibitor3 was immunoprecipitated from the hepatocyte medium and the supernatant of homogenized cells, two different forms of alpha 1-inhibitor3 were found. The intracellular form of alpha 1-inhibitor3, with an apparent Mr of 173,000, is characterized by oligosaccharide side chains of the high-mannose type. The form of alpha 1-inhibitor3 in the medium exhibited an Mr of 186,000 and carried carbohydrate side chains of the complex type. After labelling hepatocytes with radioactive sugars, [3H]mannose was found in both forms of alpha 1-inhibitor3, whereas [3H]fucose and [3H]galactose were incorporated only into the form found in the medium. In the presence of tunicamycin an unglycosylated alpha 1-inhibitor3 with an apparent Mr of 154,000 was found in cells and in the medium. In a pulse-chase experiment it was shown that inhibition of glycosylation by tunicamycin resulted in a marked delay of secretion of alpha 1-inhibitor3. Thus the oligosaccharide side chains of alpha 1-inhibitor3 play an important role during its transport into the medium.

Comparison of the effects of a preliminary hepatic washing and of saponin on the intracellular penetration of peroxidase-labeled anti-rat albumin antibodies in hepatocytes.

The effect of a preliminary hepatic washing with saline before liver fixation either by perfusion or immersion was compared to the effect of saponin, a membrane-permeabilizing agent, in order to ascertain which procedure is best to obtain a homogeneous distribution of albumin-containing hepatocytes in the hepatic lobule. Albumin was located in the hepatocytes by peroxidase-labeled antibodies using light and electron microscopy. The efficacy of the two procedures on the intracellular penetration of labeled antibodies in liver sections was judged by preparing transverse ultrathin sections. Both procedures yielded similar results. Liver fixation by perfusion with saponin and without a preliminary washing, however, distributes albumin-containing hepatocytes more homogeneously in the hepatic lobule and enables labeled antibodies to penetrate more satisfactorily. In contrast, when the liver is fixed by immersion, the preliminary washing is the only way to obtain an even distribution of albumin-containing hepatocytes, as saponin is not effective under these conditions. In conclusion, the localization of albumin in the hepatocytes must be adapted according to the technique used to fix the liver.