Unconjugated bilirubin and an increased proportion of bilirubin monoconjugates in the bile of patients with Gilbert's syndrome and Crigler-Najjar disease.

Bilirubin pigments were studied in the bile of 20 normal adults, 25 patients with Gilbert's syndrome, 9 children with Crigler-Najjar disease, and 6 patients with hemolysis, to determine how a deficiency of hepatic bilirubin UDP-glucuronosyltransferase would affect the end products of bilirubin biotransformation. In the bile from patients with Gilbert's syndrome, a striking increase was found in the proportion of bilirubin monoconjugates (48.6+/-9.8% of total conjugates) relative to that in normal bile (27.2+/-7.8%). This increase was even more pronounced in children with Crigler-Najjar disease, in whom, even in the most severe cases, glucuronide could always be demonstrated in the bile. Furthermore, unconjugated bilirubin-IXalpha was unquestionably present in the bile of these children and amounted to 30-57% of their total bilirubin pigments (<1% in the controls). It was not possible to predict from the biliary bilirubin composition whether a child would respond to phenobarbital therapy or not. Bile composition was normal in patients with hemolysis, except when there was associated deficiency of hepatic glucuronosyltransferase. Therefore, the observed alterations were not a simple consequence of unconjugated hyperbilirubinemia. The present findings suggest that Crigler-Najjar disease represents a more pronounced expression than Gilbert's syndrome of a common biochemical defect. Hepatic bilirubin UDP-glucuronosyltransferase deficiency leads to decreased formation of diconjugates with an ensuing increase in the proportion of bilirubin monoconjugates in bile; in the most severe cases, an elevated content of biliary unconjugated bilirubin is also found.

Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis.

High affinity binding sites for endothelin (ET) were identified on rat liver plasma membranes. Binding of 125I-ET-1 with its site was specific, saturable, and time dependent (kobs = 0.019 +/- 0.001 min-1), but dissociation of receptor-bound ligand was minimal. A single class of high affinity binding sites for 125I-ET-1 was identified with an apparent Kd of 32.4 +/- 9.8 pM and a Bmax of 1084 +/- 118 fmol/mg protein. ET-3 and big-ET-1 (1-38) (human) inhibited 125I-ET-1 binding with IC50 values of 1.85 +/- 1.03 nM and 43 +/- 6 nM, respectively. Aequorin measurements of cytosolic free Ca2+ in single, isolated rat hepatocytes showed that ET-1 at subnanomolar concentrations induced a series of repetitive, sustained Ca2+ transients. ET-1 had no effect on cAMP production. Finally, ET-1 caused a rapid and sustained stimulation of glycogenolysis in rat hepatocytes. A 1.8-fold maximal increase in glycogen phosphorylase alpha was observed at 1 pM ET-1, with an EC50 of 0.03 pM. Stimulation of the enzyme was specific for ET-1 since the order of potency of related peptides was similar to that in binding experiments (ET-1 greater than ET-3 greater than big ET-1). These data constitute the first demonstration of the presence of ET-1 binding sites in liver which is associated with a rise in cytosolic free Ca2+ and a potent glycogenolytic effect. We conclude that ET-1 behaves as a typical Ca2+ mobilizing hormone in liver.

Growth inhibitory properties of endothelin-1 in human hepatic myofibroblastic Ito cells. An endothelin B receptor-mediated pathway.

Ito cells play a pivotal role in the development of liver fibrosis associated with chronic liver diseases. During this process, Ito cells acquire myofibroblastic features, proliferate, and synthesize fibrosis components. Considering the reported mitogenic properties of endothelin-1 (ET-1), we investigated its effects on the proliferation of human Ito cells in their myofibroblastic phenotype. Both ET receptor A (ETA: 20%) and ET receptor B (ETB: 80%) binding sites were identified, using a selective ETA antagonist, BQ 123, and a selective ETB agonist, sarafotoxin S6C (SRTX-C). ET-1 did not stimulate proliferation of myofibroblastic Ito cells. In contrast, ET-1 inhibited by 60% DNA synthesis and proliferation of cells stimulated with either human serum or platelet-derived growth factor -BB (PDGF-BB). PD 142893, a nonselective ETA/ETB antagonist totally blunted this effect. SRTX-C was as potent as ET-1, while BQ 123 did not affect ET-1-induced growth inhibition. Analysis of the intermediate steps leading to growth-inhibition by ET-1 revealed that activation of mitogen-activated protein kinase by serum or PDGF-BB was decreased by 50% in the presence of SRTX-C. In serum-stimulated cells, SRTX-C reduced c-jun mRNA expression by 50% whereas c-fos or krox 24 mRNA expression were not affected. We conclude that ET-1 binding to ETB receptors causes a potent growth inhibition of human myofibroblastic Ito cells, which suggests that this peptide could play a key role in the negative control of liver fibrogenesis. Our results also point out that, in addition to its well known promitogenic effects, ET-1 may also exert negative control of growth on specific cells.

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

[Comparison of the effects of cimetidine and ranitidine in vivo and in vitro on the hepatic microsomal enzyme system in rats]. / Comparaison des effets de la cimétidine et de la rantidine in vivo et in vitro sur le système enzymatique microsomal hépatique chez le rat.

Cimetidine, administered intraperitoneally to male rats (20 mg/kg and 40 mg/kg) induced a 125 p. 100 and 325 p. 100 increase in the hexobarbital sleeping time, respectively. Ranitidine, at the same dosage, had no effect. Cimetidine (40 mg/kg) decreased the aminopyrine metabolic clearance by 73 p. 100, whereas ranitidine caused no change. In vitro, cimetidine (1 mM) decreased 3 oxiding enzymatic activities, ethylmorphine demethylase, aniline hydroxylase and aminopyrine demethylase, measured in a 9,000 g supernatant fraction of rat liver, by 29 p. 100, 45 p. 100 and 73 p. 100, respectively. Ranitidine (1 mM) did not modify ethylmorphine and aminopyrine demethylase activities and induced a slight decrease of aniline hydroxylase activity (10 p. 100). Bilirubin and p-nitrophenol UDP-glucuronosyltransferase activities, measured on the microsomal fraction, were slightly inhibited (5 p. 100 and 4 p. 100, respectively) by cimetidine (1 mM). Ranitidine (1 mM) did not change these enzymatic activities. The effects of cimetidine and ranitidine on both the oxidizing and conjugating enzymatic activities were not notably affected by pretreatment with phenobarbital (100 mg/kg, intraperitoneally for 3 days). These results point out that: 1) in vivo and in vitro, ranitidine, in contrast with cimetidine, does not inhibit the microsomal drug-oxidizing system; 2) neither ranitidine nor cimetidine decrease the activity of the microsomal drug-conjugating system. They clearly explain why cimetidine interferes with the disposition of drugs oxidized but not of drugs conjugated by the liver.

[Comparative study of pinocytosis and the production of prostaglandin E2 by hepatic and peritoneal resident macrophages in mice]. / Etude comparative de la pinocytose et de la production de prostaglandines E2 par les macrophages hépatiques et péritonéaux "résidents" de souris.

Pinocytosis and prostaglandin E2 production are two major functions of the mononuclear phagocyte system. The goal of this study was to compare the pinocytosis of horseradish peroxidase and the prostaglandin E2 production between the hepatic and peritoneal resident macrophages in the mouse. Hepatic resident macrophages were isolated by collagenase digestion, differential centrifugation and adherence. Peritoneal resident macrophages were isolated by peritoneal cavity washing followed by adherence. Horseradish peroxidase was endocytosed by hepatic macrophages at a significantly higher rate (118 +/- 12 ng/10(6) cells/60 min) than by peritoneal macrophages (21 +/- 4 ng/10(6) cells/60 min). Prostaglandin E2 production was measured in the culture medium of unstimulated and lymphokine-stimulated hepatic and peritoneal resident macrophages. Prostaglandin E2 concentration in the culture medium of unstimulated peritoneal macrophages was 36.6 +/- 26.8 ng/ml after a 24 h incubation. It was increased by 83 p. 100 in presence of a lymphokine-enriched secondary mixed lymphocyte culture supernatant. In contrast, hepatic macrophages did not produce any significant amount of prostaglandin E2, even if they were incubated in presence of lymphokines. This study shows that hepatic resident macrophages have a higher pinocytic capacity than peritoneal resident macrophages, suggesting that the role of the liver in the clearance of gut-derived antigens is not only due to its portal irrigation but also to the presence of macrophages highly differentiated in their endocytotic properties. The lack of prostaglandin E2 production in hepatic macrophages, in basal conditions as well as after lymphokine-stimulation, suggests that these cells play a minor role in the regulation of the immune response.(ABSTRACT TRUNCATED AT 250 WORDS)

Demonstration of a basic fibroblast growth factor-like molecule in mouse hepatic endothelial cells.

We have investigated whether isolated mouse hepatic sinusoidal endothelial cells (HSEC) synthesized basic FGF. HSEC lysate was fractionated by heparin-Sepharose chromatography. A peak of mitogenic activity for Balb/c 3T3 fibroblasts was eluted with 3M NaCl. Several arguments suggested that the mitogenic factor was related to bFGF: a) its affinity for heparin; b) the loss of its mitogenic activity by heating at 65 degrees C, which was prevented in the presence of heparin; c) the abolition of its mitogenic activity in the presence of protamine sulfate; d) finally, its mitogenic effect was reduced in the presence of antibody to bFGF. These data demonstrate the presence of a bFGF-like molecule in HSEC. This molecule could be involved in the regulation of the neighboring Ito cell proliferation.

Mouse hepatic endothelial cells in culture secrete a growth inhibitor for hepatic lipocytes and Balb/c 3T3 fibroblasts.

Hepatic lipocytes are sinusoidal cells in close contact to endothelial cells. They proliferate, switch to a fibroblastic phenotype and synthetize collagen during hepatic fibrosis. Since it is known that vascular endothelial cells can influence the proliferation of neighboring cells such as smooth muscle cells, we investigated the role of hepatic endothelial cells on the growth of lipocytes and Balb/c 3T3 fibroblasts. Concentrated conditioned medium from endothelial cells inhibited both [3H]thymidine incorporation and actual growth of lipocytes and Balb/c 3T3 fibroblasts. The inhibition was lost when conditioned medium was treated with heat or trypsin, or when medium was conditioned in the presence of cycloheximide. We conclude that hepatic endothelial cells secrete a proteic growth inhibitor for lipocytes and hepatic Balb/c 3T3 fibroblasts. This inhibitor could be of importance in limiting lipocyte proliferation in the liver and possibly in preventing hepatic fibrosis.

Characterization of microsomal bilirubin and p-nitrophenol uridine diphosphate glucuronosyltransferase activities in human liver: a comparison with rat liver.

The bilirubin and p-nitrophenol uridine diphosphate glucuronosyltransferase (UDP-GT) activities have been characterized from human liver microsomes and compared to those from rat liver. This study was performed on large samples of human liver obtained from 20 organ donors. The kinetic constants as well as sensitivities to digitonin, temperature, pH and diethylnitrosamine were determined. Our results suggest that UDP-GT activities have characteristics different in human and rat liver microsomal membranes. Moreover, differences in digitonin-induced activation, in thermodenaturation and in the response to diethyl-nitrosamine were found between bilirubin and p-nitrophenol UDP-GT activities. This supports the hypothesis of the probable heterogeneity of UDP-GT.

Effect of fasting on organic anion uptake by isolated rat liver cells.

In order to determine if the delayed clearance of organic anions observed in vivo after fasting can be related to an alteration of cell membrane carriers, kinetics of sulfobromophthalein (BSP) uptake were determined in isolated rat liver cells obtained from 48-hr starved rats. Surprisingly, in fasted rats the existence of two carriers can be directly revealed by classical kinetic plots. The high-affinity component, inhibited by Na+-taurocholate, has a Km of 0.5 +/- 0.2 microM and a Vmax of 0.2 +/- 0.1 nmole per min per 10(6) cells; the low-affinity component, which is not sensitive to Na+-taurocholate, has a Km of 21.2 +/- 3.2 microM and a Vmax of 4.8 +/- 0.9 nmoles per min per 10(6) cells. Comparison with control cells shows that fasting does not modify the total capacity of the liver cell membrane carriers to take up BSP. However, alterations in the kinetic parameters of the two uptake components were observed: a 53% decrease in the affinity of the low-affinity component and a 50% reduction in the capacity of the high-affinity uptake. These alterations, together with the observed decrease in hepatic blood flow and/or the increase in BSP efflux from the hepatocytes, could be involved in the delayed clearance of BSP and other anionic compounds occurring in vivo after fasting.

Fibroblast growth factor 2 and transforming growth factor beta 1 interactions in human liver myofibroblasts.

BACKGROUND & AIMS: During liver fibrogenesis, myofibroblastic liver cells proliferate and synthesize components of fibrosis. Fibroblast growth factor 2 (FGF-2) is expressed in vivo in myofibroblastic liver cells (MFLCs) during fibrogenesis, and exogenous FGF-2 is mitogenic for MFLCs. The aim of this study was to study the expression and role of endogenous FGF-2 in cultured human MFLCs. METHODS: FGF-2 and FGF-2 receptors were studied using immunoblotting. All RNA studies used ribonuclease protection. Growth of MFLCs was studied using [3H]thymidine incorporation and direct cell counting. RESULTS: MFLCs expressed FGF-2 and its receptors FGF receptor 1 and FGF receptor 2. An antibody to FGF-2 blocked the mitogenic effect of transforming growth factor beta 1 (TGF-beta 1) for MFLCs but not TGF-beta 1-induced increase in cellular fibronectin messenger RNA (mRNA). TGF-beta 1 increased levels of FGF-2 and FGF receptor mRNAs in MFLCs. We have previously shown that TGF-beta 1 also increased platelet-derived growth factor (PDGF) A chain mRNA in these cells and that anti-PDGF antibody blunted the mitogenic effect of TGF-beta 1. The present results show that anti-FGF-2 and anti-PDGF-AA are not additive and that FGF-2 and PDGF-AA are not sequentially induced by TGF-beta 1. CONCLUSIONS: FGF-2 mediates the mitogenic but not the profibrogenic effect of TGF-beta 1 for human MFLCs, and autocrine FGF-2 and PDGF-A interact in the mediation of the mitogenic effect of TGF-beta 1.

Decreased toxicity of polymorphonuclear neutrophils toward hepatocytes isolated from rats with acute inflammatory reaction.

We have recently demonstrated that polymorphonuclear neutrophils were toxic to hepatocytes through a protease-mediated mechanism. Since synthesis of antiproteases is markedly increased during acute inflammatory reaction, the aim of this work was to investigate the toxicity of neutrophils against normal vs. inflammatory rat hepatocytes. Acute inflammatory reaction was induced by subcutaneous injection of turpentine 24 hr before the experiments. Hepatocytes from normal and turpentine-treated rats were isolated by collagenase digestion. They were incubated with human neutrophils stimulated by 1 mg/ml opsonized zymosan. Cytotoxicity was quantified by the percentage of alanine aminotransferase activity released by hepatocytes in culture medium after an 18-hr incubation period. By comparison to normal hepatocytes, inflammatory hepatocytes were more resistant to the toxicity of neutrophils. At a neutrophil/hepatocyte ratio of 20:1, the alanine aminotransferase activity releases were 53.7% +/- 5.4% (mean +/- 1 S.E.) and 27.4% +/- 4.8% for normal and inflammatory hepatocytes, respectively. Similarly, inflammatory hepatocytes were found to be less sensitive than normal hepatocytes to the toxic effect of purified neutrophil cathepsin G. In contrast, both types of hepatocytes exhibited the same sensitivity to H2O2 generated by a system consisting of glucose and glucose oxidase. Two arguments suggested that the resistance of inflammatory hepatocytes to protease toxicity was explained by an increased production of antiproteases by these cells: (a) when tested against cathepsin G and porcine pancreatic elastase activities, the protease inhibitory capacity of conditioned medium from inflammatory hepatocytes was higher than that of conditioned medium from normal hepatocytes; (b) conditioned medium from inflammatory hepatocytes markedly reduced the toxicity of stimulated neutrophils as that of cathepsin G.(ABSTRACT TRUNCATED AT 250 WORDS)