Some observations on the morphological evidence for mechanism of the bile secretion

The morphological evidence of the intracellular route of bile secretion was investigated in the liver of goldfish (Carassius auratus) as revealed by electron microscopy. Smooth surfaced tubules or cisterns within or adjacent to the Golgi apparatus showed linear saccular forms and contained sparse particulate or cloudy materials of low electron density. The isolated vacuoles were restrictedly found between the Golgi apparatus and the intracellular bile canaliculus or hepatocytic side at the zone of transition. These vacuoles showed no reaction for acid phosphatase activity, and contained only a few cloudy materials similar to those found in the saccular tubules and within the bile canaliculus. Some of these vacuoles fused with the luminal cytolemmas of the bile canaliculus. Bases on these findings, it was assumed that these vacuoles are structures participating in transport and secretion of bile constituents and derive from the linearly sacculated tubules or cisterns in the Golgi zone. Duct cells showed no morphological evidence to suggest bile secretion.     

The functional significance of bile canalicular nucleosidephosphatase activity

Summary The histochemically demonstrable bile canalicular nucleosidephosphatase activity of the developing chicken and rat liver has been studied and correlated to the structural and functional maturation.It is concluded that the factor responsible for this activity is not bound to the bile canaliculi as anatomical structures per se. Neither does it play an active role in bile secretion but appears to be a plasma ingredient excreted in the bile.It is suggested that the transitional localization of this activity to different parts of the functional liver unit reflects the morphological route of bile secretion.This study was supported in part by a grant from the Swedish Medical Research Council     

Taurine-conjugated bile acids act as Ca2+ ionophores.

The ionophoretic properties of several taurine-conjugated bile acids have been investigated in two experimental systems: in a two-phase bulk partitioning system and in proteoliposomes. In the former, a bile acid/Ca2+ complex was extracted into the bulk organic phase and had an experimental stoichiometry of 1.75. Extraction was specific for Ca2+ over Mg2+; Na+ and K+ did not compete with the extraction of Ca2+. In the second system, bile acids at concentrations as low as 5-100 molecules/vesicle lowered the steady-state Ca2+ gradient maintained by a reconstituted sarcoplasmic reticulum Ca(2+)-ATPase. The effect was not due to nonspecific membrane perturbation. In addition to releasing intravesicular Ca2+ in a transmembraneous process, bile acids caused partition of Ca2+/bile acid complexes into the hydrophobic core of the bilayer. In both experimental systems, the Ca2+ ionophoretic activity correlated well with the concentration and the hydrophobicity of the bile acid. Taurolithocholate was most active, with a significant effect measurable at 10 microM in either system. Since bile acid concentrations equal to those used in our experiments can occur in the blood in certain liver diseases, the results support the notion that bile acids can increase the intracellular Ca2+ concentration bypassing the regulatory systems that maintain cellular Ca2+ homeostasis.     

Sources of the proteins of rat bile

The protein composition of rat bile has been studied systematically using two-dimensional agarose-polyacrylamide gel electrophoresis, with or without prior absorption by immobilised antisera, and by crossed immunoelectrophoresis. Sixteen bile proteins were distinguished. Of these, thirteen are immunologically identical to proteins present in rat serum and only one is identical to a protein present in rat liver plasma membrane but not in rat serum. Of the remaining two proteins, one is bile lipoprotein and the other has many of the properties of immunoglobulin A secretory component.The serum-related proteins in rat bile fall into two distinct groups. In the first group are immunoglobulin A and an α₂-globulin. These proteins are major constituents of bile but only minor constituents of serum. In the second group are albumin and some other major serum proteins which are found in bile at concentrations less than 1% of their concentrations in serum. The relative proportions of these proteins in bile appear to differ from their proportions in serum. It therefore appears that, although the majority of bile proteins are derived from serum, there cannot be direct leakage of serum into bile. Examination of the proteins contained within liver lysosomes indicates that, although discharge of lysosomal contents at the bile canalicular face of the hepatocyte may contribute to the bile proteins, an additional mechanism, with a considerable degree of selectivity, must also be involved in the transport of proteins from serum to bile.     

Differential regulation of cytosolic and peroxisomal bile acid amidation by PPAR alpha activation favors the formation of unconjugated bile acids

In human liver, unconjugated bile acids can be formed by the action of bile acid-CoA thioesterases (BACTEs), whereas bile acid conjugation with taurine or glycine (amidation) is catalyzed by bile acid-CoA:amino acid N-acyltransferases (BACATs). Both pathways exist in peroxisomes and cytosol. Bile acid amidation facilitates biliary excretion, whereas the accumulation of unconjugated bile acids may become hepatotoxic. We hypothesized that the formation of unconjugated and conjugated bile acids from their common substrate bile acid-CoA thioesters by BACTE and BACAT is regulated via the peroxisome proliferator-activated receptor alpha (PPARalpha). Livers from wild-type and PPARalpha-null mice either untreated or treated with the PPARalpha activator WY-14,643 were analyzed for BACTE and BACAT expression. The total liver capacity of taurochenodeoxycholate and taurocholate formation was decreased in WY-14,643-treated wild-type mice by 60% and 40%, respectively, but not in PPARalpha-null mice. Suppression of the peroxisomal BACAT activity was responsible for the decrease in liver capacity, whereas cytosolic BACAT activity was essentially unchanged by the treatment. In both cytosol and peroxisomes, the BACTE activities and protein levels were upregulated 5- to 10-fold by the treatment. These effects caused by WY-14,643 treatment were abolished in PPARalpha-null mice. The results from this study suggest that an increased formation of unconjugated bile acids occurs during PPARalpha activation.     

Electron microscopic cytochemical characterization of bile canaliculi and bile ducts in vitro.

Electron microscopic cytochemical localization of Mg++-activated adenosine triphosphatase (Mg++-ATPase) and 5-nucleotidase (AMPase) was investigated in bile canaliculus-rich and bile duct-containing fractions isolated from rat liver. Comparative cyochemical studies between prefixed and non-prefixed fractions revealed that the activity of both enzymes could be detected in the fractions under appropriate experimental conditions. However, the cytochemical activity of AMPase was much more sensitive to glutaraldehyde than that of Mg++-ATPase. Mg++-ATPase and AMPase reaction products were localized primarily on bile canalicular microvilli, that is, along the outer (luminal) surface of canalicular plasma membranes, but they were never observed on bile ductal microvilli. AMPase was also detectable on lateral hepatic plasma membranes. Mg++-ATPase demonstrated by the cytochemical technique described is a reliable enzyme marker for isolated bile canalicular membranes. At high magnification, Mg++-ATPase reaction product was also observed on the microfilaments surrounding isolated bile canaliculi. The possibility that the reaction product on the pericanalicular microfilaments may result from the hydrolysis of ATP byan actomyosin ATPase-like enzyme associated with these filaments is briefly discussed.     

Molecular aspects of bile formation and cholestasis

Recent insights into the cellular and molecular mechanisms that control the function and regulation of hepatobiliary transport have led to a greater understanding of the physiological significance of bile secretion. Individual carriers for bile acids and other organic anions in both liver and intestine have now been cloned from several species. In addition, complex networks of signals that regulate key enzymes and membrane transporters located in cells that participate in the metabolism or transport of biliary constituents are being unraveled. This knowledge has major implications for the pathogenesis of cholestatic liver diseases. Here, we review recent information on molecular aspects of hepatobiliary secretory function and its regulation in cholestasis. Potential implications of this knowledge for the design of new therapies of cholestatic disorders are also discussed.     

Inhibition of glutathione S-transferase by bile acids.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.     

Immune defence mechanisms presented in liver homogenates and bile of gilthead seabream (Sparus aurata)

Because the role of the liver of fishes in providing possible immunity remains largely unknown, the aim of this work was to identify and characterize different humoral defence mechanisms in the liver homogenates and bile of gilthead seabream (Sparus aurata) for the first time. Total protein levels and several immune parameters (complement activity, lysozyme and immunoglobulin M level) were studied. Furthermore, the activity of some lytic (proteases, antiproteases, esterase, alkaline phosphatase) and antioxidant (superoxide dismutase, catalase and peroxidase) enzymes was determined. Finally, bacteriostatic activity on three opportunist fish pathogens (Vibrio harveyi, Vibrio angillarum and Photobacterium damselae) was measured. Lysozyme and antiprotease activity were undetected in liver and bile, while natural haemolytic complement activity was only detected in bile, and immunoglobulin M was detected in both samples. The levels of proteases, esterase and antioxidant enzymes were greater in bile than in liver homogenates, while the level of alkaline phosphatase was very low in both samples. In addition, while no bacteriostatic activity was detected on liver homogenates, the bile revealed a very potent bacteriostatic activity against all the tested pathogenic bacteria. These results corroborate that fish liver – especially fish bile – contains many factors involved in innate immunity that could be useful for better understanding the role of the liver as an organ involved in fish immune functions as well as the possible contribution of bile to gut mucosal immunity.     

Evidence for the hepatic origin of a major azo dye metabolite-binding protein from rat bile

Small amounts of metabolite-binding protein (MBP) originally characterized from the bile were detected in rat serum and cytosol by an indirect enzyme-linked immunoabsorbant assay. The site of MBP synthesis was shown to be the liver based upon results of (1) the in vitro translation of liver poly(A)+ mRNA, followed by immunoprecipitation with anti-MBP sera and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate, and (2) immunoprecipitation of bile collected from [3H]leucine perfused liver in situ and SDS-polyacrylamide gel electrophoresis and fluorography of the immunoprecipitate. To determine whether part of the MBP in bile is derived from the circulation, [125I]MBP was injected intravenously and bile was collected and subjected to SDS-polyacrylamide gel electrophoresis and fluorography. Intact [125]MBP was not detected in bile even though several other iodinated bile proteins were taken up by the liver from the circulation and secreted intact into the bile under similar experimental conditions. These data indicate that MBP is synthesized in the liver and secreted into the bile and circulation independently. In addition, MBP was not found in brain, spleen or kidneys.     

Free radical signal of bile pigment in paraffin embedded liver tissue

A linear correlation have been found between the amplitude of the free radical signal of the electron spin resonance (ESR) spectrum of paraffin embedded liver blocks and the number of bile casts in the histological sections made from these blocks. It has been suggested that the major part of the ESR free radical signal arises from bile pigment, but the contribution of "age pigment" cannot be excluded. On the basis of model experiments the majority of these radical centers was assigned to protein bound bilirubin. In the course of histological processing more than 80% of various free radical centers arising in air dried liver tissue is extracted.     

Molecular regulation of sinusoidal liver bile acid transporters during cholestasis.

Impairment of the hepatic transport of bile acids and other organic anions will result in the clinically important syndrome of cholestasis. Cloning of a number of specific hepatic organic anion transporters has enabled studies of their molecular regulation during cholestasis. The best characterized transport system is a 50-51 kDa sodium-dependent taurocholate cotransporting polypeptide (ntcp), which mediates the sodium-dependent uptake of conjugated bile acids at the sinusoidal plasma membrane of hepatocytes. Under physiologic conditions and after depletion of biliary constituents, ntcp remains constitutively expressed throughout the liver acinus. However, both function and expression of ntcp are rapidly down-regulated in rat liver in various models of experimental cholestasis, such as cholestasis induced by common bile duct ligation, estrogen, endotoxin or cytokine treatment. In addition to ntcp, the sinusoidal organic anion transporting polypeptide oatp-1 is also down-regulated at the protein and steady-state mRNA levels in estrogen-cholestasis, but does not affect sodium-independent uptake of taurocholate. The regulation of a recently cloned member of the organic anion transporter family (oatp-2), which is highly expressed in liver, remains to be studied under cholestatic conditions.     

Xanthine oxidoreductase is present in bile ducts of normal and cirrhotic liver

Xanthine oxidoreductase (XOR) is a widely distributed enzyme, involved in the metabolism of purines, which generates superoxide and is thought to be involved in free radical-generated tissue injury. It is present at high concentrations in the liver, from where it may be released during liver injury into the circulation, binding to vascular endothelium and causing vascular dysfunction. The cellular localization of the enzyme, essential to understanding its function, is, however, still debated. The present study has used a highly specific mouse monoclonal antibody to define the cellular distribution of XOR in normal and cirrhotic human liver. As shown previously, XOR is present in hepatocytes. However, the novel finding of this study is that XOR is present in bile duct epithelial cells, where it is concentrated toward the luminal surface. Moreover, in liver disease, proliferating bile ducts are also strongly positive for XOR. These findings suggest that the enzyme is secreted into bile, and this was confirmed by analysis of human and rat bile. Xanthine oxidase activity was 10 to 20-fold higher in liver tissue obtained from patients with liver disease, than in healthy liver. We conclude that XOR is expressed primarily in hepatocytes, but is also present in bile duct epithelial cells and is secreted into bile. Its role in bile is unknown but it may be involved in innate immunity of the bowel muscosa.     

Regulation of bile acid synthesis. IV. Interrelationship between cholesterol and bile acid biosynthesis pathways

Under most experimental conditions, the activities of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA reductase) and cholesterol 7 alpha-hydroxylase, change together in parallel directions. It has been suggested that newly synthesized cholesterol may be the preferred substrate for cholesterol 7 alpha-hydroxylase, which may account for the observed synchronous behavior of the two enzymes. To test this hypothesis, mevinolinic acid, a potent competitive inhibitor of HMG-CoA reductase, was administered as a single intravenous bolus (10 mg/kg) to rats with a chronic bile fistula. Bile acid synthesis was determined following inhibition of HMG-CoA reductase by mevinolinic acid over a 27-h time course and specific activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase were determined in liver microsomes. At 3, 6, and 27 h after a bolus dose of mevinolinic acid, bile acid synthesis was reduced by 54 +/- 5%, 42 +/- 8%, and 23 +/- 13%, respectively, from preinfusion baseline. Within 30 min after administration of mevinolinic acid, HMG-CoA reductase activity was inhibited by at least 87%. At 0.5, 1.5, 3, 6, and 27 h after mevinolinic acid injection, cholesterol 7 alpha-hydroxylase activity was decreased by 6%, 25%, 54%, 41%, and 17%, respectively. By 27 h, the activities of both enzymes had returned to baseline levels. The reduction of bile acid synthesis correlated closely with the observed changes in the activities of cholesterol 7 alpha-hydroxylase. In vitro addition of mevinolinic acid (up to 20 microM) to rat liver microsomes failed to inhibit cholesterol 7 alpha-hydroxylase activity, suggesting no direct effect of mevinolinic acid on enzyme activity. When a bolus dose of mevinolinic acid was coupled with a continuous infusion of mevalonate, the product of the reaction catalyzed by HMG-CoA reductase, the mevinolinic acid-induced decrease in cholesterol 7 alpha-hydroxylase activity and bile acid synthesis was prevented. The results of this study provide evidence that, under the experimental conditions described, there is a linkage between the rates of cholesterol synthesis and the activities of cholesterol 7 alpha-hydroxylase. The data also emphasize the importance of the newly synthesized cholesterol in the regulation of cholesterol 7 alpha-hydroxylase activity.     

Low level transport of IgA to bile via the asialoglycoprotein receptor

The rat and rabbit transport IgA from blood to bile by a highly efficient transcellular pathway mediated by secretory component (SC). Other mammals do not express SC on liver hepatocytes, but they do transport a small amount of IgA to bile. In the first part of this study, human polymeric IgA was radiolabeled and depleted of SC binding activity by successive affinity adsorption. Transport of this preparation intact to rat bile was 4%, but was reduced to 2% when 50 mg unlabeled asialoglycoprotein was preadministered. The 2% decline corresponds to the percent of asialo-orosomucoid diverted to bile from the lysosomal pathway. In guinea-pigs, missorting of asialo-orosomucoid intact to bile was 10% of the injected dose. Transport of normal human IgA to bile was 1-2%, even though guinea-pigs do not express SC in the liver. Excess unlabeled asialofetuin reduced the transport of asialo-orosomucoid by 10-fold and IgA by 6-fold. This demonstrates that the asialoglycoprotein receptor can mediate transport of IgA to bile in small amounts, but that this transport may be only a biological artifact resulting from limited fidelity of intracellular protein sorting.     

Hydrolysis of retinyl palmitate by enzymes of rat pancreas and liver. Differentiation of bile salt-dependent and bile salt-independent, neutral retinyl ester hydrolases in rat liver

Previous studies have demonstrated that homogenates of the livers of rats contain a neutral retinyl ester hydrolase activity that requires millimolar concentrations of bile salts for maximal in vitro activity. The enzymatic properties of this neutral, bile salt-dependent retinyl ester hydrolase activity in liver homogenates are nearly identical to those observed in the present report for the in vitro hydrolysis of retinyl palmitate by purified rat pancreatic cholesteryl ester hydrolase (EC 3.1.1.13). Moreover, anti-rat pancreatic cholesteryl ester hydrolase IgG completely inhibits the bile salt-dependent retinyl ester hydrolase activity of rat liver homogenates whereas normal rabbit IgG does not. We also show that liver homogenates contain a neutral, bile salt-independent retinyl ester hydrolase activity that differs from the bile salt-dependent activity in that 1) its absolute activity does not vary markedly among individual rats, 2) it is not inhibited by antibodies to pancreatic cholesteryl ester hydrolase, and 3) it is localized in the microsomal fraction of liver homogenates. Subfractionation of microsomes demonstrates that the neutral, bile salt-independent retinyl ester hydrolase activity is associated with liver cell plasma membranes and thus may play a role in the hydrolysis of retinyl esters delivered to the liver by chylomicron remnants.     

Identification of unconjugated bile acids in human bile

Unconjugated bile acids in the bile of healthy and diseased humans were determined qualitatively and quantitatively by means of gas-liquid chromatography and gas-liquid chromatography-mass spectrometry, after their isolation by ion-exchange chromatography. In a healthy person and three patients with cholelithiasis, unconjugated bile acids comprised 0.1-0.4% of total biliary bile acids. The bile acid composition of the unconjugated fraction was quite different from that of the glycine- or taurine-conjugate fraction, in that it contained a relatively large proportion of unusual bile acids including C23 and C27 bile acids. In two patients with cerebrotendinous xanthomatosis, C22 and C23 bile acids were the major constituents of the biliary unconjugated bile acids, and comprised about 0.8% of total bile acids; no detectable amounts of C27 bile acids were found in their bile. The analysis of biliary unconjugated bile acids may be useful for the diagnosis of metabolic diseases concerning bile acids, particularly the accumulation or disappearance of unusual bile acids.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from rat liver

An in vitro study of bile acid-CoA:amino acid N-acyltransferase activity of rat liver was undertaken in order to determine whether separate amino acid-specific enzymes catalyzed the formation of glycine and taurine conjugates of bile acids as postulated by others. Polyacrylamide gel electrophoresis of 200-fold purified enzyme localized the glycine- and taurine-dependent activities to a single band. Both activities were optimal at pH 7.8 and showed similar loss of activity at pH 6.0, pH 9.0, in the presence of 5,5'-dithiobis(2-nitrobenzoic acid), and at temperatures exceeding 50 degrees. With the purified fraction, Km for glycine was 31 mM and Km for taurine was 0.8 mM. Km for several bile acid-CoA substrates was approximately 20 micron and independent of the amino acid acceptor. Only amino acids with terminal alpha- or beta-amino groups were active as acyl acceptors. Acyl donors were limited to bile acid-CoA derivatives. The data support the conclusion that the rat has a single bile acid-CoA:amino acid N-acyltransferase. The substrate kinetics are consistent with previous observations that taurine conjugates predominate in rat bile at normal hepatocellular concentrations of glycine and taurine.     

Regulation of bile acid synthesis under reconstructed enterohepatic circulation in rats

Cholesterol 7alpha-hydroxylase (CYP7A1) is regulated by bile acids through the farnesoid X receptor (FXR) mechanism in a negative feedback fashion. However, the fact that CYP7A1 is down-regulated by intraduodenal administration of bile acid, but not by intravenous administration may not be explained only by this mechanism. The aim of this study was to establish a new rat model with reconstructed or simulated enterohepatic circulation to examine if intravenous or portal administration of bile acid can regulate CYP7A1. Under biliary drainage, taurocholate (0 or 6 micromol/h/100g body weight) was administered continuously for 48h into the duodenum (ID-0/ID-6), femoral vein (IV-0/IV-6), or portal vein (IP-0/IP-6) to create a condition in which biliary bile acids were continuously lost, and a similar dose of taurocholate was supplied to the liver simultaneously. CYP7A1 activity and mRNA expression of the ID-0 group were significantly increased compared with the no treatment (NT) group. CYP7A1 activity and mRNA expression of the ID-6 group were suppressed significantly to 41 and 46% of those of the ID-0 group, respectively. In the IV-6 and IP-6 groups, however, enzyme activity and mRNA expression were decreased slightly, but the suppression was not statistically significant. The results suggested that portal as well as intravenous administration of bile acids cannot suppress bile acid synthesis as effectively as intraduodenal administration. It was concluded that an unidentified regulatory factor other than the nuclear receptors may be involved in bile acid synthesis in vivo.     

Studies on the mechanism of the increase in serum alkaline phosphatase activity in cholestasis: significance of the hepatic bile acid concentration for the leakage of alkaline phosphatase from rat liver

In experimental bile obstruction the serum activities of the membrane-bound liver enzymes, alkaline phosphatase, 5'-nucleotidase and gamma-glutamyltransferase are greatly increased, whereas in the liver only the alkaline phosphatase activity is elevated. After partial hepatectomy or tetrachloride poisoning the alkaline phosphatase activity in the regenerating live is increased to the same extent as in cholestasis without an accompanying elevation in serum activity. The following results support the hypothesis of a bile salt-mediated solubilization of membrane-bound enzymes in cholestatic liver: (1) 30 min after bile duct ligation the total bile acids in the liver were increased 5-fold, 2 h later as much as 10-fold. After 1 day, the bile acid concentration was still 4 times above normal. (2) Isolated plasma membranes from normal and obstructed livers were incubated in vitro with increasing amounts of tri- and dihydroxycholanic acids. At a final concentration of 1 mmol/l taurochenodeoxycholate significant amounts of membrane-bound enzymes were released into the 12,000-g supernatant. (3) In the regenerating liver, where tissue phsophatase activity was high and serum phosphatase activity unchanged, the bile salt concentration was not increased.