The bile salt export pump: molecular properties, function and regulation

Secretion of bile salts from the hepatocyte into bile is the major driving force for the generation of bile flow. Identification of the bile salt export pump (BSEP, ABCB11) as the main adenosine-triphosphate-dependent bile salt transporter in mammalian liver has led to a greater understanding of the biliary bile salt secretory process and its regulation. The biology and pathobiology of BSEP have been the subject of many recent studies. Thus, it has been recognized that while mutations in the gene encoding BSEP are responsible for a subgroup of progressive familial cholestasis (progressive familial intrahepatic cholestasis subtype 2), a pediatric cholestatic disorder that may progress to cirrhosis, defective expression or function of BSEP may underlie some forms of drug-induced cholestasis. The present review summarizes recent data on the molecular properties and regulation of BSEP, as well as the clinical implications of absent or defective function of this hepatic efflux pump.     

豚鼠胆汁酸盐输出泵（BSEP）基因克隆及其在胆结石豚鼠肝组织中的表达分析

 目的：克隆并分析豚鼠BSEP基因全长cDNA序列,检测BSEP基因在胆结石豚鼠肝组织中的表达。方法：采用3'、5'RACE方法扩增获得豚鼠肝组织BSEP基因全长cDNA,用生物信息学方法对其进行分析鉴定。实时荧光定量PCR检测BSEP基因在胆结石豚鼠肝组织的表达。结果：豚鼠BSEP基因全长cDNA5579bp,共包含28个外显子,开放性阅读框（ORF）长为3963bp,编码蛋白长1320aa。该基因在胆结石豚鼠肝组织的表达较正常豚鼠显著下调（P<0.01）。结论：豚鼠BSEP基因在胆结石豚鼠肝组织中表达下调可能与结石形成有关。     

Significance of liver canalicular changes after experimental bile duct ligation.

An ultrastructural morphologic, histochemical, and morphometric study was made on the canalicular structures in rat liver after experimental bile duct ligation (BDL). Four different canalicular structures are distinguished, which are proved by morphometry to correspond to the different types observed during perinatal maturation.Canaliculus type 4, corresponding to the excretory pole of a normal adult hepatocyte, is characterized by a lumen filled with regular, slender microvilli. Canaliculus type 3, corresponding to the canalicular structure described in cholestasis, has a wide lumen devoid of microvilli and is found 2–3 days after BDL. A transitional canalicular structure between type 3 and 4, partly provided with microvilli, is found from the first day after BDL. After 3 days of BDL an increasing number of other canalicular types is observed.Canaliculi type 2, showing an irregular lumen with irregular microvillus-like projections, and canaliculi type 1, representing intracytoplasmic invaginations of two adjacent cell membranes, are found together with canaliculi type 3 and type 3–4.After incubation for alkaline phosphatase, the enzyme activity is detected in canaliculi type 1, 3–4, and type 4.The striking analogy of the sequential changes in canalicular structures after BDL and during perinatal maturation leads to the following interpretation: (1) Canaliculi type 1 and 2, more frequently observed in longstanding cholestasis correspond to newly formed excretory poles of the hepatocytes. (2) Canaliculus type 3, or the so-called cholestatic canalicular type, reflects an inactive secretory function. Morphometrically this canalicular type shows a very low S/V relation which may be partly responsible for this secretory inactivity. (3) Canaliculus type 3–4 reflects a disturbed secretory function after BDL and a beginning secretory activity during perinatal maturation.     

The protective effect of boric acid on cholestatic rat liver ischemia reperfusion injury

Background/aim To evaluate the potential protective effects of boric acid (BA) in experimental cholestatic liver ischemia reperfusion (IR) injury model.Materials and methods The study included 24 female rats which were divided into 3 groups each containing 8 rats. The control group (Group 1) only received laparotomy. In the IR group (Group 2) biliary tract ligation was applied and 1 week later 45 min ischemia and 1 h reperfusion with relaparotomy without any treatment was implemented. In the treatment BA+IR group (Group 3). 1 week after the biliary ligation intraperitoneal administration of 200 mg/kg BA was given 10 min before the ischemia for 45 min and reperfusion for 1 h with relaparotomy. Liver tissue and blood samples were taken for histopathological and biochemical examination. Ischemia modified albumin (IMA), SCUBE1, total antioxidant status (TAS), and total oxidant status (TOS) levels were also examined.Results Compared to control, groups IR and BA+IR had higher serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), total, and direct bilirubin levels. Albumin value was high in the control group and low in the other groups. In terms of IMA levels there was no significant difference between groups (p > 0.05). When SCUBE-1 levels were examined groups IR and BA+IR were significantly higher than the group 1. TAS was highest in the group BA+IR whereas TOS was highest in the group IR and lower in the group BA+IR. In histopathological analysis, loss of intercellular border loss in hepatocytes, diffuse nuclear pycnosis and mild to moderate neutrophilic cell infiltration were observed in the IR group. Statistically significant dissociation, hemorrhage and severe neutrophilic cell infiltration were seen in hepatocytes of rats with IR (p < 0.05). Conclusion BA has promising results in the treatment of experimental IR injury of the cholestatic liver because of its antioxidant effects. It may be used in clinical practice after more extensive studies about the effects of BA on IR injury of the cholestatic liver.     

Neuroendocrine features of reactive bile ductules in cholestatic liver disease.

Various cholestatic liver diseases are accompanied by a striking increase in the number of bile ductules. This so-called ductular reaction is thought to arise both from ductular metaplasia of hepatocytes and from proliferation of pre-existing bile ductules. Previous studies have shown that these reactive bile ductules differ from their normal counterpart in enzyme and immunohistochemical make-up. Using monoclonal antibodies directed to neuroendocrine markers and immunohistochemistry, we found that reactive bile ductules in cholestatic liver disease display neuroendocrine features. In all cases of primary biliary cirrhosis (PBC), primary sclerosing cholangitis (PSC), extrahepatic obstruction, and acute hepatitis A, reactive bile ductules expressed the neural cell adhesion molecule (N-CAM) and reacted with monoclonal antibody A2B5. Both N-CAM and the ganglioside, recognized by A2B5, are restricted to neuroendocrine cells and tissues. In all but four of these cases, the same bile ductules expressed chromogranin-A, present in the matrix of neuroendocrine granules. Furthermore, in three cases of longstanding cholestasis, scattered periportal hepatocytes expressed chromogranin-A but not N-CAM. Other neuroendocrine markers such as neuron-specific enolase, synaptophysin, or myelin-associated glycoprotein were lacking from both bile ductules and hepatocytes. The neuroendocrine phenotype of bile ductules and hepatocytes was confirmed on electronmicroscopy, demonstrating various numbers of dense-cored, neuroendocrine granules near the peripheral cell membrane in bile ductules as well as in cells intermediate between hepatocytes and bile ductular cells. In 10 cases of normal liver tissue without ductular reaction, bile ductules lacked neuroendocrine markers except in two cases in which very weak reactivity for chromogranin-A was observed. These findings illustrate the presence of a new, neuroendocrine cell type that emerges in the liver during cholestasis. Elucidation of the significance of the neuroendocrine substance(s) produced in the dense cored granules of reactive bile ductules awaits their isolation and characterization. We can speculate that this molecule plays an autocrine or paracrine regulatory role in the process of ductular metaplasia of hepatocytes or growth of bile ductules.     

Effect of cholecystokinin-pancreozymin on bile salt secretion into the duodenal juice in patients with liver cirrhosis

Summary Bile salt secretion into duodenal juice was investigated in eight normal subjects and in 12 patients with hepatic cirrhosis. Duodenal juice was quantitatively collected during a fast and following intravenous injecton of secretin and of cholecystokinin-pancreozymin (CCK).Fasting bile salt output was higher in the cirrhotics than in the controls. Following secretin administration the volume of juice increased more marked by in patients with cirrhosis of the liver than in the normal group. After injection of CCK the bile salt output increased 2–35 fold in the latter, while there was no significant alteration in the cirrhotics. It is suggested that this lack of stimulation of bile salt output after CCK is caused by impaired gall bladder function in patients with chronic liver disease.These results were presented at the International Bile-Salt Meeting, Freiburg i. Br., October 1970.     

The effects of bile salt and raw bile on the intestinal absorption of micellar fatty acid in the rat in vitro

1. The uptake, esterification and transport of [(14)C]oleic acid were studied using sacs of rat everted small intestine incubated in 25 ml. of a buffered mixture of sodium taurocholate, glyceryl mono-oleate and (14)C-labelled oleic acid in micellar form.2. Intestine obtained from bile fistula rats (bile duct cannulated 48 hr previously) showed elevated rates of (14)C uptake into the tissue total lipid compared with sham-operated controls.3. Nearly all of the excess (14)C uptake in the bile fistula group was in the form of free fatty acid. Both groups showed similar rates of [(14)C]oleic acid incorporation into tissue triglyceride and also similar, though small, amounts transported into the serosal fluid.4. In further experiments using intestine from bile fistula rats the addition of 1 ml. of fresh rat bile to the incubation mixture reduced the (14)C uptake to approximately control levels. The addition of 2-3 ml. of fresh bile similarly reduced the uptake and increased (14)C incorporation into the triglycerides of mucosal tissue and serosal fluid.5. These responses were not entirely the result of the bile salts contained in fresh bile since increasing the taurocholate concentration per se caused uptake, esterification and transport all to increase. In the presence of the higher taurocholate concentration the addition of fresh bile still caused a decrease in (14)C uptake.6. There was no significant effect of either fresh bile or taurocholate on the transport of the 3-O-methyl analogue of D-glucose under comparable conditions.7. It is concluded that raw bile contains one or more components other than bile salts which may be important in determining fatty acid absorption.     

Relationship between biliary calcium and bile acid secretion in the regenerating liver of the rat

Biliary calcium secretion during liver regeneration following two-thirds hepatectomy was studied in Wistar rats. Calcium output per 100 g body weight was significantly reduced at 24 h post-hepatectomy but not at other times. Values per gram of liver were significantly increased at 48 h and 96 h. A significant relationship was found between bile acid output and calcium output into bile both in controls and hepatectomized animals. The number of nmol of calcium secreted per nmol of bile acids was increased at 24 h post-hepatectomy parallel to an increased choleretic capacity of bile acids and a decrease in the fraction of bile acid independent flow. Our data confirm that bile acid secretion is an important determinant in the secretion of calcium into bile after partial hepatectomy in the rat.     

Reduced cholestatic potency of taurolithocholate during backward perfusion of the rat liver

Rat livers perfused backward are less susceptible to taurolithocholate (TLC) cholestasis than livers perfused forward. To understand this phenomenon, the uptake, metabolism, and excretion of TLC were studied in rat livers perfused forward and backward with TLC. Bolus injections of [3H]TLC were administered 20 minutes after the onset of unlabeled TLC infusion. Biliary bile acids were measured enzymatically and bile acid species were separated and quantified by thin-layer chromatography. Determination of radioactivity in each spot yielded the percentage distribution of various biliary bile acid metabolites. After perfusion, livers were examined by light and transmission electron microscopy. TLC infusion at 32 nmoles/minute/gm of liver reduced bile flow by 80% or more within 30 minutes of forward perfusion but not at all during backward perfusion. TLC uptake was over 95% regardless of perfusion direction. Although the distribution of biliary metabolites of TLC was the same during forward and backward perfusion, maximal rate of total biliary bile acid excretion was four-fold higher and total recovery of radioactivity in bile was six-fold higher during backward perfusion. Less than 6% of excreted radioactivity was in the form of unmetabolized TLC. Severe cholestasis induced by forward TLC perfusion was associated with extensive structural distortion of bile canalicular membranes almost exclusively in the periportal zone but with little hepatocellular necrosis. Livers perfused backward manifested no cholestasis. They showed cell necrosis in the pericentral zone which became extensive (10%) by 60 minutes, but the canalicular changes occurred only in a small proportion of canaliculi in the pericentral region. Bile canalicular membrane changes developed extensively only when very high doses of TLC were perfused backward. Even then, bile flow fell only 60%. We conclude: the lesser susceptibility to TLC cholestasis of livers perfused backward is in part related to the greater biotransformation and consequent excretion of TLC by pericentral hepatocytes. TLC-induced cholestasis is associated more closely with bile canalicular membrane changes than with extent of hepatocellular necrosis. The greater reduction of bile flow with periportal than with pericentral canalicular change is compatible with current concepts of biliary microanatomy which postulate a flow of bile from pericentral through periportal regions of the lobule into the bile ductules.     

Protective effects of hepatocellular canalicular conjugate export pump (Mrp2) on sodium arsenite-induced hepatic dysfunction in rats

Arsenic is a double-edged sword to human health. The excretion of various organic anions into bile is mediated by an adenosine triphosphate-dependent conjugate export pump, which has been identified as the canalicular isoform of the multidrug resistance protein 2 (Mrp2). It has been proved that Mrp2 can transport arsenite in vitro, but its effects in vivo are not clear. The aim of this study was to investigate whether Mrp2 plays a role in exportation of arsenic in vivo and its protective effects on liver function. Mrp2 protein level in rat liver was determined by Western blot analysis. Total arsenic concentrations in whole blood and bile were measured using hydride generation atomic absorption spectrometry. Alanine aminotransferase (ALT) activity, aspartate aminotransferase activity (AST), glutathione peroxidase (GSH-PX) activity, malon dialdehyde (MDA) and total bilirubin were measured by biochemical assays. The morphological changes were observed by electron microscopy. Total arsenic levels in blood and bile of arsenite-treated rats were significantly higher than those of control rats (P<0.05) at all three different time points. The overexpression of Mrp2 was 36.61%, 32.36% and 12.73% at 2, 4 and 6 weeks, respectively (percentage of controls, P<0.05), which was significantly higher than controls. A positive correlation between Mrp2 expression level and total arsenic concentration in bile indicated that Mrp2 accelerated the transport of arsenic. Electron microscopy showed that microvilli of bile canaliculi became swollen and sparse. ALT and AST activities in serum were markedly raised at 6 weeks. MDA level in serum increased (P<0.05) and GSH-PX activity in serum decreased except for 2 weeks. Damage of liver function became worse following decreased expression of Mrp2. In conclusion, overexpression of Mrp2 may explain increased biliary excretion of arsenic and it may protect liver function.     

Relationship between biliary calcium and bile acid secretion in the regenerating liver of the rat.

Biliary calcium secretion during liver regeneration following two-thirds hepatectomy was studied in Wistar rats. Calcium output per 100 g body weight was significantly reduced at 24 h post-hepatectomy but not at other times. Values per gram of liver were significantly increased at 48 h and 96 h. A significant relationship was found between bile acid output and calcium output into bile both in controls and hepatectomized animals. The number of nmol of calcium secreted per nmol of bile acids was increased at 24 h post-hepatectomy parallel to an increased choleretic capacity of bile acids and a decrease in the fraction of bile acid independent flow. Our data confirm that bile acid secretion is an important determinant in the secretion of calcium into bile after partial hepatectomy in the rat.     

Cellular sources of matrix proteins in experimentally induced cholestatic rat liver

Collagens (I, III, and IV), fibronectin, and laminin were localized using the indirect immunoperoxidase technique 14 days after bile duct ligation, i.e., when extensive fibrosis and numerous neoformed bile ducts were observed. Extensive fibrous septa in enlarged portal spaces were stained for collagens I, III and IV, fibronectin, and laminin. Collagen IV and laminin were abundant around proliferative bile ducts. In addition, collagen IV was nearly continuous in the sinusoids. At the ultrastructural level, antigens were localized in the endoplasmic reticulum of several liver cell types. In portal spaces, bile duct cells and cells that form the transitional canal of Hering were strongly labelled for basement membrane components, particularly laminin, but not for collagens I and III and fibronectin, which were abundant in fibroblast-like cells. Inside the lobule, only Ito cells and, to a lesser extent, endothelial cells contained collagens, fibronectin, and laminin. Ito cells were found to be heavily stained for collagens III and IV, and laminin. Except for fibronectin, which was always abundant, precursors of extracellular matrix proteins were only slightly detectable in the endoplasmic reticulum of some hepatocytes, particularly those located close to altered areas. This study demonstrates that experimental extrahepatic cholestasis in the rat induces periportal fibrosis and continuous deposition of collagen IV in the sinusoids. Several cell types participate in the formation of extracellular matrix components, particularly bile duct cells and Ito cells, with a possible involvement of hepatocytes, thus suggesting that cholestasis provokes changes in the pattern of matrix protein production in liver cells.     

The protective effect of allopurinol on cholestatic liver injury induced by bile duct ligation.

To determine whether oxygen free radicals are responsible for the pathogenesis of the cholestasis induced by ligation of common bile duct (CBD) variables which reflect the hepatic function in the serum, the amount of superoxide radical production, and xanthine oxidase(XO) activity were studied. The activity of serum alanine aminotransferase, bilirubin level in the serum and the amount of superoxide radical production were lower in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Abnormalities of the microscopic structures were reduced in a CBD ligation with allopurinol treated group than in a CBD ligation without allopurinol treated group. Allopurinol, an inhibitor of XO, prevented the hepatic damage induced by CBD ligation through the inhibition of XO. These experiments demonstrate that oxygen free radicals are responsible for the pathogenesis of the cholestatic liver.     

Electron microscopy and morphometry of canalicular differentiation in fetal and neonatal rat liver

In order to complement previous electron microscopic studies on the differentiation of the biliary pole of the rat hepatocyte during perinatal maturation, a morphometric analysis was undertaken of the bile canaliculi in fetal and adult rat liver. Four morphologic canalicular types were found and morphometrically characterized using a surface/volume relation (s/v index) as key parameter. The surface/volume relation of the canaliculus in fetal and postnatal maturation as well as in the adult rat liver supports the previously formulated hypothesis that these different types correspond to successive maturation stages of the biliary pole of the hepaocyte. Furthermore, the s/v index of type 3 canaliculus supports the concept that this canalicular type represents a canalicular structure inefficient for secretion.