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.     

Feedback regulation of bile acid biosynthesis in the rat

The hepatic biosynthesis of bile salts in the rat has been shown to be controlled homeostatically by the quantity of bile salt returning to the liver via the portal circulation. The feedback mechanism was demonstrated in two kinds of experiments. In the first, rats with bile fistulas were infused intraduodenally with sodium taurocholate 12 hr after surgery. If the rate of infusion was greater than 10 mg per 100 g rat per hr, the increase in bile acid output normally observed in bile fistula rats was prevented. In the second type of experiment, the rats were infused with taurocholate 48-72 hr after biliary diversion, when bile acid output had reached a maximal value. Provided the rate of infusion exceeded 10 mg per 100 g rat per hr, bile acid secretion returned to the low levels observed in intact rats. Previous attempts to demonstrate the feedback control have been unsuccessful because too little bile salt was infused. The taurocholate pool of the experimental animals was measured as approximately 15 mg per 100 g rat; it was calculated from this and the above results that this pool circulated 10-13 times daily.     

Bilirubin conjugates in bile of man, rat and dog. Semi-quantitative analysis of bile composition by thin-layer chromatography.

1. Conjugated bile pigments, separated in two fractions by semi-quantitative t.l.c. performed on silicic acid with phenol/water as the developing solvent, were treated with diazotized ethyl anthranilate. Resulting dipyrrylazo derivatives were analysed by quantitative t.l.c. 2. The tentative structure elucidation of tetrapyrrolic bilirubin conjugates and semi-quantitative evaluation of rat bile, post-obstructive human bile and dog bile composition is presented. 3. Homogeneous and mixed hexuronic acid diesters of bilirubin containing glucuronic acid constitute 51% of the total conjugates in normal rat bile, 45% of those in human post-obstructive bile and 38% of those in obstructed rat biles. 4. Monoconjugated bilirubin amounts to 33% of total conjugated bile pigments in normal rat bile, and 17 and 14% in post-obstructive hepatic human bile and gall-bladder bile of dog respectively. After loading with unconjugated bilirubin a greater amount of monoconjugates (56%) occur in the rat bile, whereas bilirubin diglucuronide excretion is decreased (34%). 5. In gall-bladder bile of normal dog, 40% of glucose-containing diconjugates, 32% of homogeneous and/or mixed hexuronic acid (mainly glucuronic acid) diesters of bilirubin and 14% of xylose-containing diconjugates are estimated. 6. Increased amounts of bilirubin conjugates, including some with unidentified uronic acid groups, were observed in cholestatic rat biles and quantities of conjugates with glucuronic acid were decreased.     

Effects of rat bile infusion on renal function in rats.

The influence of rat bile infusion on renal function in rats and the possible role of bile-induced hemolysis in these effects were examined. The hemolytic action of rat bile and some bile salts were determined in vitro. After the i.v. infusion of rat bile (70 mg freeze-dried powder/2.55 ml) into pentobarbitone-anesthetized rats, the urine, sodium and potassium excretion rates were reduced more than half, which was due to the decrease of glomerular filtration rate and increase of tubular water and sodium reabsorption. A fall in blood pressure, a rise in hematocrit, and hemolysis were also found. Infusion of hemolysed (30 microliters RBC) solution produced by distilled water and then made isotonic caused a short-duration increase in renal excretion and glomerular filtration rate, and the blood pressure was unchanged. Infusion of a rat bile-hemolysed solution after removal of bile acids with cholestyramine increased renal excretions at first with reduction thereafter. Infusion of the rat bile-hemolysed solution treated with barium sulfate produced a renal response very similar to rat bile alone. It is proposed that two factors are involved in the renal response after bile infusion, namely bile acid-induced hemolysis producing diuresis with natriuresis, and bile acid-induced antidiuresis and antinatriuresis, possibly due to a direct renal effect.     

Effects of ursodeoxycholic acid, analogues of ursodeoxycholic acid and combination of bile acids on bile acid synthesis in cultured rat hepatocytes

The effect of individual 7 beta-hydroxy bile acids (ursodeoxycholic and ursocholic acid), bile acid analogues of ursodeoxycholic acid, combination of bile acids (taurochenodeoxycholate and taurocholate), and mixtures of bile acids, phospholipids and cholesterol in proportions found in rat bile, on bile acids synthesis was studied in cultured rat hepatocytes. Individual steroids tested included ursodeoxycholate (UDCA), ursocholate (UCA), glycoursodeoxycholate (GUDCA) and tauroursodeoxycholate (TUDCA). Analogues of UDCA (7-methylursodeoxycholate, sarcosylursodeoxycholate and ursooxazoline) and allochenodeoxycholate, a representative of 5 alpha-cholanoic bile acid were also tested in order to determine the specificity of the bile acid biofeedback. Each individual steroid was added to the culture media at concentrations ranging from 10 to 200 microM. Mixtures of taurochenodeoxycholate (TDCA) and taurocholate in concentrations ranging from 150 to 600 microM alone and in combination with phosphatidylcholine (10-125 microM) and cholesterol (3-13 microM) were also tested for their effects on bile acid synthesis. Rates of bile acid synthesis were determined as the conversion of added lipoprotein [4-14C]cholesterol or [2-14C]mevalonate into 14C-labeled bile acids and by GLC quantitation of bile acids secreted into the culture media. Individual bile acids, bile acid analogues, combination of bile acids and mixture of bile acids with phosphatidylcholine and cholesterol failed to inhibit bile acid synthesis in cultured hepatocytes. The addition of UDCA or UCA to the culture medium resulted in a marked increase in the intracellular level of both bile acids, and in the case of UDCA there was a 4-fold increase in beta-muricholate. These results demonstrate effective uptake and metabolism of these bile acids by the rat hepatocytes. UDCA, UCA, TUDCA and GUDCA also failed to inhibit cholesterol-7 alpha-hydroxylase activity in microsomes prepared from cholestyramine-fed rats. The current data confirm and extend our previous observations that, under conditions employed, neither single bile acid nor a mixture of bile acids with or without phosphatidylcholine and cholesterol inhibits bile acid synthesis in primary rat hepatocyte cultures. We postulate that mechanisms other than a direct effect of bile acids on cholesterol-7 alpha-hydroxylase might play a role in the regulation of bile acid synthesis.     

Effect of streptozotocin-induced diabetes in neonatal rat on bile acid pool changes in adult life: selective sensitivity in females

The effect of streptozotocin-induced diabetes in neonatal rat on the bile acid pool and composition during adult life was investigated. Unlike the effect of diabetes in adult rats (where bile acid pool increases markedly), neonatal diabetes caused a reduction in bile acid pool in adult life in females (but not in males) with significant reduction in both cholic and chenodeoxycholic acids. Upon challenge with dietary cholesterol, only the female diabetic rat responded with a further reduction in total bile acid pool. These studies demonstrate a selective sensitivity in the female diabetic rat with regard to diabetes-induced changes in bile acid pool.     

Influence of ischaemic time on the production of bile by perfused rat liver

Production of bile has been studied in rat livers in situ and in livers perfused with rat blood or with bovine erythrocytes in Krebs Ringer buffer containing bovine albumin. The mean rate bile flow in four in situ livers remained almost constant for 3 hr following cannulation while bile flow in vitro decreased gradually throughout the 3-hr perfusion period. The total amount of bile produced in vitro decreased linearly with increase in ischaemic time. This decrease was greater in livers perfused with rat blood than with bovine erythrocytes. The length of the ischaemic time had no effect on other indices of liver function which were measured, i.e., urea synthesis, the ability to maintain a low concentration of ammonia in the perfusate, and the ability to retain potassium within the cells.     

Further studies on the properties of bile acids. I. Bile acid-induced changes of the action of drugs acting on the autonomic nervous system on the heart and blood vessels

The action of certain drugs on the autonomic system was studied under the influence of bile acids: 3,12-dihydroxycholanic acid (deoxycholic), 3,7,12-trihydroxycholanic acid (cholic), and 3,7,12-triketocholanic acid (dehydrocholic). The experiments were carried out on rat heart in vivo and on isolated rat vessel preparation. It was shown that bile acids reduced the stimulating effect of isoprenaline on rat heart and the relaxing effect on the blood vessels. An inversion of the effects of adrenaline and noradrenaline on the rat heart was observed, and an enhancement of the vasoconstrictor effect of these substances. Under the conditions of this experiment an inversion was noted of the action of acetylcholine on the heart and blood vessels.     

Partitioning of bile acids into subcellular organelles and the in vivo distribution of bile acids in rat liver.

1. The subcellular distribution of conjugates of cholic acid and chenodeoxycholic acid between cytosol, nuclei, mitochondria and microsomes in rat liver has been determined. 2. The partition coefficients for the distribution of these bile acids between subcellular fractions and buffer have been measured and used to construct a compartmental model of the amounts of conjugated bile acids present in the different subcellular organelles in vivo. 3. This model indicates that a large percentage of the bile acid in the rat liver is found in the nuclear fraction; 42% of the cholic acid conjugates and 27% of the chenodeoxycholic acid conjugates. Substantial amounts of bile acid are also present in microsomes and mitochondria suggesting that published estimates of the amounts of bile acids in these fractions are underestimates. 4. The model also allows the amount of bile acid which is in free solution in cytosol to be determined; 10.9% of the cholic acid conjugates and 4.1% of the chenodeoxycholic acid conjugates in rat liver were present in this fraction. Knowlege of the amount of free bile acid allows possible roles of the cytosolic bile binding proteins to be assessed.     

Liver receptor homologue-1 mediates species- and cell line-specific bile acid-dependent negative feedback regulation of the apical sodium-dependent bile acid transporter

Intestinal reclamation of bile salts is mediated in large part by the apical sodium-dependent bile acid transporter (ASBT). The bile acid responsiveness of ASBT is controversial. Bile acid feeding in mice results in decreased expression of ASBT protein and mRNA. Mouse but not rat ASBT promoter activity was repressed in Caco-2, but not IEC-6, cells by chenodeoxycholic acid. A potential liver receptor homologue-1 (LRH-1) cis-acting element was identified in the bile acid-responsive region of the mouse but not rat promoter. The mouse, but not rat, promoter was activated by LRH-1, and this correlated with nuclear protein binding to the mouse but not rat LRH-1 element. The short heterodimer partner diminished the activity of the mouse promoter and could partially offset its activation by LRH-1. Interconversion of the potential LRH-1 cis-elements between the mouse and rat ASBT promoters was associated with an interconversion of LRH-1 and bile acid responsiveness. LRH-1 protein was found in Caco-2 cells and mouse ileum, but not IEC-6 cells or rat ileum. Bile acid response was mediated by the farnesoid X receptor, as shown by the fact that overexpression of a dominant-negative farnesoid X-receptor eliminated the bile acid mediated down-regulation of ASBT. In addition, ASBT expression in farnesoid X receptor null mice was unresponsive to bile acid feeding. In summary cell line- and species-specific negative feedback regulation of ASBT by bile acids is mediated by farnesoid X receptor via small heterodimer partner-dependent repression of LRH-1 activation of the ASBT promoter.     

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.     

Biliary protein output by isolated perfused rat livers. Effects of bile salts.

The output of proteins into bile was studied by using isolated perfused rat livers. Replacement of rat blood with defined perfusion media deprived the liver of rat serum proteins (albumin, immunoglobulin A) and resulted in a rapid decline in the amounts of these proteins in bile. When bovine serum albumin was incorporated into the perfusion medium it appeared in bile within 20 min and the amount in the bile was determined by the concentration of the protein in the perfusion medium. The use of a defined perfusion medium also deprived the livers of bile salts and the amounts of these, and of plasma-membrane enzymes [5'-nucleotidase (EC 3.1.3.5) and phosphodiesterase I], in bile declined rapidly. Introduction of micelle-forming bile salts (taurocholate or glycodeoxycholate) to the perfusion medium 80 min after liver isolation markedly increased the output of plasma-membrane enzymes but had no effect on the other proteins. The magnitude of this response was dependent on the bile salt used and its concentration in bile; there was little effect on plasma-membrane enzyme output until the critical micellar concentration of the bile salt had been exceeded in the bile. A bile salt analogue, taurodehydrocholate, which does not form micelles, did not produce the enhanced output of plasma-membrane enzymes. This work supports the view that the output of plasma-membrane enzymes in bile is a consequence of bile salt output and also provides evidence for mechanisms by which serum proteins enter the bile.     

Biochemical site of regulation of bile acid biosynthesis in the rat

The production of bile salts by rat liver is regulated by a feedback mechanism, but it is not known which enzyme controls endogenous bile acid synthesis. In order to demonstrate the biochemical site of this control mechanism, bile fistula rats were infused intravenously with (14)C-labeled bile acid precursors, and bile acid biosynthesis was inhibited as required by intraduodenal infusion of sodium taurocholate. The infusion of taurocholate (11-14 mg/100 g of rat per hr) inhibited the incorporation of acetate-1-(14)C, mevalonolactone-2-(14)C, and cholesterol-4-(14)C into bile acids by approximately 90%. In contrast, the incorporation of 7alpha-hydroxycholesterol-4-(14)C into bile acids was reduced by less than 10% during taurocholate infusion. These results indicate that the regulation of bile acid biosynthesis is exerted via cholesterol 7alpha-hydroxylase provided that hepatic cholesterol synthesis is adequate.     

Biliary excretion of inorganic electrolytes: its role in hepatic bile formation

To define the role of inorganic electrolyte secretion in hepatic bile formation, the effects of secretin, glucagon, and differently structured bile acids on bile flow and composition were studied in the dog, guinea pig, and rat. In the dog and guinea pig, secretin (2.5-10 clinical units X kg-1 X 30 min-1) increased bile flow and bicarbonate concentration in bile, a finding consistent with the hypothesis that the hormone stimulates a bicarbonate-dependent secretion possibly at the level of the bile ductule-duct. In the rat, secretin (5-15 CU X kg-1 X 30 min-1) failed to increase bile secretion. Glucagon (1.25-300 micrograms X kg-1 X 30 min-1) increased bile flow in all the three species, and produced no changes in biliary bicarbonate concentrations in the dog and rat. In the guinea pig, however, glucagon choleresis was associated with an increase in bicarbonate concentration in bile, similar to that observed with secretin. The choleretic activities of various bile acids (taurocholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, and ursodeoxycholic acid, infused at 30-360 mumol X kg-1 X 30 min-1) were similar in the rat (6.9-7.2 microL/mumol), but different in the guinea pig (11-31 microL/mumol). In the latter species, the more hydrophobic the bile acid, the greater was its choleretic activity. In all instances, bile acid choleresis was associated with a decline in the biliary concentrations of chloride, but with no major change in bicarbonate levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification of LDL cholesteryl ester contribution to biliary steroids in the rat

The proportion of LDL cholesteryl ester converted to biliary steroids was quantified in the rat. The pre-existing pool of bile was allowed to drain for 10-12 h through a bile duct cannula. A single intravenous pulse injection of LDL labelled with [3H]cholesterol linoleyl ester was made, followed by a constant infusion of the same material in order to maintain constant specific radioactivity in plasma. A new steady state was achieved within 6 h and bile samples were then collected hourly until 12 h. Although substantial amounts (53-61 micrograms/h) of cholesteryl ester were released into the liver during LDL catabolism, only a very small fraction (0.8-1.90 micrograms/h) was found in biliary steroids. The proportion of LDL cholesteryl esters contributing to biliary steroids was only 1-2%. These results perhaps explain why perturbations to accelerate bile acid excretion have no effect on plasma LDL cholesterol concentration in the rat.     

Molecular cloning and characterization of the murine bile salt export pump

Hepatic bile salt secretion and bile formation are essential functions of the mammalian liver, and the rate-limiting step of hepatocellular secretion of bile salts is canalicular secretion. Recently, the rat sister-of-p-glycoprotein/bile salt export pump (spgp/BSEP) was demonstrated to encode for the rat ATP-dependent canalicular bile salt export protein, and mutations of human BSEP were identified as the cause of PFIC 2. Since mouse models are vital for studies in hepatocellular transport and metabolism, cloning and characterization of the murine gene are essential. In this study, we have cloned a full-length, functional cDNA for the mBsep. The deduced amino acid sequence encodes for a 1321-amino-acid protein and is 94% similar to rat and 89% similar to human bsep. Western immunoblotting using an antibody directed against a carboxy-terminal peptide of mbsep protein reveals a 160kDa protein, which is highly enriched in mouse canalicular membranes. Transfection of mBSEP into Sf-9 insect cells or mammalian Balb-3T3 cells confers functional transport of the bile salt taurocholate. The mBsep mRNA is expressed in murine liver, but not in other tissues. Hepatic mBsep levels appear highly regulated, being markedly diminished in both LPS and estrogen models of cholestasis. These data are important for further murine studies of hepatocellular transport physiology and metabolism.     

Identification of the 14 kDa bile acid transport protein of rat ileal cytosol as gastrotropin

The 14 kDa bile acid binding protein of rat ileal cytosol (I-BABP), previously shown to be the major intracellular transporter of bile acids in enterocytes, was purified by affinity chromatography and gel electrophoresis. Enzymatic digestion of I-BABP which had been electroblotted to nitrocellulose led to the recovery and sequence analysis of four peptides representing 47 residues of sequence (approximately 35% of the full sequence). All the peptide sequences displayed high levels of identity (greater than 60%) and homology (greater than 80%) to the sequences of porcine and canine gastrotropin. This high level of homology together with other features of I-BABP identify it as rat gastrotropin, establishing gastrotropin as the major intracellular bile acid carrier of rat enterocytes.     

Human IgA as a heterovalent ligand: switching from the asialoglycoprotein receptor to secretory component during transport across the rat hepatocyte

Asialoglycoproteins are taken up by the rat liver for degradation; rat polymeric IgA is taken up via a separate receptor, secretory component (SC), for quantitative delivery to bile. There is negligible uptake of these ligands by the converse receptor, and only a low level of missorting of ligands to opposite destinations. The two pathways are not cross-inhibitable and operate independently (Schiff, J.M., M. M. Fisher, and B. J. Underdown, 1984, J. Cell Biol., 98:79-89). We report here that when human IgA is presented as a ligand in the rat, it is processed using elements of both pathways. To study this in detail, different IgA fractions were prepared using two radiolabeling methods that provide separate probes for degradation or re-secretion. Behavior of intravenously injected human polymeric IgA in the rat depended on its binding properties. If deprived of SC binding activity by affinity adsorption or by reduction and alkylation, greater than 80% of human IgA was degraded in hepatic lysosomes; radioactive catabolites were released into bile by a leupeptin-inhibitable process. If prevented from binding to the asialoglycoprotein receptor by competition or by treatment with galactose oxidase, human IgA was cleared and transported to bile directly via SC, but its uptake was about fivefold slower than rat IgA. Untreated human IgA was taken up rapidly by the asialoglycoprotein receptor, but depended on SC binding to get to bile: the proportion secreted correlated 1:1 with SC binding activity determined in vitro, and the IgA was released into bile with SC still attached. These results demonstrate that human IgA is normally heterovalent: it is first captured from blood by the asialoglycoprotein receptor, but escapes the usual fate of asialoglycoproteins by switching to SC during transport. Since the biliary transit times of native human and rat IgA are the same, it is probable that the receptor switching event occurs en route. This implies that the two receptors briefly share a common intracellular compartment.