Role of the hepatocyte microtubular system in the excretion of bile salts and biliary lipid: implications for intracellular vesicular transport

The role of the hepatocyte microtubular system in the transport and excretion of bile salts and biliary lipid has not been defined. In this study the effects of microtubule inhibition on biliary excretion of micelle- and non-micelle-forming bile salts and associated lipid were examined in rats. Low-dose colchicine pretreatment had no effect on the baseline excretion of biliary bile salts and phospholipid in animals studied 1 hr after surgery (basal animals), but slightly retarded the excretion of tracer [14C]taurocholate relative to that of lumicolchicine-pretreated (control) rats. However, colchicine pretreatment resulted in a marked reduction in the excretion of 2 mumol/100 g doses of a series of four micelle-forming bile salts of differing hydrophilicity, but had no significant effect on the excretion of the non-micelle-forming bile salt, taurodehydrocholate. Continuous infusion of 0.2 mumol of taurocholate/(100 g.min) following 24 hr of biliary drainage (depleted/reinfused animals) resulted in physiologic bile flow with biliary excretion rates of bile salts, phospholipid, and cholesterol that were markedly inhibited (mean 33, 39, and 42%, respectively) by colchicine or vinblastine pretreatment. Excretion of tracer [14C]taurocholate also was markedly delayed by colchicine in these bile salt-depleted/reinfused animals. In contrast, colchicine did not inhibit bile salt excretion in response to reinfusion of taurodehydrocholate. Thus, under basal conditions, the microtubular system appears to play a minor role in hepatic transport and excretion of bile salts and biliary lipid. However, biliary excretion of micelle-forming bile salts and associated phospholipid and cholesterol becomes increasingly dependent on microtubular integrity as the transcellular flux and biliary excretion of bile salts increases, in both bile salt-depleted and basal animals. We postulate that cotransport of micelle-forming bile salts and lipids destined for biliary excretion, via an intracellular vesicular pathway, forms the basis for this microtubule dependence.     

Characterization of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells.

The unstirred water layer has been shown to lead to an underestimation of apparent Km (Km(app)) values for active transport processes in intestinal whole tissue preparations. Isolated cells offer several potential advantages in the study of transport processes including a decreased diffusion layer of water adjacent to their absorptive membranes. Initial studies in cells isolated from rat intestine involving measurements of CO2 and lactate production and O2 consumption showed that overall metabolic pathways were functioning. Next, unidirectional uptake rates of bile acids across the isolated cell membrane were determined following correction for extracellular fluid contamination with a non-absorbable marker. Using epithelial cells isolated from jejunum P(app) for eight bile acid monomers varied from 24.9 (taurocholate) to 1563 (deoxycholate) nmol/min/100 mg protein/mM. From these data the incremental free energy changes for the addition of a hydroxyl, glycine and taurine group to the bile acid molecule were calculated to be 982, 1040 and 1464 cal/mol, respectively, values similar to those obtained after correction for unstirred water layer resistance in whole tissue preparations. Following subtraction of the passive component in isolated ileal cells complete kinetic curves for taurocholate and taurodeoxycholate yielded V(app) values of 109 and 70 nmol/min per 100 mg, respectively. Km(app) values of 0.24 mM (taurocholate) and 0.10 mM (taurodeoxycholate) are lower than usually recorded in whole tissue. Bile acid uptake into cells from ileum, but not jejunum, was affected by temperature, metabolic and competitive inhibition. These studies indicate that isolated epithelial cells are a metabolically viable, relatively purified intestinal preparation which discriminates between active and passive transport processes for bile acids under conditions where unstirred water layer artifacts are minimized.     

Regulatory effects of sterols and bile acids on hepatic 3-hydroxy-3-methylglutaryl CoA reductase and cholesterol 7alpha-hydroxylase in the rat

Specific activities of the hepatic microsomal enzymes 3-hydroxy-3-methylglutaryl CoA (HMG CoA) reductase and cholesterol 7alpha-hydroxylase were studied in rats fed sterols and bile acids. The administration of bile acids (taurocholate, taurodeoxycholate, taurochenodeoxycholate) at a level of 1% of the diet for 1 wk reduced the activity of HMG CoA reductase. Taurocholate and taurodeoxycholate, but not taurochenodeoxycholate, inhibited cholesterol 7alpha-hydroxylase. Dietary sitosterol produced increases in the specific activity of HMG CoA reductase (3.6-fold) and cholesterol 7alpha-hydroxylase (1.4-fold), and biliary cholesterol concentrations in this group more than doubled. Compared with controls fed the stock diet, the simultaneous administration of sitosterol and taurochenodeoxycholate resulted in a 60% decrease of HMG CoA reductase activity and no change in cholesterol 7alpha-hydroxylase activity or biliary cholesterol concentration. Rats fed sitosterol plus taurocholate had nearly normal HMG CoA reductase activity, but cholesterol 7alpha-hydroxylase was inhibited and biliary cholesterol remained high. Bile acid secretion rates and biliary bile acid composition were similar in controls and sterol-fed animals. In all groups receiving bile acids, biliary secretion of bile acids was nearly doubled and bile acid composition was shifted in the direction of the administered bile acid. It is concluded that the composition of the bile acid pool influences the hepatic concentrations of the rate-controlling enzymes of bile acid synthesis.     

Absence of negative feedback control of bile acid biosynthesis in cultured rat hepatocytes

The effect of individual bile acids on bile acid synthesis was studied in primary hepatocyte cultures. Relative rates of bile acid synthesis were measured as the conversion of lipoprotein [4-14C]cholesterol into 4-14C-labeled bile acids. Additions to the culture media of cholate, taurocholate, glycocholate, chenodeoxycholate, taurochenodeoxycholate, glycochenodeoxycholate, deoxycholate, and taurodeoxycholate (10-200 microM) did not inhibit bile acid synthesis. The addition of cholate (100 microM) to the medium raised the intracellular level of cholate 10-fold, documenting effective uptake of added bile acid by cultured hepatocytes. The addition of 200 microM taurocholate to cultured hepatocytes prelabeled with [4-14C]cholesterol did not result in inhibition of bile acid synthesis. Taurocholate (10-200 microM) also failed to inhibit bile acid synthesis in suspensions of freshly isolated hepatocytes after 2, 4, and 6 h of incubation. Surprisingly, the addition of taurocholate and taurochenodeoxycholate (10-200 microM) stimulated taurocholate synthesis from [2-14C]mevalonate-labeled cholesterol (p less than 0.05). Neither taurocholate nor taurochenodeoxycholate directly inhibited cholesterol 7 alpha-hydroxylase activity in the microsomes prepared from cholestyramine-fed rats. By contrast, 7-ketocholesterol and 20 alpha-hydroxycholesterol strongly inhibited cholesterol 7 alpha-hydroxylase activity at low concentrations (10 microM). In conclusion, these data strongly suggest that bile acids, at the level of the hepatocyte, do not directly inhibit bile acid synthesis from exogenous or endogenous cholesterol even at concentrations 3-6-fold higher than those found in rat portal blood.     

Tauroursodeoxycholate prevents taurocholate induced cholestasis

The nature of transport pathway(s) for the biliary excretion of taurocholate and tauroursodeoxycholate was examined by comparing the biliary transport maximum (Tm) value for taurocholate during the infusion of taurocholate alone with that of taurocholate combined with tauroursodeoxycholate. The combined infusion of tauroursodeoxycholate resulted in an appreaciable excretion of tauroursodeoxycholate while the excretion rate of taurocholate was not reduced in comparison with the Tm value of taurocholate alone. Furthermore, the Tm state of taurocholate was maintained for a much longer period with the simultaneous infusion of tauroursodeoxycholate than by the infusion of taurocholate alone. The cholestasis usually produced by the excess infusion of taurocholate was also prevented when tauroursodeoxycholate was simultaneously infused. Since plasma taurocholate concentration was not significantly different between the two rat groups, the results suggest the presence of the facilitative interaction of tauroursodeoxycholate with the taurocholate excretion.     

Mechanisms by which cAMP increases bile acid secretion in rat liver and canalicular membrane vesicles

Bile acid secretion induced by cAMP and taurocholate is associated with recruitment of several ATP binding cassette (ABC) transporters to the canalicular membrane. Taurocholate-mediated bile acid secretion and recruitment of ABC transporters are phosphatidylinositol 3-kinase (PI3K) dependent and require an intact microtubular apparatus. We examined mechanisms involved in cAMP-mediated bile acid secretion. Bile acid secretion induced by perfusion of rat liver with dibutyryl cAMP was blocked by colchicine and wortmannin, a PI3K inhibitor. Canalicular membrane vesicles isolated from cAMP-treated rats manifested increased ATP-dependent transport of taurocholate and PI3K activity that were reduced by prior in vivo administration of colchicine or wortmannin. Addition of a PI3K lipid product, phosphoinositide 3,4-bisphosphate, but not its isomer, phosphoinositide 4,5-bisphosphate, restored ATP-dependent taurocholate in these vesicles. Addition of a decapeptide that activates PI3K to canalicular membrane vesicles increased ATP-dependent transport above baseline activity. In contrast to effects induced by taurocholate, cAMP-stimulated intracellular trafficking of the canalicular ABC transporters was unaffected by wortmannin, and recruitment of multidrug resistance protein 2, but not bile salt excretory protein (bsep), was partially decreased by colchicine. These studies indicate that trafficking of bsep and other canalicular ABC transporters to the canalicular membrane in response to cAMP is independent of PI3K activity. In addition, PI3K lipid products are required for activation of bsep in the canalicular membrane. These observations prompt revision of current concepts regarding the role of cAMP and PI3K in intracellular trafficking, regulation of canalicular bsep, and bile acid secretion.     

The role of tubular reabsorption in the renal excretion of bile acids.

1. The renal excretion of bile acids was studied in an isolated rat kidney preparation perfused with a protein-free medium. 2. Tubular reabsorption exceeded 95% for both non-sulphated and sulphated bile acids at filtered loads of less than 30 nmol/min. 3. At low filtered loads the reabsorption of taurocholate and taurochenodeoxycholate was almost complete. Efficient reabsorption of taurochenodeoxycholate was maintained over a wider range of filtered loads than for taurocholate. These observations suggest that active transport may occur. 4. At high filtered loads saturation of reabsorption of taurocholate and taurochenodeoxycholate did not occur, which indicates that passive diffusion is involved in reabsorption. 5. Active proximal-tubular secretion of bile acids was not demonstrated in competition experiments with p-aminohippurate. 6. The fractional reabsorption of taurocholate, chenodeoxycholate 3,7-disulphate and chenodeoxycholate 7-monosulphate was decreased by the addition of taurochenodeoxycholate to the perfusate, so that their renal excretion was enhanced. This interaction between the bile acids for reabsorption may explain the different composition of bile acids in urine compared with that in plasma in cholestasis in man. 7. Conjugated bilirubin decreased the fractional reabsorption of both taurocholate and taurochenodeoxycholate at low filtered loads (less than 30 nmol/min) but not at high filtered loads (400 nmol/min).     

Renal dipeptidase is one of the membrane proteins released by phosphatidylinositol-specific phospholipase C.

4,4-Di-isothiocyanostilbene-2,2'-disulphonic acid inhibition of taurocholate efflux from canalicular vesicles was used to demonstrate that potential driven and 'carrier'-mediated canalicular excretion of taurocholate occur via a common, rather than two separate, pathways. This electrogenic canalicular bile acid 'carrier' preferentially transports trihydroxylated and conjugated dihydroxylated bile acids, but not the unphysiological oxo bile acids, and possibly extends its substrate specificity to other amphipathic molecules such as sulphobromophthalein.     

The effect of colchicine on the development of lithocholic acid-induced cholestasis. A study of the role of microtubules in intracellular cholesterol transport.

The pathogenesis of lithocholic acid (LCA-Na)-induced cholestasis involves a rapid accumulation of cholesterol in the bile canalicular membrane. Since microtubules play an important role in the intracellular transport of many materials, including cholesterol, the present study was undertaken to assess the extent to which they participate in the development of LCA-Na-induced cholestasis. Rats were pretreated with either colchicine (0.2 mumol/100 g body wt.) or saline solution 90 min before injection with LCA-Na (12 mumol/100 g body wt.). Colchicine, although not increasing bile flow by itself, significantly reduced the cholestasis caused by LCA-Na (57-32% reduction in bile flow) without affecting its metabolism into less toxic bile acids or its distribution in blood, liver or bile. Bile canalicular membranes isolated from animals treated with a combination of colchicine and LCA-Na contained less cholesterol than those treated with LCA-Na alone. However, membranes obtained from rats treated with colchicine alone contained much less cholesterol than did controls. It was found that the total amount of cholesterol accumulated within the bile canalicular membrane following LCA-Na treatment (LCA-Na + colchicine versus colchicine alone compared with LCA-Na versus controls) was unchanged by colchicine treatment. In view of these findings it is suggested that the total amount of cholesterol present within the bile canalicular membrane determines the extent of LCA-Na-induced cholestasis, LCA-Na probably moves cholesterol to the bile canalicular membrane via a microtubule independent pathway, and microtubules are unlikely to function in the transcellular transport of LCA-Na.     

Hydrolysis of bile acid conjugates by Clostridium bifermentans

Summary Two strains of Clostridium bifermentans have been investigated for their ability to hydrolyse bile acid conjugates under conditions suited to further transformation of the free acids liberated. In batch fermentation at 0.5 g/l substrate concentration, growing cells effected the near-quantitative hydrolysis of glycodeoxycholate, taurodeoxycholate and taurocholate within 48 h; glycocholate was 88% hydrolysed. At substrate concentration greater than 1.0 g/l however, taurine conjugates were less well hydrolysed. Further transformation of the liberated cholic acid to deoxycholic acid and/or 7-ketodeoxycholic acid was achieved, but quantitative conversion was not observed.     

Alteration of the enterohepatic recirculation of bile acids in rats after exposure to ionizing radiation

The aim of this work was to study acute alterations of the enterohepatic recirculation (EHR) of bile acids 3 days after an 8-Gy radiation exposure in vivo in the rat by a washout technique. Using this technique in association with HPLC analysis, the EHR of the major individual bile acids was determined in control and irradiated animals. Ex vivo ileal taurocholate absorption was also studied in Ussing chambers. Major hepatic enzyme activities involved in bile acid synthesis were also measured. Measurements of bile acid intestinal content and intestinal absorption efficiency calculation from washout showed reduced intestinal absorption with significant differences from one bile acid to another: absorption of taurocholate and tauromuricholate was decreased, whereas absorption of the more hydrophobic taurochenodeoxycholate was increased, suggesting that intestinal passive diffusion was enhanced, whereas ileal active transport might be reduced. Basal hepatic secretion was increased only for taurocholate, in accordance with the marked increase of CYP8B1 activity in the liver. The results are clearly demonstrate that concomitantly with radiation-induced intestinal bile acid malabsorption, hepatic bile acid synthesis and secretion are also changed. A current working model for pathophysiological changes in enterohepatic recycling after irradiation is thus proposed.     

Effects of bile acids on biliary epithelial cells: proliferation,cytotoxicity, and cytokine secretion

Hydrophobic bile acids, which are known to be cytotoxic for hepatocytes, are retained in high amount in the liver during cholestasis. Thus, we have investigated the effects of bile acids with various hydrophobicities on biliary epithelial cells. Biliary epithelial cells were cultured in the presence of tauroursodeoxycholate (TUDC), taurocholate (TC), taurodeoxycholate (TDC), taurochenodeoxycholate (TCDC), or taurolithocholate (TLC). Cell proliferation, viability, apoptosis and secretion of monocyte chemotactic protein-1 (MCP-1) and of interleukin-6 (IL-6) were studied. Cell proliferation was increased by TDC, and markedly decreased by TLC in a dose dependent manner (50-500 microM). Cell viability was significantly decreased by TLC and TCDC at 500 microM. TLC, TDC and TCDC induced apoptosis at high concentrations. The secretion of MCP-1 and IL-6 was markedly stimulated by TC. TUDC had no significant effect on any parameter. These findings demonstrate that hydrophobic bile acids were cytotoxic and induced apoptosis of biliary epithelial cells. Furthermore, TC, a major biliary acid in human bile, stimulated secretion of cytokines involved in the inflammatory and fibrotic processes occurring during cholestatic liver diseases.     

Effects of acute administration of taurocholic and taurochenodeoxycholic acid on biliary lipid excretion in the rat.

Comparison of the effects of biliary lipid excretion produced by infusion of taurochenodeoxycholate and taurocholate showed no significant difference when the bile acids were infused for a relatively short period of time. Cholesterol excretion rates measured during depletion of the bile acid pool were significantly higher than cholesterol excretion rates measured during infusion of bile acids at various rates. These data indicate that there is some mechanism in addition to bile acid excretion that is responsible for biliary excretion of cholesterol when the enterohepatic circulation is intact.     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

Examination of bile acid negative feedback regulation in rats

Recent data obtained using cultured rat hepatocytes showed that bile acids do not inhibit bile acid synthesis, whereas cholesterol concentrations vary in parallel with bile acid synthesis (Davis et al. (1983. J. Biol. Chem. 258: 4079-4082). This led us to re-evaluate in vivo experiments upon which the consensus that bile acid synthesis is primarily regulated by bile acid "negative feedback" is based. Infusion of taurocholate into either the jugular vein or duodenum of bile-diverted rats stimulated biliary cholesterol secretion and bile flow, but it did not inhibit bile acid synthesis. The lack of an inhibitory effect was evident using several different infusion rates of taurocholate. Even at the greatest rate of taurocholate infusion (25 mumol/(100 g.hr] there was no significant inhibition of bile acid synthesis. In contrast, infusing mevinolin (1 mg/hr), a potent competitive inhibitor of HMG-CoA reductase, almost completely inhibited bile acid synthesis and biliary cholesterol secretion. Since mevinolin did not affect bile flow, these results cannot be ascribed to bile secretory failure. Thus, while these studies suggest that taurocholate may not regulate bile acid synthesis directly via negative feedback, cholesterol is likely to act as a positive effector of bile acid synthesis.     

Weanling, but not adult, rabbit colon absorbs bile acids: flux is linked to expression of putative bile acid transporters

Intestinal handling of bile acids is age dependent; adult, but not newborn, ileum absorbs bile acids, and adult, but not weanling or newborn, distal colon secretes Cl(-) in response to bile acids. Bile acid transport involving the apical Na(+)-dependent bile acid transporter (Asbt) and lipid-binding protein (LBP) is well characterized in the ileum, but little is known about colonic bile acid transport. We investigated colonic bile acid transport and the nature of the underlying transporters and receptors. Colon from adult, weanling, and newborn rabbits was screened by semiquantitative RT-PCR for Asbt, its truncated variant t-Asbt, LBP, multidrug resistance-associated protein 3, organic solute transporter-alpha, and farnesoid X receptor. Asbt and LBP showed maximal expression in weanling and significantly less expression in adult and newborn rabbits. The ileum, but not the colon, expressed t-Asbt. Asbt, LBP, and farnesoid X receptor mRNA expression in weanling colon parallel the profile in adult ileum, a tissue designed for high bile acid absorption. To examine their functional role, transepithelial [(3)H]taurocholate transport was measured in weanling and adult colon and ileum. Under short-circuit conditions, weanling colon and ileum and adult ileum showed net bile acid absorption: 1.23 +/- 0.62, 5.53 +/- 1.20, and 11.41 +/- 3.45 nmol x cm(-2) x h(-1), respectively. However, adult colon secreted bile acids (-1.39 +/- 0.47 nmol x cm(-2) x h(-1)). We demonstrate for the first time that weanling, but not adult, distal colon shows net bile acid absorption. Thus increased expression of Asbt and LBP in weanling colon, which is associated with parallel increases in taurocholate absorption, has relevance in enterohepatic conservation of bile acids when ileal bile acid recycling is not fully developed.     

Characterization of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells

The unstirred water layer has been shown to lead to an underestimation of apparent passive permeability coefficients ($\text{P(}\text{app}\text{)}$) and cause a significant overestimation of apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{(K}{}_{\mathrm{<mtext>m</mtext>}}\text{(}\text{app}\text{))}$ values for active transport processes in intestinal whole tissue preparations. Isolated cells offer several potential advantages in the study of transport processes including a decreased diffusion layer of water adjacent to their absorptive membranes. Initial studies in cells isolated from rat intestine involving measurements of CO₂ and lactate production and O₂ consumption showed that overall metabolic pathways were functioning. Next, unidirectional uptake rates of bile acids across the isolated cell membrane were determined following correction for extracellular fluid contamination with a non-absorbable marker. Using epithelial cells isolated from jejunum $\text{P(}\text{app}\text{)}$ for eight bile acid monomers varied from 24.9 (taurocholate) to 1563 (deoxycholate) nmol/min/100 mg protein/mM. From these data the incremental free energy changes for the addition of a hydroxyl, glycine and taurine group to the bile acid molecule were calculated to be 982, 1040 and 1464 cal/mol, respectively, values similar to those obtained after correction for unstirred water layer resistance in whole tissue preparations. Following subtraction of the passive component in isolated ileal cells complete kinetic curves for taurocholate and taurodeoxycholate yielded $\text{V(}\text{app}\text{)}$ values of 109 and 70 nmol/min per 100 mg, respectively. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{(}\text{app}\text{)}$ values of 0.24 mM (taurocholate) and 0.10 mM (taurodeoxycholate) are lower than usually recorded in whole tissue. Bile acid uptake into cells from ileum, but not jejunum, was affected by temperature, metabolic and competitive inhibition. These studies indicate that isolated epithelial cells are a metabolically viable, relatively purified intestinal preparation which discriminates between active and passive transport processes for bile acids under conditions where unstirred water layer artifacts are are minimized.     

Synthesis and biliary excretion of tyrosine-conjugated bile salts in Wistar rats

Tyrosine-labelled free and glycine-conjugated bile acids were synthesized and radiolabelled with 125I to high purity. The synthetic method utilized excess tyrosine methyl ester hydrochloride (1.4 equiv.) and bile acid (one equiv.) via dicyclohexylcarbodiimide (1.4 equiv.) with yields of 90-93% for tyrosine bile acid conjugates and glycyltyrosine conjugates and 56-60% yields for the glycylglycyltyrosine conjugates. All of the eight iodinated tyrosine bile acids tested were rapidly excreted into bile following intravenous injection. In bile duct-cannulated rats with ligated renal pedicles under pentobarbital anaesthesia the percentages of injected dose recovered from bile within 20 min were as follows: cholylglycine ([14C]cholylGly), 81.2 +/- 1.3%; taurocholate ([14C]taurocholate), 94.3 +/- 1.0%; cholyltyrosine (125I-cholylTyr), 85.5 +/- 3.3%; deoxycholyltyrosine (125I-deoxycholylTyr), 87.9 +/- 6.3%; chenodeoxycholyltyrosine (125I-chenodeoxycholylTyr), 93.4 +/- 2.9; cholylglycyltyrosine (125I-cholylGlyTyr), 95.7 +/- 6.7%; deoxycholylglycyltyrosine (125I-deoxylcholylGlyTyr), 92.5 +/- 3.2%; chenodeoxycholylglycyltyrosine (125I-chenodeoxycholylGlyTyr), 94.1 +/- 3.1%; cholyldiglycyltyrosine (125I-cholylGlyGlyTyr), 85.2 +/- 3.6%, and deoxycholyldiglycyltyrosine (125I-deoxycholylGlyGlyTyr), 85.5 +/- 2.7%. Values are means +/- SD. Thus the biliary excretion of 125I-chenodeoxycholylGlyTyr, 125I-chenodeoxycholylTyr, 125I-deoxycholylGlyTyr and 125I-cholylGlyTyr was similar to that of [14C]taurocholate, the major naturally occurring bile acid in the rat, and the biliary excretion of all the tyrosine conjugates was similar to or exceeded that of [14C]cholylglycine. Conjugation with tyrosine enhanced the efficiency of plasma-to-bile transport of most naturally occurring bile acids. Comparison of glycyltyrosine conjugates with glycylglycyltyrosine conjugates suggests that any additional benefit derived by elongation of the side-chain is probably negated by obscuring the 12 alpha-hydroxyl function on the steroid nucleus in the bile acid glycylglycyltyrosine conjugates.     

Relation between glutathione redox changes and biliary excretion of taurocholate in perfused rat liver

The influence of the intracellular glutathione status on bile acid excretion was studied in the perfused rat liver. Perturbation of the thiol redox state by short term additions of diamide (100 microM) or hydrogen peroxide (250 microM) or t-butyl hydroperoxide (250 microM) led to a reversible inhibition of biliary taurocholate release without affecting hepatic uptake; inhibition amounted to 45% for diamide and 90% for the hydroperoxides. Concomitantly, the bile acid accumulated intracellularly. Bile flow increased from 1.3 to 2.0 microliters X min-1 X g liver-1 upon infusion of taurocholate (10 microM); the latter value was suppressed to 1.2 microliters X min-1 X g liver-1 by the addition of t-butyl hydroperoxide (250 microM). Similarly, the hepatic disposition of another bile constituent, bilirubin, was suppressed by 70% upon addition of hydrogen peroxide. While the addition of hydrogen peroxide inhibited also the endogenous release of bile acids almost completely, endogenous bile flow was much less affected, decreasing from 1.3 to 1.0 microliters X min-1 X g liver-1. Measurement of [14C]erythritol clearance showed bile/perfusate ratios of about unity both in the absence and presence of hydrogen peroxide, suggesting canalicular origin of the bile under both conditions. In livers from Se-deficient rats low in Se-GSH peroxidase (less than 5% of controls), hydrogen peroxide inhibited taurocholate transport substantially less, providing evidence for the involvement of glutathione in mediating the inhibition observed in normal livers. The percentage inhibition of taurocholate release and intracellular glutathione disulfide (GSSG) content were closely correlated. The addition of t-butyl hydroperoxide caused a several-fold increase of biliary GSSG release, whereas biliary GSH release was even decreased. The results establish a role of glutathione in canalicular taurocholate disposition.     

Bile salt related secretion of acid phosphatase in rat bile

The biliary excretion of bile salts, lysosomal acid phosphatase, and total proteins were studied in rats under different experimental conditions: during bile salt loss through a bile fistula and after loading with exogenous sodium taurocholate. The experimental models were suitable to demonstrate that variations in the excretion of bile salts were associated with those of acid phosphatase output. During bile salt depletion, acid phosphatase output showed a decrease parallel to that of bile salts. Following a single i.v. injection of sodium taurocholate and during its i.v. infusion, a rapid increase of acid phosphatase excretion in bile was seen. The patterns of enzyme outputs observed after administration of sodium taurocholate suggested a bulk discharge in bile of lysosomal contents. The profiles of protein output were similar to those of acid phosphatase suggesting an association between the secretory mechanism of these bile constituents. In contrast to sodium taurocholate, 4-methylumbelliferone, which also increases canalicular bile flow, did not produce changes in the excretory patterns of the bile components studied. Therefore, the results suggested a bile salt related secretion of acid phosphatase in the rat, which may involve protein secretion in bile.