Bile secretory characteristics of beta-muricholate and its taurine conjugate are similar to those of ursodeoxycholate in the rat

Ursodeoxycholate (UDC) has very high biliary transport maxima values (Tm) for its conjugates as well as the capability of inducing choleresis rich in bicarbonate concentration in the bile in rats. We examined in the present study whether these properties are shared by beta-muricholate (beta-MC), using beta-MC, alpha-muricholate (alpha-MC) and tauro-beta-MC (T beta-MC) in the rat. Bile samples were collected every 20 min for 2 hr in male rats under the infusion of alpha- or beta-MC (1.2 mumol/min/100g). The choleretic response was quicker in beta-MC infused rats than in rats infused with alpha-MC. Bile salt excretion rates increased radically in both experiments. However, in beta-MC infused rats, the bile salt excretion rate began to decrease after 40 min, whereas in alpha-MC infused rats, it continued to increase after 1 hr. Bile bicarbonate concentration significantly increased in beta-MC infused rats but not in alpha-MC infused rats. The Tm of T beta-MC was 2 times higher than the Tm value for taurocholate and was comparable to that of tauroursodeoxycholate (TUDC) which was previously found by the authors. The bile flow (Y, microliter/min/100 g) was significantly correlated with the bile salt excretion rate (X, mumol/min/100 g) [Y = (6.90 +/- 0.24) X + (5.5 + 1.06), n = 41, -0.98, P less than 0.01)], the slope value being higher than that found for TUDC. The results suggest that UDC and beta-MC (and their conjugates) have very similar bile secretory characteristics and may probably share the same transport system in the rat.     

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.     

Effect of taurochenodeoxycholate or tauroursodeoxycholate upon biliary output of phospholipids and plasma-membrane enzymes, and the extent of cell damage, in isolated perfused rat livers.

Isolated perfused rat livers were used to study the effects of taurochenodeoxycholate (TCDC) and tauroursodeoxycholate (TUDC) upon some aspects of biliary composition. After depletion of the endogenous bile salt pool of the liver, introduction of either bile salt brought about increases in bile flow, bile salt output and biliary phospholipid output. Taurochenodeoxycholate needed a lower biliary concentration to produce phospholipid output than did tauroursodeoxycholate. TCDC perfusion caused a substantial output of plasma-membrane enzymes (5'-nucleotidase and alkaline phosphodiesterase) into the bile, whereas TUDC caused little output of either enzyme; this may represent a characteristic difference between the effects of the two bile salts on the hepatobiliary system. The results from TUDC perfusion indicate also that much of the output of biliary phospholipid promoted by bile salts, may be independent of the output of plasma-membrane enzymes promoted by bile salts.     

Tauroursodeoxycholate counteracts hepatocellular lysis induced by tensioactive bile salts by preventing plasma membrane-micelle transition

Ursodeoxycholic acid is widely used as a therapeutic agent for the treatment of cholestatic liver diseases. In these hepatopathies, the bile secretory failure produces accumulation of endogenous, tensioactive bile salts, leading to plasma membrane damage and, eventually, hepatocellular lysis. In the present study, we analyzed the capacity of the ursodeoxycholic acid endogenous metabolite, tauroursodeoxycholate (TUDC), to stabilize the hepatocellular plasma membrane against its transition to the micellar phase induced by the tensioactive bile salt taurochenodeoxycholate (TCDC), the main endogenous bile salt accumulated in cholestasis. The disruption of the plasma membrane was evaluated (i) in isolated hepatocytes, through the release of the enzyme lactate dehydrogenase to the incubation medium and (ii) in isolated plasma membranes, through the self-quenching assay of the membranotropic probe octadecylrhodamine B; this assay allows for detergent-induced transition from membrane bilayer to micelle to be monitored. Our results showed that isolated hepatocytes treated with TUDC are more resistant to TCDC-induced cell lysis. When this effect was evaluated in isolated plasma membranes, the TCDC concentration necessary to reach half of the transition from bilayer to micelle was increased by 22% (p < 0.05). This difference remained even when TUDC was removed from the incubation medium before adding TCDC, thus indicating that TUDC exerted its effect directly on the plasma membrane. When the same experiments were carried out using the non-ionic detergent TX-100 or the cholesterol-complexing detergent digitonin, no protective effect was observed. In conclusion, TUDC prevents selectively the bilayer to micelle transition of the hepatocellular plasma membrane induced by hydrophobic bile salts that typically build up and accumulate in cholestatic processes. Our results suggest that formation of a complex between negatively charged TUDC and cholesterol in the membrane favours repulsion of negatively charged detergent bile salts, thus providing a basis for the understanding of the TUDC protective effects.     

Effects of bile salts on bile formation in rabbits

The effects of different species of bile salts: deoxycholate, taurochenodeoxycholate, ursodeoxycholate, glycodeoxycholate, tauroursodeoxycholate, chenodeoxycholate and cholate (DCA, TCDC, UDCA, GDCA, TUDC, CDCA, CA) on bile secretion were examined in anesthetized rabbits using two different infusion routes. When bile salts were infused intravenously, all bile salts (except for TCDC) significantly increased the volume of bile and bile salt excretion, but their respective efficiency for bile formation was different. The concentration of bicarbonate ion in the bile significantly increased during the choleretic periods induced by DCA, UDCA, GDCA and CDCA but remained unchanged with the other bile salts (CA, TCDC, TUDC). In rabbits, where a bile salt solution was infused in the duodenum and then drained from the intestine through an incision in the distal part of duodenum, none of these bile salts affected bile secretion. The effects of intravenously administered bile salts on rabbit bile secretion are different in terms of their choleretic potency and bicarbonate excretion depending on the species of bile salts used. Furthermore, it was concluded that the intraduodenal infusion of UDCA, which was found to stimulate the pancreatic exocrine function, did not affect bile secretion.     

Effect of bile acids on calcium efflux from isolated rat hepatocytes and perfused rat livers

The changes in intracellular Ca2+ concentration [( Ca2+]i) of hepatocytes induced by certain bile acids are biphasic: an initial increase is followed by a more gradual decrease. This latter decline in [Ca2+]i may be due to an efflux of Ca2+ across the plasma membrane. This hypothesis was tested by studying the effect of different bile acids on the efflux of 45Ca from preloaded rat hepatocytes and isolated perfused rat livers. The following bile acids were studied: cholic (C), ursodeoxycholic (UDC), chenodeoxycholic (CDC), and deoxycholic (DC) acids; their taurine (T) conjugates (TC, TUDC, TCDC, and TDC); and the taurine, sulfate (S), and glucuronide (Glu) derivatives of lithocholic acid (TLC, LS, TLS, and LGlu, respectively). At 0.3 mM, all bile acids except C, TC, TCDC, UDC, and TUDC significantly increased 45Ca efflux from preloaded hepatocytes without affecting cell viability. Dose-response studies revealed that the minimum effective concentration needed to induce 45Ca efflux was 0.06 mM for LS, 0.8 mM for TCDC, and 10 mM for TC. Efflux of 86Rb from preloaded hepatocytes was not significantly altered by 0.1 mM LS, indicating relative specificity for calcium. TDC and DC, but not TC, increased 45Ca efflux from preloaded perfused rat livers. These results showed that bile acids known to increase [Ca2+]i (CDC, DC, TDC, and TLC) also increased 45Ca efflux from hepatocytes and perfused livers and that efflux was also stimulated by LS, TLS, and LGlu. The extent of this efflux was related to the hydrophobicity of the steroid nucleus of the bile acid. It is speculated that bile acid-induced increases in [Ca2+]i activate the plasma membrane Ca2+ pump resulting in increased Ca2+ efflux.     

Cholestasis, metabolism and biliary lipid secretion during perfusion of rat liver with different bile salts.

The biological effects of bile acids depend largely upon their molecular structure. When bile acid uptake exceeds the maximal biliary secretory rate (SRm) cholestasis occurs. In order to characterize the influence of bile acid structure on its cholestatic potency we systematically studied SRm, maximal bile flow, maximal and cumulative phospholipid and cholesterol secretion with different taurine-conjugated tri-, di- and keto bile acids (Table I) in the isolated perfused rat liver. Bile acids with a high critical micellar concentration (CMC) promoted the greatest bile flow; a positive non-linear correlation between CMC and maximal bile flow was found. 3 alpha-Hydroxylated bile acids with a hydroxyl group in 6 alpha and/or 7 beta position and lacking a 12 alpha hydroxy group had a high SRm. SRm was not related to CMC or maximal bile flow, respectively. Phospholipids and cholesterol were secreted in a nearly fixed ratio of 12:1; a strong linear relationship could be observed. Cumulative phospholipid secretion over 48 min was significantly lower for non and poor micelle forming bile acids (TDHC and TUC) than for those with comparatively low CMC values (TUDC, TC, THC, THDC, TCDC) (70-140 vs. 210-450 nmol/g liver). At SRm all bile acids with good micelle forming properties showed a similar cumulative biliary lipid output. However, when biliary lipid output was related to 1 mumol bile acid secreted bile acids with a low SRm induced the highest lipid secretion (TCDC, TC). These data (1) demonstrate that a 6 alpha and/or a 7 beta hydroxy group on the steroid nucleus reduce cholestatic potency if the 12 alpha hydroxy group is absent, (2) suggest that in the case of micelle forming bile acids the total amount of phospholipids secreted in bile (depletion of cellular phospholipids) is associated with the occurrence of cholestasis whereby bile acids with a low SRm deplete the cellular phospholipid content at much lower bile acid concentrations than those with a higher SRm and (3) imply that bile acids with non and poor micelle forming properties (TDHC, TUC) presumably do not cause cholestasis (solely) by depletion of cellular phospholipids.     

Biliary secretion of cefoperazone and its inter-relationship with bile acid transport in the rat

Cefoperazone is a third generation cephalosporin which is secreted predominantly in bile. This study set out to examine the effect of stimulating bile choleresis on the biliary secretion of cefoperazone. Stimulation of both bile acid-dependent and independent bile flow (phenobarbitone pretreatment) hastened the peak appearance of a pulse of cefoperazone into bile. Although the biliary secretion rate of cefoperazone was enhanced by bile acid infusion, the % recovery and maximal biliary concentration were reduced. The reciprocal effect of continuous cefoperazone infusion on the rate of biliary transport of a pulse of bile acid was examined. Cefoperazone infusion hastened the biliary transport of glycocholate. Net recovery of glycocholate was unaffected.     

Effects of bile acids on the muscle functions of guinea pig gallbladder

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.     

Effects of bile salts on cholestan-3beta,5alpha,6beta-triol-induced apoptosis in dog gallbladder epithelial cells

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3beta,5alpha,6beta-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14+/-11% (control) to 48+/-12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63+/-16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31+/-12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.     

Effects of bile salt infusion on chlorpromazine-induced cholestasis in the isolated perfused rat liver.

The present study has demonstrated that tauroursodeoxycholate (TUDC), but not taurocholate, can reverse chlorpromazine (CPZ)-induced cholestasis in the isolated perfused rat liver. At an infusion rate of 1.5 mumol/min, TUDC led to restoration of bile flow in the perfused rat liver made cholestatic by the addition of 250 microM CPZ. This reversal was accompanied by an increased excretion of CPZ and its metabolites. A higher infusion rate of 5.0 mumols TUDC/min, however, led to only a transient increase in bile flow and to no increase in CPZ excretion. In contrast to the effects of TUDC, infusion of taurocholate led to an exacerbation of CPZ-induced cholestasis. The differences in the efficacy of the two bile salts may be due to their relative detergent (hydrophobic) properties.     

Ryanodine receptors contribute to bile acid-induced pathological calcium signaling and pancreatitis in mice

Biliary pancreatitis is the most common etiology for acute pancreatitis, yet its pathophysiological mechanism remains unclear. Ca(2+) signals generated within the pancreatic acinar cell initiate the early phase of pancreatitis, and bile acids can elicit anomalous acinar cell intracellular Ca(2+) release. We previously demonstrated that Ca(2+) released via the intracellular Ca(2+) channel, the ryanodine receptor (RyR), contributes to the aberrant Ca(2+) signal. In this study, we examined whether RyR inhibition protects against pathological Ca(2+) signals, acinar cell injury, and pancreatitis from bile acid exposure. The bile acid tauro-lithocholic acid-3-sulfate (TLCS) induced intracellular Ca(2+) oscillations at 50 μM and a peak-plateau signal at 500 μM, and only the latter induced acinar cell injury, as determined by lactate dehydrogenase (LDH) leakage. Pretreatment with the RyR inhibitors dantrolene or ryanodine converted the peak-plateau signal to a mostly oscillatory pattern (P < 0.05). They also reduced acinar cell LDH leakage, basolateral blebbing, and propidium iodide uptake (P < 0.05). In vivo, a single dose of dantrolene (5 mg/kg), given either 1 h before or 2 h after intraductal TLCS infusion, reduced the severity of pancreatitis down to the level of the control (P < 0.05). These results suggest that the severity of biliary pancreatitis may be ameliorated by the clinical use of RyR inhibitors.     

Effects of bile salts on cholestan-3β,5α,6β-triol-induced apoptosis in dog gallbladder epithelial cells

Oxysterols are cytotoxic agents. The gallbladder epithelium is exposed to high concentrations of oxysterols, and so elucidating the mechanisms of cytotoxicity in this organ may enhance our understanding of the pathogenesis of biliary tract disorders. We investigated the cytotoxic effects of the oxysterol cholestan-3β,5α,6β-triol (TriolC) on dog gallbladder epithelial cells. Apoptosis was the major form of cytotoxicity, as determined by analysis of nuclear morphologic changes and by multiparameter flow cytometry. Hydrophobic bile salts are known to have cytotoxic effects, whereas hydrophilic bile salts have cytoprotective effects. We therefore examined whether the hydrophobic bile acid taurodeoxycholic acid (TDC) and the hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had modifying effects on oxysterol-induced cytotoxicity. TriolC caused an increase in the number of apoptotic cells from 14±11% (control) to 48±12% of total cells (P<0.01). After combining TriolC with TDC, cell apoptosis increased to 63±16% (P<0.05), whereas after addition of TUDC, the number of apoptotic cells decreased to 31±12% (P<0.05) of total cells. In summary, oxysterols such as TriolC induce apoptosis. Hydrophobic bile salts enhance TriolC-induced apoptosis, whereas hydrophilic bile salts diminish TriolC-induced apoptosis. These results suggest that interactions between oxysterols and bile salts play a role in the pathophysiology of biliary tract disorders.     

Thyroid hormone differentially augments biliary sterol secretion in the rat. I. The isolated-perfused liver model.

Thyroid hormone lowers serum cholesterol and alters sterol metabolic processes. This laboratory has previously reported increased biliary lipid secretion as an early effect of triiodothyronine (T3) in the rat. To evaluate whether the bile lipid action of T3 is a primary or secondary effect, the isolated-perfused rat liver model was used. Red blood cells in lipid-free buffer were used to perfuse livers of euthyroid and methimazole-hypothyroid rats, as well as hypothyroid rats given T3 at intervals before perfusion. Bile flow was maintained by taurocholate perfusion. Hypothyroid rats had elevated pre-perfusion serum cholesterol compared to euthyroid (107 +/- 4 vs. 65 +/- 2 mg/dl) and decreased biliary cholesterol (0.016 +/- 0.001 vs. 0.031 +/- 0.004 mumol/g liver/h) secretion. Serum cholesterol decreased to euthyroid levels by 18 h after T3, an effect that was prevented by bile duct ligation. Bile cholesterol secretion doubled by 18 h, and reached levels twice euthyroid by 42 h, while phospholipid secretion doubled to levels just above euthyroid. The fourfold increase in biliary cholesterol secretion occurred with lipid-free perfusion and unchanging bile acid uptake or output. It occurred without a fall in hepatic lipoprotein cholesterol secretion. Blockade of cholesterol synthesis with lovastatin failed to alter T3-augmented bile cholesterol secretion. We conclude that T3 induces biliary cholesterol secretion concomitantly with the fall in serum cholesterol. This augmented biliary secretion did not appear to depend upon lipoprotein uptake, increased bile acid transport, or cholesterol synthesis. It did not occur at the expense of hepatic lipoprotein secretion. Facilitated biliary lipid secretion may be a primary effect of T3.     

Structural alterations in lecithin-cholesterol vesicles following interactions with monomeric and micellar bile salts: physical-chemical basis for subselection of biliary lecithin species and aggregative states of biliary lipids during bile formation

Using complementary physical-chemical methods including turbidimetry, quasielastic light scattering, gel filtration, and phase analysis, we examined the interactions between dilute concentrations of the common bile salt, taurochenodeoxycholate (TCDC), and uni- and multilamellar vesicles (MLVs) composed of defined molecular species of lecithin (L) and varying contents of cholesterol (Ch). Dissolution rates of MLVs with micellar TCDC, as assessed by turbidimetry, were more rapid with vesicles composed of sn-1 palmitoyl species, typical of biliary L, compared with those composed of the more hydrophobic sn-1 stearoyl species. Incorporation of Ch retarded MLV dissolution rates in proportion to the Ch content, and only at high Ch contents were dissolution rates appreciably influenced by the sn-2 fatty acid composition of L. When MLVs contained Ch in amounts characteristic of intracellular membranes (Ch/L approximately 0.1), the dissolution rates of the individual L species by TCDC accurately predicted the steady state L composition of human bile. TCDC interacted with small unilamellar L/Ch vesicles (SUVs) at concentrations well below, as well as appreciably above, its critical micellar concentration. In accordance with the TCDC-egg yolk L-H2O phase diagram, perimicellar concentrations of TCDC interacted with SUVs to form aggregates that were approximately twice the size of the SUVs. These were consistent with the formation of a dispersed hexagonal (rod-like) phase, which co-existed with aqueous bile salt (BS) monomers and either micellar or unilamellar SUV phases. Micellar TCDC completely solubilized SUVs as mixed micelles, putatively via this transient hexagonal phase. With modest Ch-supersaturation, dissolution was followed by the reemergence of a new vesicle population that coexisted metastably with mixed micelles. With high Ch supersaturation, TCDC extracted L and Ch molecules from SUVs in different proportions to form Ch-supersaturated mixed micelles and Ch-enriched SUVs, in accordance with the metastable phase diagram. These experiments are consistent with the hypothesis that sn-1 palmitoyl L species are subselected for bile, in part, by physical-chemical interactions of intracellular BS concentrations with Ch-poor membranes and that the subsequent evolution of Ch-rich vesicles and Ch-saturated mixed micelles occurs via a transitional hexagonal (rod) phase. These liquid-crystalline states are likely to be transient in Ch-unsaturated biles, but may persist in Ch-supersaturated human biles because of their high Ch contents which retard or inhibit these phase transitions.     

Inhibitory action of cyclobutyrol on the secretion of biliary cholesterol and phospholipids.

A number of organic anions are known to decrease biliary secretion of cholesterol and phospholipid without affecting bile acid secretion. Cyclobutyrol (CB) is a choleretic agent which also inhibits biliary lipid secretion. Using isolated perfused rat liver we have studied this inhibition in relation to possible mechanisms suggested for other anions. Shortly after its administration to the isolated perfused liver, CB decreases biliary outputs of cholesterol and phospholipid, without changes in bile acid secretion, at low (450 nmol/min), high (1350 nmol/min) and nil taurocholate infusion rates. The absolute inhibition does not appear to be decreased by elevated bile acid secretion. There is a differential effect on secretion of cholesterol and phospholipid, more marked at low bile acid secretion rates. Biliary outputs of the canalicular membrane enzymes 5'-nucleotidase and alkaline phosphodiesterase I are also depressed by CB administration, but the anion does not affect the biliary output of bovine serum albumin or the output of rat serum albumin into the perfusion fluid. Since CB does not inhibit intracellular vesicular transport or apparently inhibit intracanalicular events, its effect is different from the effect of several other anions. From these studies it appears that the most likely effect of CB is exerted at the level of the canalicular membrane.     

Studies on the origin of biliary phospholipid. Effect of dehydrocholic acid and cholic acid infusions on hepatic and biliary phospholipids.

The correlation between the secretion of biliary phospholipid (PL) and bile acid suggests a regulatory effect of bile acid on PL secretion. Bile acids may influence PL synthesis and/or the mobilization of a preformed PL pool. The objective of this study was to determine the contribution of these two sources to biliary PL, by using an experimental protocol in which dehydrocholic acid (DHCA) and cholic acid (CA) were infused to manipulate biliary PL secretion. In control rats, there was a steady state in bile flow. PL secretion and the biliary secretion of newly synthesized phosphatidylcholine (PC). The specific radioactivity of PC in bile was significantly higher than in plasma, microsomes and canalicular membranes. DHCA infusion decreased biliary PC secretion rate by 80%, and secretion returned to normal values at the transport maximum of CA. The specific radioactivity of biliary PC was decreased by 30% by DHCA infusion and reached normal values during CA infusion. There were no significant changes in the specific radioactivity of PC in plasma or cellular organelles during infusion of bile acids. These data indicate that: (1) newly synthesized PC contributes a small percentage to biliary PC; thus a preformed pool (microsomal and extrahepatic) is a major source of biliary PL; (2) the contribution of the extrahepatic pool to the biliary PL may be more important than the microsomal pool.     

Diabetes and intestinal cholesterol uptake from bile salt solutions

A previously validated in vitro technique was used to determine the effect of diabetes mellitus on the intestinal uptake of cholesterol from various micellar bile salt solutions. The bile salts studied included cholic (C), taurocholic (TC), glycocolic (GC), chenodeoxycholic (CDC), taurochenodeoxycholic (TCDC), glycochenodeoxycholic (GCDC), deoxycholic (DC), taurodeoxycholic (TDC), and glycodeoxycholic (GDC). In control rats there was a reciprocal decline in cholesterol uptake with increasing concentrations of these nine bile acids, and cholesterol uptake was greater from the conjugated primary bile acids than from the unconjugated ones. With a 5 mM concentration of bile acids, the ratios of the uptake of 0.2 mM cholesterol in control rats were C = CDC = DC, TCDC greater than TC greater than TDC, and GC = GCDC greater than GDC; with 20 mM concentrations, the ratios of cholesterol uptake in control rats were C greater than CDC greater than DC, TC greater than TCDC greater than TDC, and GC = GCDC greater than GDC. In the diabetic animals cholesterol uptake was higher than in control rats when using 5 or 20 mM of each of the conjugated bile acids and with cholic acid. In contrast, cholesterol uptake was similar in diabetic and control animals when cholesterol was solubilized with 5 or 20 mM CDC or DC. These differences in cholesterol uptake using the various bile acids and the failure of CDC and DC to facilitate the enhanced uptake of cholesterol in diabetic animals remains unexplained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential stimulation of S-adenosylmethionine decarboxylase by difluoromethylornithine in the rat colon and small intestine.

The effects of sodium cyclobutyrate, a synthetic hydrocholeretic drug, on biliary lipid secretion and on the biliary outputs of several plasma-membrane enzymes were investigated in anaesthetized rats. Administration of a single oral dose of cyclobutyrol (0.72 mmol/kg body wt.) reduced biliary concentration and output of cholesterol and phospholipid. However, bile acid secretion was not significantly modified. This uncoupling effect of lipid secretion remained even when the choleretic response to the drug had ceased. It additionally led to a statistically significant decrease in the cholesterol/bile acid and phospholipid/bile acid molar ratios and in the lithogenic index of the bile. The biliary outputs of the plasma-membrane enzymes alkaline phosphatase and gamma-glutamyltransferase were markedly reduced by the drug. When cyclobutyrol was administered to rats which had been previously fed with a high-cholesterol diet, the effects of cyclobutyrol persisted, but were less marked. Our results demonstrate that the bile acid-independent choleresis induced by cyclobutyrol (related to its pharmacokinetic effect) is accompanied by a pharmacodynamic action that selectively reduces the secretion of biliary lipids. This is due to an uncoupling of the secretion of cholesterol and phospholipids from that of bile acids. Possible explanations for the biliary response to cyclobutyrol are discussed.     

The bile acid taurochenodeoxycholate activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade

Liver injury during cholestasis reflects a balance between the effects of toxic and nontoxic bile acids. However, the critical distinction between a toxic and nontoxic bile acid remains subtle and unclear. For example, the glycine conjugate of chenodeoxycholate (GCDC) induces hepatocyte apoptosis, whereas the taurine conjugate (TCDC) does not. We hypothesized that the dissimilar cellular responses may reflect differential activation of a phosphatidylinositol 3-kinase (PI3K)-dependent signaling pathway. In the bile acid-transporting McNtcp.24 rat hepatoma cell line, TCDC, but not GCDC, stimulated PI3K activity. Consistent with this observation, inhibition of PI3K rendered TCDC cytotoxic, and constitutive activation of PI3K rendered GCDC nontoxic. Both Akt and the atypical protein kinase C isoform zeta (PKCzeta) have been implicated in PI3K-dependent survival signaling. However, TCDC activated PKCzeta, but not Akt. Moreover, inhibition of PKCzeta converted TCDC into a cytotoxic agent, whereas overexpression of wild-type PKCzeta blocked GCDC-induced apoptosis. We also demonstrate that TCDC activated nuclear factor kappaB (NF-kappaB) in a PI3K- and PKCzeta-dependent manner. Moreover, inhibition of NF-kappaB by an IkappaB super-repressor rendered TCDC cytotoxic, suggesting that NF-kappaB is also necessary to prevent the cytotoxic effects of TCDC. Collectively, these data suggest that some hydrophobic bile acids such as TCDC activate PI3K-dependent survival pathways, which prevent their otherwise inherent toxicity.