Increases in biliary cholesterol-to-bile acid ratio in pregnant hamsters fed low and high levels of cholesterol

Gallstones develop when the secretion of cholesterol is elevated compared with the secretion of bile acids into bile. One of the risk factors for the formation of gallstones is pregnancy. Because the pregnancy-induced increase in hepatic cholesterol synthesis rates could play a critical role in the development of cholesterol stones, the aim of the present study was to determine whether stone formation, as assessed by the ratio of cholesterol to bile acids in bile, could be ablated by blocking the pregnancy-induced increase in hepatic sterol synthesis rates. Golden Syrian hamsters were fed either ground chow or chow supplemented with 0.5% cholesterol for 3 wk and studied in the nonpregnant state or in late gestation. In chow-fed animals, a 1.6-fold increase in the ratio of cholesterol to bile acids occurred simultaneously with a sevenfold increase in hepatic sterol synthesis rate and a ninefold increase in the amount of newly synthesized cholesterol secreted into the bile in late gestation. In the cholesterol-fed dams, an increase in the ratio of cholesterol to bile acids occurred even with the lack of induction of hepatic sterol synthesis rates during pregnancy. Thus it appears that the marked induction of hepatic sterol synthesis rates during gestation is not essential for the pregnancy-induced cholesterol saturation of bile when cholesterol is fed to animals.     

The effect of taurine depletion with guanidinoethanesulfonate on bile acid metabolism in the rat

Administration of guanidinoethanesulfonate (GES) to male rats for 5 weeks resulted in a 90% decrease in the hepatic taurine concentration. This depletion of hepatic taurine was associated with a 570% increase in the concentration of glycine-conjugated bile acids, a 30% decrease in the concentration of taurine-conjugated bile acids, and an increase in the ratio of glycine- to taurine-conjugated bile acids from 0.046 to 0.45. The total concentration of bile salts in the bile and the turnover of cholic acid were not affected by administration of GES. The data indicate that the taurine-depleted rat conserves taurine to some extent by using glycine instead of taurine for bile salt synthesis but not by decreasing the daily fractional turnover of bile acids.     

Thyroid hormone differentially augments biliary sterol secretion in the rat. II. The chronic bile fistula model.

To further define thyroid hormone effects on bile acid synthesis and biliary lipid secretion, studies were done in chronic bile fistula rats. Euthyroid and methimazole-hypothyroid rats, with and without triiodothyronine (T3) injection, had total bile diversion for timed bile collections. With interrupted enterohepatic circulation, cholesterol absorption is negligible and bile acid secretion equals bile acid synthesis rate. Hypothyroid rats had diminished levels of bile acid synthesis and biliary secretion of cholesterol and phospholipid. Single dose T3 injection produced a 13-fold increase in bile cholesterol secretion and a 3-fold increase in phospholipid secretion, both initiated 12 h after T3. Bile acid synthesis increased by 50%, but the increase did not begin until 24 h after T3. Neither hypothyroidism nor T3 treatment abolished diurnal rhythms of bile acid synthesis and biliary lipid secretion. Inhibition of cholesterol synthesis with lovastatin resulted in a persistent 33% decrease in bile acid synthesis in euthyroid and hypothyroid rats, while bile cholesterol secretion only transiently decreased. Inhibition of cholesterol synthesis did not alter T3-induced bile cholesterol secretion, with a 10-fold increase seen. However, bile acid synthesis was not stimulated by T3 in the presence of lovastatin. We conclude that facilitated bile acid synthesis and biliary cholesterol secretion are early effects of T3 and may account for the hypocholesterolemia of T3. Cholesterol synthesis does not appear to be required for the T3-induced bile cholesterol secretion.     

Transport of cholesterol into mitochondria is rate-limiting for bile acid synthesis via the alternative pathway in primary rat hepatocytes

Bile acid synthesis occurs mainly via two pathways: the "classic" pathway, initiated by microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" (acidic) pathway, initiated by sterol 27-hydroxylase (CYP27). CYP27 is located in the inner mitochondrial membrane, where cholesterol content is very low. We hypothesized that cholesterol transport into mitochondria may be rate-limiting for bile acid synthesis via the "alternative" pathway. Overexpression of the gene encoding steroidogenic acute regulatory (StAR) protein, a known mitochondrial cholesterol transport protein, led to a 5-fold increase in bile acid synthesis. An increase in StAR protein coincided with an increase in bile acid synthesis. CYP27 overexpression increased bile acid synthesis by <2-fold. The rates of bile acid synthesis following a combination of StAR plus CYP27 overexpression were similar to those obtained with StAR alone. TLC analysis of (14)C-labeled bile acids synthesized in cells overexpressing StAR showed a 5-fold increase in muricholic acid; in chloroform-extractable products, a dramatic increase was seen in bile acid biosynthesis intermediates (27- and 7,27-hydroxycholesterol). High-performance liquid chromatography analysis showed that 27-hydroxycholesterol accumulated in the mitochondria of StAR-overexpressing cells only. These findings suggest that cholesterol delivery to the inner mitochondrial membrane is the predominant rate-determining step for bile acid synthesis via the alternative pathway.     

Regulation of biliary protein secretion in dogs

The biliary secretion of protein in response to bile acids and other agents known to increase bile flow was examined in a chronic bile fistula dog model. Infusion of 25, 50, or 75 mumole/kg/hr sodium taurocholate after 3 hr of bile fistulization increased biliary protein output significantly by 52, 86, and 108% respectively compared to preinfusion values. A proportionate increase in biliary albumin output during taurocholate choleresis was demonstrated. Protein outputs during bile fistulization without taurocholate replacement were unchanged. The non-micelle-forming bile acid dehydrocholate markedly increased bile flow but did not change protein output. Similarly, the hormonal choleretics glucagon and secretin caused significant decreases in biliary protein concentration but no change in protein output. These data indicate a correlation between biliary protein secretion and bile acid-dependent bile flow. It is likely that regulation of certain proteins is dependent on the micelle-forming properties of bile acids.     

Bile salts and calcium absorption

1. The study of the effect of bile salts on enhancing calcium absorption in the rachitic chick has been extended to bile salts not present in chick bile, e.g. glycine conjugates and bile alcohol sulphates. 2. Bile and bile salts cause an increase in calcium absorption from sparingly soluble calcium hydrogen phosphate when compared with a suspension of calcium hydrogen phosphate in saline. 3. If the bile ducts of normal rats are tied the absorption of calcium from calcium hydrogen phosphate decreases but can be restored by giving bile salts with the calcium salt. 4. Bile salts increase solubility in water of the sparingly soluble calcium salts, phytate and phosphate at pH values between 6 and 8. 5. Bile salts increase the solubility in lipid solvents of calcium in approximately the same proportion as they increase the absorption of calcium from the gut. 6. The physiological role of bile in calcium absorption and its mode of action are discussed.     

Biliary lipid secretion in hypercholesterolemia.

A report on the effects of primary bile acid ingestion alone or in combination with plant sterols on serum cholesterol levels, biliary lipid secretion, and bile acid metabolism. Biliary bile acid and cholesterol secretion were measured in four patients with type IIa hypercholesterolemia before and after randomized treatment periods. During these periods either a bile acid mixture (cholic-chenodeoxycholic 2:1, a proportion similar to that endogenously synthesized in health), at a level of 20 mg/kg, or the same mixture plus sitosterols, 200 mg/kg, was fed. Serum cholesterol and the cholesterol saturation of fasting-state bile was also measured. Pretreatment biliary lipid secretion was within normal limits. Bile acid kinetic measurements were also recorded before treatment and showed that cholic acid synthesis was disproportionately decreased relative to that of chenodeoxycholic acid, a finding previously reported by others. Administration of the bile acid mixture increased biliary bile acid secretion in 3 of 4 patients, but did not influence biliary cholesterol secretion. The combination of sitosterol-bile acid, however, caused a relative decrease in cholesterol secretion in bile, and fasting-state bile became unsaturated in all patients. No change in fecal neutral sterol excretion occurred during the beta-sitosterol-bile acid regimen, suggesting that simultaneous bile acid feeding blocks the compensatory increase in cholesterol synthesis known to be induced by beta-sitosterol feeding in hypercholesterolemic patients. Serum cholesterol levels also fell modestly during the sitosterol-bile acid regimen, the decrease averaging 15%. We conclude that the abnormally low rate of bile acid synthesis in patients with type IIa hyperlipoproteinemia does not influence biliary lipid secretion; that increasing the input of the two primary bile acids into the enterohepatic circulation does not increase biliary cholesterol secretion or lower serum cholesterol levels in such patients; and that the usual increase in cholesterol synthesis induced by beta-sitosterol feeding does not occur if bile acids are administered simultaneously.     

Alkaline phosphatase in cholestatic and cirrhotic rats. A biochemical and histochemical study

Alkaline phosphatase (ALP) was studied by enzyme histochemical methods and by biochemical quantitations in rat livers with chronic bile duct obstruction and experimental cirrhosis. The most evident ALP increase was histochemically found in portal tracts of rats with bile duct obstruction and localized to the walls of proliferating blood vessels. Furthermore, a slight canalicular membrane enzyme increase was histochemically found in both groups, most evident in cirrhosis, whereas the biochemical assay of ALP in serum and liver from both pathological groups showed 3 times higher values compared to controls. The portal tracts did not seem to contribute to the serum increase, since the rise of serum ALP was similar in chronic bile duct obstruction and in experimental cirrhosis without changes of the portal tracts. It is concluded that the increase ALP activity in serum from rats with bile duct obstruction and cirrhosis mainly has a hepatocytic origin.     

Alpha-asarone inhibits HMG-CoA reductase, lowers serum LDL-cholesterol levels and reduces biliary CSI in hypercholesterolemic rats.

Our results showed that alpha-asarone was an inhibitor of hepatic HMG-CoA reductase and that the administration of alpha-asarone at 80 mg/kg body wt. for 8 days decreased serum cholesterol by 38% (p < 0.001) in hypercholesterolemic rats. This alpha-asarone treatment affected mainly the serum LDL-cholesterol levels, leaving serum HDL-cholesterol lipoproteins unaffected, with a consequent decrease of 74% in the LDL/HDL ratio. In addition, alpha-asarone especially stimulated bile flow in hypercholesterolemic rats (60%), increasing the secretion of bile salts, phospholipids and bile cholesterol. The drug also reduced the cholesterol levels of gallbladder bile, whereas the concentration of phospholipids and bile salts increased only slightly, leading to a decrease in the cholesterol saturation index (CSI) of bile in the hypercholesterolemic rats. This CSI decrease and the increase in bile flow induced by alpha-asarone may account for the cholelitholytic effect of alpha-asarone. It seems that alpha-asarone induced clearance of cholesterol from the bloodstream and that the excess of hepatic cholesterol provided by LDL-cholesterol is diverted to bile sterol secretion via a bile choleresis process. The inhibition of HMG-CoA reductase and the increase in bile flow induced by alpha-asarone, as well as the decrease in the CSI, could then explain the hypocholesterolemic and cholelitholytic effects of alpha-asarone.     

The common bile duct ligation in rat: a relevant in vivo model to study the role of mechanical stress on cell and matrix behaviour

Common bile duct ligation leads to bile accumulation and liver fibrosis. In this model, little attention has been dedicated to the modification of the common bile duct. We have studied by histochemistry and immunohistochemistry, 3 and 5 days after ligation, the connective tissue modifications of the common bile duct wall. After bile duct ligation, compared with normal bile duct, a strong increase of the bile duct diameter, due to bile stasis, and a thickness of the bile duct wall were observed; numerous myofibroblasts expressing α-smooth muscle actin appeared in parallel with the detection of many proliferating connective tissue cells. These myofibroblasts secreted very early high amount of elastic fibre components, elastin and fibrillin-1. Elastic fibre increase was also observed close to the epithelial cell layer. Procollagen type III deposition was also induced 3 days after ligation but decreased thereafter, underlining that myofibroblasts modify their synthesis of extracellular matrix components to comply with the request. We show here that common bile duct ligation represents an invaluable model to study myofibroblastic differentiation and extracellular matrix adaptation produced by an acute mechanical stress.     

Natural bile acids and synthetic analogues modulate large conductance Ca2+-activated K+ (BKCa) channel activity in smooth muscle cells

Bile acids have been reported to produce relaxation of smooth muscle both in vitro and in vivo. The cellular mechanisms underlying bile acid-induced relaxation are largely unknown. Here we demonstrate, using patch-clamp techniques, that natural bile acids and synthetic analogues reversibly increase BK(Ca) channel activity in rabbit mesenteric artery smooth muscle cells. In excised inside-out patches bile acid-induced increases in channel activity are characterized by a parallel leftward shift in the activity-voltage relationship. This increase in BK(Ca) channel activity is not due to Ca(2+)-dependent mechanism(s) or changes in freely diffusible messengers, but to a direct action of the bile acid on the channel protein itself or some closely associated component in the cell membrane. For naturally occurring bile acids, the magnitude of bile acid-induced increase in BK(Ca) channel activity is inversely related to the number of hydroxyl groups in the bile acid molecule. By using synthetic analogues, we demonstrate that such increase in activity is not affected by several chemical modifications in the lateral chain of the molecule, but is markedly favored by polar groups in the side of the steroid rings opposite to the side where the methyl groups are located, which stresses the importance of the planar polarity of the molecule. Bile acid-induced increases in BK(Ca) channel activity are also observed in smooth muscle cells freshly dissociated from rabbit main pulmonary artery and gallbladder, raising the possibility that a direct activation of BK(Ca) channels by these planar steroids is a widespread phenomenon in many smooth muscle cell types. Bile acid concentrations that increase BK(Ca) channel activity in mesenteric artery smooth muscle cells are found in the systemic circulation under a variety of human pathophysiological conditions, and their ability to enhance BK(Ca) channel activity may explain their relaxing effect on smooth muscle.     

Choleretic activity and biliary elimination of lipids and bile acids induced by an artichoke leaf extract in rats

The therapeutic properties of artichoke (Cynara scolymus L.) preparations have been known since ancient times. The traditional use of artichoke leaf extract (ALE) in gastroenterology is mainly based upon its strong antidyspeptic actions which are mediated by its choleretic activity. The aim of this study was to investigate the effects of ALE on bile flow and the formation of bile compounds in anaesthetised Wistar rats after acute and repeated (twice a day for 7 consecutive days) oral administration. A significant increase in bile flow was observed after acute treatment with ALE as well as after repeated administration. The choleretic effects of ALE were similar to those of the reference compound dehydrocholic acid (DHCA). Total bile acids, cholesterol and phospholipid were determined by enzymatic assays. There was a strong ALE-induced increase in total bile acid concentration over the entire experiment. With the highest dose (400 mg/kg), a significant increase was obtained after single and repeated administration. The bile acids-increased effects of ALE were much more pronounced than those of reference (DHCA). No significant differences in cholesterol and phospholipid content could be found.     

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.     

Diabetes-induced bile acid composition changes in rat bile determined by high performance liquid chromatography.

The distribution of glycine and taurine conjugated bile acids in bile from streptozotocin-induced diabetic rats were determined by high performance liquid chromatography (HPLC). Biliary bile acid output in diabetic rats was significantly greater compared to control (p less than 0.001). The increase is not a generalized effect of diabetes, but is the preferential increased production of taurochenodeoxycholic acid. These observed changes in bile acid composition may represent greater capacity of bile from diabetic rats to solubilize cholesterol. In the absence of a gallbladder, however, rat bile undergo continuous enterohepatic circulation, and consequently is not subjected to modifications by gallbladder epithelial cells that would potentiate cholesterol precipitation.     

Increased cholesterol 7alpha-hydroxylase expression and size of the bile acid pool in the lactating rat

Maximal bile acid secretory rates and expression of bile acid transporters in liver and ileum are increased in lactation, possibly to facilitate increased enterohepatic recirculation of bile acids. We determined changes in the size and composition of the bile acid pool and key enzymes of the bile acid synthetic pathway [cholesterol 7alpha-hydroxylase (Cyp7a1), sterol 27-hydroxylase (Cyp27a1), and sterol 12alpha-hydroxylase (Cyp8b1)] in lactating rats relative to female virgin controls. The bile acid pool increased 1.9 to 2.5-fold [postpartum (PP) days 10, 14, and 19-23], compared with controls. A 1.5-fold increase in cholic acids and a 14 to 20% decrease in muricholic acids in lactation significantly increased the hydrophobicity index. In contrast, the hepatic concentration of bile acids and small heterodimer partner mRNA were unchanged in lactation. A 2.8-fold increase in Cyp7a1 mRNA expression at 16 h (10 h of light) demonstrated a shift in the diurnal rhythm at day 10 PP; Cyp7a1 protein expression and cholesterol 7alpha-hydroxylase activity were significantly increased at this time and remained elevated at day 14 PP but decreased to control levels by day 21 PP. There was an overall decrease in Cyp27a1 mRNA expression and a 20% decrease in Cyp27a1 protein expression, but there was no change in Cyp8b1 mRNA or protein expression at day 10 PP. The increase in Cyp7a1 expression PP provides a mechanism for the increase in the bile acid pool.     

Effect of bile on Vibrio parahaemolyticus.

Many enteric pathogens are thought to enter a viable but nonculturable state when deprived of nutrients. Virulent strains of the enteric pathogen Vibrio parahaemolyticus are rarely isolated from their low-nutrient aquatic environments, possibly due to their nonculturability. Host factors such as bile may trigger release from dormancy and increase virulence in these strains. In this study, the addition of bile or the bile acid deoxycholic acid to estuarine water-cultured bacteria led to an increase in the direct viable count and colony counts among the virulent strains. This effect was not demonstrated in the nonvirulent strains, and it was reversed by extraction of bile acids with cholestyramine. Bile-treated V. parahaemolyticus had lower levels of intracellular calcium than untreated cells, and this effect coincided with an increase in the number of metabolically active cells. Chelation of intracellular calcium with BAPTA/AM (R. Y. Tsien, Biochemistry 19:2396-2402, 1980) produced similar results. Addition of bile to V. parahaemolyticus cultures in laboratory medium enhanced factors associated with virulence such as Congo red binding, bacterial capsule size, and adherence to epithelial cells. These results suggest that a bile acid-containing environment such as that found in the human host favors growth of virulent strains of V. parahaemolyticus and that bile acids enhance the expression of virulence factors. These effects seem to be mediated by a decrease in intracellular calcium.     

Altered bile acid physiology during lactation in the rat

There is evidence that the rate of bile acid secretion increases significantly during lactation in the rat. We show that this increase in secretion rate is accompanied by an expanded bile acid pool and that occasioning the enhancement of both pool size and secretion is an increase in bile acid synthesis. The hypothesis is advanced that maternal prolactin, promoted by suckling young, amplifies cholesterol-7-alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis, and perhaps HMG-CoA reductase, the rate-limiting enzyme in cholesterogenesis.     

Regulatory effects of dietary sterols and bile acids on rat intestinal HMG CoA reductase

The specific activity (concentration) of microsomal HMG CoA reductase of intestinal crypt cells was studied in rats fed sterols and bile acids, either singly or in combination. It was found that the basal activity of the reductase was not suppressed by the administration of relatively large amounts of bile acid (taurocholate or taurochenodeoxycholate). Bile acids reduced the specific activity of the reductase only in rats in which the activity of the enzyme had first been enhanced by biliary diversion or by sitosterol feeding. In addition, bile acid feeding abolished the diurnal elevation of reductase activity that normally occurs between midnight and 2 a.m. In no case did bile acids reduce enzyme activity below basal levels. A pronounced (60%) reduction of intestinal HMG CoA reductase activity was observed in rats fed cholesterol and bile acid in combination. This reduction in activity could not be ascribed to an increase in intestinal bile acid flux but was associated with an increase in sterol concentration within the intestinal crypt cells. These results indicate that dietary sterols and bile acids both play a role in the regulation of intestinal HMG CoA reductase.     

Retrograde intrabiliary injection of amphipathic materials causes phospholipid secretion into bile. Taurocholate causes phosphatidylcholine secretion, 3-[(3-cholamidopropyl)dimethylammonio]-propane-1-sulphonate (CHAPS) causes mixed phospholipid secretion.

The control of biliary phospholipid and cholesterol secretions by bile acid was studied by using the technique of retrograde intrabiliary injection. Taurocholate (TC), a moderately hydrophobic bile acid, taurodehydrocholate (TDHC), a hydrophilic non-micelle-forming bile acid, and 3-[(3-cholamidopropyl)-dimethylammonio]propane-1-sulphonate (CHAPS), a detergent, were individually administered by retrograde intrabiliary injection (RII) into the biliary tree, and bile acids, phospholipids and cholesterol subsequently appearing in the bile were measured. TC (1.3 mumol; 45 microliters) injected retrogradely provoked a 3.5-fold increase in biliary phospholipid output for 40 min, as compared with the saline control. Injection of 2.7 mumol of TC (90 microliters) caused a 7.5-fold increase in phospholipid output, which reached a peak at 12 min after RII, and phospholipid output continued for 40 min. Cholesterol output was also elicited under these conditions, showing both dose-dependency and extended secretion. Injection of 1.8 mumol of TDHC caused very little increase in either biliary phospholipid or cholesterol. Injection of 0.9 mumol of CHAPS (45 microliters) provoked a single substantial peak of phospholipid output in the 3 min bile sample. T.l.c. analysis of the phospholipid extracts of the bile collected after each compound showed, for TC, a single compound which co-migrated with the phosphatidylcholine standard, whereas for CHAPS substantial amounts of other phospholipids were present.     

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.