Prolonged stimulation of pancreatic serous secretions by bile and sodium taurocholate in anaesthetized rats.

There have been numerous reports that infusion of either natural bile or bile salts into the duodenum evokes a rapid increase in pancreatic secretion through the release of the hormone secretin from the duodenal mucosa. We have extended this observation by the demonstration of an additional late increase in secretion which persisted for many hours and have sought to identify the processes underlying this increase. In anaesthetised rats, infusion of 20 mM taurocholate into the duodenum caused a staircase-like increase in the weight of pancreatic secretion which extended over many hours during which, the HCO[Formula: see text] and protein output of the secretion showed only minimal changes. This effect was also reproduced with intra-duodenal infusion of natural bile which was inferred to act though its taurocholate content. Since the stimulatory action was also obtained with superfusion of taurocholate or natural bile onto the small intestine and by intravenous injection of taurocholate, it was concluded that taurocholate acted by being absorbed into the bloodstream and then by exerting a stimulatory action on the exocrine pancreas. This action was inhibited by puromycin (a protein synthesis inhibitor), by furosemide (a Na( + )/K( + )/2Cl(-) cotransporter inhibitor), though not by SITS (an inhibitor of Cl(-)/HCO[Formula: see text] exchange). The long lasting increase in pancreatic serous secretion would be consistent with the possible activation of gene transcription by taurocholate leading to increased activity of the Na( + )/K( + )/2Cl(-) cotransporter through which the acinar cells increased their secretions.

Fibrogenesis in pediatric cholestatic liver disease: role of taurocholate and hepatocyte-derived monocyte chemotaxis protein-1 in hepatic stellate cell recruitment.

UNLABELLED: Cholestatic liver diseases, such as cystic fibrosis (CF) liver disease and biliary atresia, predominate as causes of childhood cirrhosis. Despite diverse etiologies, the stereotypic final pathway involves fibrogenesis where hepatic stellate cells (HSCs) are recruited, producing excess collagen which initiates biliary fibrosis. A possible molecular determinant of this recruitment, monocyte chemotaxis protein-1 (MCP-1), an HSC-responsive chemokine, was investigated in CF liver disease and biliary atresia. The bile-duct-ligated rat and in vitro coculture models of cholestatic liver injury were used to further explore the role of MCP-1 in HSC recruitment and proposed mechanism of induction via bile acids. In both CF liver disease and biliary atresia, elevated hepatic MCP-1 expression predominated in scar margin hepatocytes, closely associated with activated HSCs, and was also expressed in cholangiocytes. Serum MCP-1 was elevated during early fibrogenesis. Similar observations were made in bile-duct-ligated rat liver and serum. Hepatocytes isolated from cholestatic rats secreted increased MCP-1 which avidly recruited HSCs in coculture. This HSC chemotaxis was markedly inhibited in interventional studies using anti-MCP-1 neutralizing antibody. In CF liver disease, biliary MCP-1 was increased, positively correlating with levels of the hydrophobic bile acid, taurocholate. In cholestatic rats, increased MCP-1 positively correlated with taurocholate in serum and liver, and negatively correlated in bile. In normal human and rat hepatocytes, taurocholate induced MCP-1 expression. CONCLUSION: These observations support the hypothesis that up-regulation of hepatocyte-derived MCP-1, induced by bile acids, results in HSC recruitment in diverse causes of cholestatic liver injury, and is a key early event in liver fibrogenesis in these conditions. Therapies aimed at neutralizing MCP-1 or bile acids may help reduce fibro-obliterative liver injury in childhood cholestatic diseases.

The role of NF-kappaB activation in the pathogenesis of acute pancreatitis.

Acute pancreatitis is an inflammatory disease of the pancreas which, in its most severe form, is associated with multi-organ failure and death. Recently, signalling molecules and pathways which are responsible for the initiation and progression of this disease have been under intense scrutiny. One important signalling molecule, nuclear factor kappaB (NF-kappaB), has been shown to play a critical role in the development of acute pancreatitis. NF-kappaB is a nuclear transcription factor responsible for regulating the transcription of a wide variety of genes involved in immunity and inflammation. Many of these genes have been implicated as central players in the development and progression of acute pancreatitis. This review discusses recent advances in the investigation of pancreatic and extrapancreatic (lungs, liver, monocytes and macrophages, and endothelial cells) NF-kappaB activation as it relates to acute pancreatitis.

Bicarbonate-rich choleresis induced by secretin in normal rat is taurocholate-dependent and involves AE2 anion exchanger.

Canalicular bile is modified along bile ducts through reabsorptive and secretory processes regulated by nerves, bile salts, and hormones such as secretin. Secretin stimulates ductular cystic fibrosis transmembrane conductance regulator (CFTR)-dependent Cl- efflux and subsequent biliary HCO3- secretion, possibly via Cl-/HCO3- anion exchange (AE). However, the contribution of secretin to bile regulation in the normal rat, the significance of choleretic bile salts in secretin effects, and the role of Cl-/HCO3- exchange in secretin-stimulated HCO3- secretion all remain unclear. Here, secretin was administered to normal rats with maintained bile acid pool via continuous taurocholate infusion. Bile flow and biliary HCO3- and Cl- excretion were monitored following intrabiliary retrograde fluxes of saline solutions with and without the Cl- channel inhibitor 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) or the Cl-/HCO3- exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). Secretin increased bile flow and biliary excretion of HCO3- and Cl-. Interestingly, secretin effects were not observed in the absence of taurocholate. Whereas secretin effects were all blocked by intrabiliary NPPB, DIDS only inhibited secretin-induced increases in bile flow and HCO3- excretion but not the increased Cl- excretion, revealing a role of biliary Cl-/HCO3- exchange in secretin-induced, bicarbonate-rich choleresis in normal rats. Finally, small hairpin RNA adenoviral constructs were used to demonstrate the involvement of the Na+-independent anion exchanger 2 (AE2) through gene silencing in normal rat cholangiocytes. AE2 gene silencing caused a marked inhibition of unstimulated and secretin-stimulated Cl-/HCO3- exchange. In conclusion, maintenance of the bile acid pool is crucial for secretin to induce bicarbonate-rich choleresis in the normal rat and that this occurs via a chloride-bicarbonate exchange process consistent with AE2 function.

Bile salts regulate intestinal epithelial cell migration by nuclear factor-kappa B-induced expression of transforming growth factor-beta.

BACKGROUND: Mucosal restitution is an important repair modality in the gastrointestinal tract. We have shown that taurodeoxycholate increases intestinal epithelial cell migration by increasing TGF-beta expression, and that the transcription factor NF-kappa B regulates TDCA induced cell migration after injury. The objectives of this study were to determine if this is a property shared by other bile salts or an effect specific to TDCA, and to determine if NF-kappa B regulates TGF-beta expression. STUDY DESIGN: Studies were conducted in IEC-6 cells. Cell migration was examined using an in vitro model. TGF-beta protein and mRNA expression was determined by ELISA and Northern blot analysis. Sequence-specific NF-kappa B binding activity was measured by gel shift assays. RESULTS: Taurocholate and deoxycholate at physiologic concentrations significantly increased intestinal epithelial cell migration 6 hours after wounding (p < 0.01), and was associated with a significant increase in specific NF-kappa B binding activity. Inhibition of NF-kappa B activity significantly inhibited cell migration during restitution and resulted in a significant decrease in TGF-beta mRNA expression and protein expression. CONCLUSIONS: We conclude that bile salts at physiologic conditions increase cell migration after injury, an effect regulated by NF-kappa B. Further, NF-kappa B elicits TGF-beta gene transcription during cell migration. These data support a physiologic role of bile salts in the maintenance of intestinal mucosal integrity.

Effects of high taurocholate load on activities of hepatic alcohol metabolizing enzymes.

Membrane-associated cytotoxicity induced by hydrophobic bile salts is a major contributing factor leading to liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases but the nature of this effect is still unclear. Using alcohol metabolising enzymes as cellular markers, the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate was examined. Two animal models of biliary retention, bile duct obstruction and choledochocaval fistula was used to investigate the effect of taurocholate on the hepatic alcohol metabolizing enzymes: cytosolic alcohol dehydrogenase, microsomal ethanol oxidizing system, catalase and aldehyde dehydrogenase before and after the infusion of taurocholic acid or tauroursodeoxycholic acid for two days period. Bile duct obstruction was found to be similar to or slightly exceeds choledochocaval fistula in the degree of retention. Following the taurocholic acid infusion, the serum alcohol dehydrogenase activity as well as microsomal ethanol oxidizing system and aldehyde dehydrogenase were greatly increased but the level of cytosolic alcohol dehydrogenase and catalase activities was found to be lower in either or both models in comparison with the control animals. However, the tauroursodeoxycholic acid infusion did not induce any significant changes in the levels of all the alcohol metabolizing enzyme activities in either or both models. These findings suggest that hydrophobic taurocholic acid (7alpha) affects the plasmalemma to allow leakage of cytosolic alcohol dehydrogenase into the blood circulation, stimulates the biosynthesis of microsomal ethanol oxidizing system and aldehyde dehydrogenase, and suppresses the biosynthesis of alcohol dehydrogenase and catalase. But in contrast, the hydrophilic tauroursodeoxycholic acid (7beta) provided hepatoprotective effect.

Intraduodenal conjugated bile salts exert negative feedback control on gall bladder emptying in the fasting state without affecting cholecystokinin release or antroduodenal motility.

BACKGROUND: Intraduodenal bile salts exert negative feedback control on postprandial gall bladder emptying. AIMS: We wished to examine whether a similar control mechanism occurs in the fasting state. METHODS: Intraduodenal bile salt depletion was achieved by 12 g of cholestyramine. Thereafter, in study A (seven subjects), the effects on gall bladder volume (by ultrasound) and antroduodenal motility of intraduodenal infusions of taurocholate egg yolk-phosphatidylcholine micelles were assessed. In study B (nine subjects), the effects on gall bladder volume of infusing mixed micelles composed of taurocholate (100 mM) and low (26 mM) or high (68 mM) amounts of egg yolk-phosphatidylcholine, or low amounts of dipalmitoylphosphatidylcholine were determined. RESULTS: Cholestyramine induced strong and prolonged gall bladder contraction without cholecystokinin release. In study A, micellar infusions increased gall bladder volume without affecting migrating motor complex cycle length. In study B, intraduodenal infusion induced strong increases in gall bladder volume in the case of taurocholate micelles containing low amounts of egg yolk-phosphatidylcholine, moderate increases in micelles containing low amounts of dipalmitoylphosphatidylcholine but no change in micelles containing high amounts of egg yolk-phosphatidylcholine, in all cases without altered plasma cholecystokinin levels. Phosphatidylcholine hydrolysis was significantly higher after infusion of egg yolk-phosphatidylcholine compared with infusion of dipalmitoylphosphatidylcholine containing micelles. Intermixed micellar-vesicular bile salt concentrations (responsible for detergent effects) were higher in egg yolk-phosphatidylcholine than in dipalmitoylphosphatidylcholine containing model biles and if lyso-phosphatidylcholine was included. CONCLUSIONS: Intraduodenal bile salts exert negative feedback on fasting gall bladder volume. The modulating effects of various phospholipids may relate to their effects on intermixed micellar-vesicular bile salt concentrations.

Intracellular trafficking and regulation of canalicular ATP-binding cassette transporters.

The bile canaliculus contains at least four ATP-binding cassette (ABC) proteins responsible for ATP-dependent transport of bile acids (spgp), nonbile acid organic anions (mrp2), organic cations (mdr1), and phosphatidylcholine (mdr2). Other ABC transporters (including mrp3) have also been partially localized to the canaliculus; however, their function has not been fully delineated. The specific amount and function of spgp and mrp2 in the canalicular membrane increases in response to taurocholate and cAMP. The mechanism involves increased recruitment of spgp and mrp2 from Golgi to the canalicular membrane by a microtubular and PI3 kinase-dependent vesicular trafficking system. Because the effects of taurocholate and cAMP summate, two distinct pathways are proposed. Mdr family members traffic either directly to the apical plasma membrane or, in the case of spgp, through a separate intracellular pool(s); in either case, there is no direct evidence for transcytosis of ABC transporters from Golgi to basolateral plasma membrane and subsequently to the canalicular plasma membrane. Direct transfer from Golgi to apical membrane was demonstrated by in vivo pulse labeling, in vitro membrane localization, and on-line video microscopy in WIFB9 cells that were stably transfected with mdr1-GFP. A critical role for 3'-phosphoinositide products of PI3 kinase was demonstrated in the intracellular trafficking of canalicular ABC transporters and for optimal transporter activity within the canalicular membrane. These studies suggest that many intracellular components, including ATP, Ca2+, numerous GTPases, microtubules, cytoplasmic motors, and other unknown factors, are required for physiologic regulation of ABC transporter traffic from Golgi to the canalicular membrane. Defects in this complex system are postulated to produce an "intrahepatic traffic jam" that results in defective ABC transporter function in the canalicular membrane and, consequently, in cholestasis.

Solubilization of liver alkaline phosphatase isoenzyme during cholestasis in dogs.

OBJECTIVE: To determine the mechanism by which liver alkaline phosphatase (LALP) isoenzyme is converted from a membrane-bound enzyme to the soluble enzyme during cholestasis. SAMPLE POPULATION: Serum and tissues from 2 dogs. PROCEDURE: The LALP was purified by use of affinity chromatography in samples of serum from dogs with complete bile duct obstruction. Gas chromatography/mass spectrometry was used to detect myo-inositol residues that would be evident when serum LALP had been membrane-attached and released by phospholipase activity. Exclusion chromatography, gel electrophoresis, and octyl-sepharose phase separation of the serum isolate were used to confirm cleavage of the hydrophobic membrane anchor. Western immunoblot analysis was used to distinguish release by glycosylphosphatidylinositol phospholipase D (GPI-PLD) from that by glycosylphosphatidylinositol phospholipase C (GPI-PLC). Intact hepatocytes were incubated with canine serum GPI-PLD to test sensitivity of LALP to release by GPI-PLD. Hepatocyte membrane fragments were treated with serum GPI-PLD and mixtures of taurocholate and taurodeoxycholate to test effects of bile acids on LALP release. RESULTS: Amounts of myo-inositol per mole of serum LALP isolate were equal to amounts detected with LALP isolated from hepatic tissue. Evaluation of results of western immunoblot analysis and electrophoretic mobility suggested release by GPI-PLD rather than by GPI-PLC. Membrane-bound LALP was resistant to serum GPI-PLD activity in the absence of bile acids; however, incubation in the presence of bile acids caused release of LALP. CONCLUSIONS: Solubilization of LALP during cholestasis involves cleavage of its membrane anchor by endogenous GPI-PLD activity. Action of GPI-PLD is likely enhanced by increased concentrations of hepatic bile acids during cholestasis.

Partial characterization of mechanisms of cytoprotective action of hydrophilic bile salts against hydrophobic bile salts in rats: relation to canalicular membrane fluidity and packing density.

Bile salts regulate the subselection of phosphatidylcholine species secreted into bile and thereby modulate bile metastability. The aim of this study was to determine whether bile salts alter phosphatidylcholine species of the canalicular membrane, and if they do, to clarify whether the cytoprotective action of hydrophilic bile salts is associated with modulation of phosphatidylcholine composition in cell membrane bilayers. Bile salt-pool-depleted rats were infused intravenously with sodium taurocholate at a constant rate (200 nmol/min/100 g body wt) for 2 hr, followed by infusion of either sodium tauroursodeoxycholate, sodium tauroalphamuricholate, or sodium taurobetamuricholate (200 nmol/min/100 g) for 2 hr. Biliary outputs of cholesterol and phosphatidylcholine and phosphatidylcholine hydrophobicity in bile and subcellular fractions were determined. The cytoprotective action of hydrophilic bile salts was determined by the release of canalicular membrane-localizing enzymes (alkaline phosphatase, leucine aminopeptidase) into bile. Tauroursodeoxycholate, taurobetamuricholate, and tauroalphamuricholate decreased the release of these enzymes when compared to values under taurocholate infusion. Bile phosphatidylcholine hydrophobicity was also decreased by the bile salts, whereas the cholesterol/phosphatidylcholine ratio was increased. In contrast, phosphatidylcholine hydrophobicity in the canalicular membrane was increased by these three bile salts. In conclusion, hydrophilic bile salts promote biliary secretion of relatively hydrophilic phosphatidylcholine secretion into bile, and consequently phosphatidylcholine hydrophobicity in canalicular membranes increased. Such an alteration in phosphatidylcholine species within canalicular membrane enhances its lateral packing density with less fluidity, and this may account, in part, for the cytoprotective action of hydrophilic bile salts against hydrophobic bile salts.

Acute ethanol hepatotoxicity is modulated by bile salt hydrophilic-hydrophobic properties.

The influence of the hydrophobic-hydrophilic properties of bile salts (BS) on acute ethanol hepatotoxicity was investigated. Bile flow, biliary BS secretion and enzyme (LDH,AST) release in the perfusate were measured before and after exposure to low (0.1%) or high (1%) doses of ethanol in in vitro isolated livers perfused with 1 microM/min taurocholate (TCA), tauroursodeoxycholate (TUDCA) or taurodeoxycholate (TDCA). Ethanol promotes a rapid decrease of basal bile flow and BS secretion in TCA-perfused livers [-28% of basal values with 0.1% (N = 6), and -35% with 1% ethanol (N = 6)]. Bile flow and BS secretion were minimally decreased by ethanol in livers perfused with a hydrophilic BS (TUDCA) [-8% decrease of basal values with 0.1% ethanol (N = 6), and -10% with 1% ethanol (N = 9); p < 0.02 vs TCA-perfused livers]. In contrast, when livers were perfused with a hydrophobic BS (TDCA), ethanol showed a higher cholestatic effect than either TCA- or TUDCA-perfused livers. Enzyme release in the perfusate was not modified by 0.1% ethanol, while 1% ethanol promoted a 4-5 fold increase in LDH and AST release in the perfusate of TCA-perfused livers with respect to a mere 2-fold increase in TUDCA-perfused livers and a 6-7 fold increase in TDCA perfused livers (p < 0.03). In conclusion, we showed that TUDCA almost completely counteracts the cholestatic and cytolitic effects promoted by ethanol in the isolated perfused rat liver.

Direct, concentration-dependent inhibition by taurocholate of pancreatic exocrine secretion and CCK release in conscious rats.

In conscious rats, bile inhibits pancreatic secretion. The role of luminal taurocholate (TC), a major component of rat bile, in the regulation of pancreatic secretion was studied in conscious rats with external bile and pancreatic fistulae. On the fourth postoperative day, after the basal collection of bile and pancreatic juice (PJ) returned to the duodenum, graded doses of TC (0, 0.4, 4, 40 mM) containing 10 mM CaCl2 were infused into the duodenum instead of bile and PJ for 2 hr (1 ml/hr), with or without 1 mg/ml of porcine trypsin. Luminal trypsin activities were not affected by any dose of TC. The increases in pancreatic secretion in response to diversion of bile and PJ were progressively inhibited with increasing doses of infused TC from 0 mM to 4 mM both with and without trypsin infusion. The effects with 4 and 40 mM TC were not significantly different. Changes in plasma cholecystokinin concentrations roughly correlated with changes in protein output in rats without trypsin infusion. We concluded that TC directly inhibited pancreatic secretion independent of the luminal trypsin activity and that its inhibitory action was concentration dependent.

Regulation of apolipoprotein B secretion by biliary lipids in CaCo-2 cells.

The regulation of apoB synthesis and secretion by lipids present within bile was investigated in CaCo-2 cells grown on semipermeable filters. Bile acids decreased the basolateral secretion of immunoreactive apoB. Taurocholic acid decreased the secretion of newly synthesized apoB by increasing the rate of apoB degradation, but had no effect on the synthesis and secretion of apoA-I or trichloroacetic acid-precipitable proteins. The calcium ionophore, A23187, decreased apoB secretion similar to that observed for taurocholate. The addition of the ionophore and taurocholate together did not cause a further decrease in apoB secretion. Cholesterol or its hydroxylated derivative, 25-hydroxycholesterol, did not alter secretion of immunoreactive or newly synthesized apoB. Phosphatidylcholine increased apoB synthesis and secretion without affecting the synthesis or secretion of apoA-I. Phosphatidylcholine also reversed the effect of A23187 on apoB secretion. When phosphatidylcholine was added to the basolateral medium, apoB secretion was not altered. ApoB secretion was not increased by phospholipids of other classes. Dioleoylphosphatidylcholine increased apoB secretion, whereas dipalmitoylphosphatidylcholine did not. Fatty acid-labeled phosphatidylcholine was not hydrolyzed in the apical medium. Only 2% of the added phosphatidylcholine was cell-associated, and of this, 80% of the label remained as phosphatidylcholine with most of the remainder in triacylglycerols, fatty acids, and phosphatidylethanolamine. The results suggest that bile acids decrease apoB secretion by increasing its rate of degradation. This effect may be related to their ionophoric property. Cholesterol flux does not regulate apoB secretion. Phosphatidylcholine, independent of triacylglycerol flux and independent of its hydrolysis, increases the secretion of apoB by increasing apoB synthesis. Luminal phosphatidylcholine may play a role in apoB secretion in the intestine.

Tauro alpha-muricholate has a biliary transport maximum (Tm) value equivalent to that for tauroursodeoxycholate and tauro beta-muricholate in the rat.

Our previous studies have shown that Tm values for tauroursodeoxycholate (TUDC) and tauro beta-muricholate (T beta-MC) are more than two-fold higher than that for taurocholate (TC) in the rat. The present study attempted to clarify whether tauro alpha-muricholate (T alpha-MC) also has such an unusually large Tm value in the rat. Under nembutal anesthesia, male Wistar derived rats (body weight 280-300 g, 13 wks in age) were continuously infused with T alpha-MC solution. The infusion rate was raised stepwise every 20 min, until the bile flow began to decline. Bile was collected every 10 min and bile salt excretion rate was determined. The average of the highest three excretion values was assumed to be the Tm in each animal. The Tm value of T alpha-MC was found to be 2.86 +/- 0.36 mumol/min/100 g (mean +/- SD, n = 4), which was even greater than Tm values for TUDC (2.59 +/- 0.39 mumol/min/100 g, n = 4) and T beta-MC (1.93 +/- 0.31 mumol/min/100 g, n = 4) as we reported previously. The relationship between the bile flow rate (microliter/min/100 g, Y axis) and bile salt excretion rate (mumol/min/100 g, X axis) was highly linear [Y = (6.00 +/- 0.29) x +(6.60 +/- 1.88), P < 0.001, r = 0.95, n = 54]. The slope value for T alpha-MC (6.00 +/- 0.29 microliters/mumol) was significantly higher than that for TUDC (4.76 +/- 0.71 microliters/mumol) and was comparable to that for T beta-MC as we previously found for these bile salts in this rat strain. The results suggest that T alpha-MC has a very efficient transport system in this species as was observed for the other two bile salts that have a 7 beta-hydroxy group (TUDC and T beta-MC). This efficient transport system thus appears to be shared not only by bile salts specifically having a 7 beta-hydroxy group, but also by other bile salts such as T alpha-MC that have a 6 beta-hydroxy group but not a 7 beta-hydroxy group.

Effects of bile acids and bilirubin on bicarbonate secretion of isolated guinea pig antrum.

The effects of bile acids and bilirubin, which increase in blood in obstructive jaundice, on bicarbonate secretion were studied experimentally using isolated antral mucosa of the guinea pig. Antral mucosal preparations were mounted between Ussing chambers. Basal and bethanechol-stimulated secretion were measured by a pH stat device using 5 mM HCl. After 10(-4), 10(-3), and 5 x 10(-3) M taurocholic acid, 10(-4) and 10(-3) M cholic acid, and 1.2 x 10(-4) and 2.4 x 10(-4) M bilirubin conjugate were added to the serosal solution, basal and bethanechol-stimulated secretion were also measured. Taurocholic acid, at any dose, did not affect basal secretion, but bethanechol-stimulated secretion was inhibited dose dependently. Cholic acid and bilirubin conjugate did not affect basal secretion, but bethanechol-stimulated secretion was significantly inhibited. Although the addition of bile acid or bilirubin into the serosal solution under experimental conditions is not the same as obstructive jaundice, these studies suggest that the inhibition of bicarbonate secretion in the gastric mucosa may have an important role in the formation of acute gastric mucosal lesions in obstructive jaundice.

Functional and morphological features of isolated hepatocytes preserved in University of Wisconsin solution.

Although several studies suggest that hepatic graft failure after cold ischemia results from nonparenchymal cell damage, other data indicate that hepatocellular ATP content is significantly correlated with the transplantation success rate. In this study, we have conducted a systematic investigation of various aspects of cell viability and function of isolated hepatocytes stored at 4 degrees C for 24 and 48 hr in either University of Wisconsin solution or Hanks' HEPES buffer, a control solution clinically unsuitable for organ preservation. After 24 hr, hepatocytes stored in Hanks' HEPES buffer had viability (measured by trypan blue exclusion and ALT and lactic dehydrogenase leakage), transport function (measured by 22Na+ and [3H]taurocholate uptake) and cell size similar or only slightly altered when compared with freshly isolated and University of Wisconsin solution-stored hepatocytes. ATP content was decreased in both groups; however, the reduction was much greater in Hanks' HEPES buffer-stored cells. Furthermore, ATP regenerating capacity was greatly reduced in Hanks' HEPES buffer- stored but not in University of Wisconsin solution-stored hepatocytes. By 48 hr viability and function of Hanks' HEPES buffer-stored hepatocytes were decreased; University of Wisconsin solution afforded partial protection. When examined by light and electron microscopy, cells stored in both University of Wisconsin solution and Hanks' HEPES buffer for 24 hr appeared essentially normal except for the presence of numerous membrane blebs in the Hanks' HEPES buffer group. Tissue sections of livers preserved in Hanks' HEPES buffer but not in University of Wisconsin solution revealed the presence of extensive amounts of blebs in the sinusoidal lumen and loss of endothelial elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of cholesterol 7 alpha-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat.

Cholesterol 7 alpha-hydroxylase catalyzes the rate-limiting step in the bile acid biosynthetic pathway. Regulation of this pathway is thought to occur solely as a result of a negative feedback control mechanism that is dependent upon the flux and composition of bile salts undergoing enterohepatic circulation. We have used the chronic biliary diverted (CBD) rat model to study the mechanism of regulation of cholesterol 7 alpha-hydroxylase by taurocholate. As compared to nonoperated controls, CBD rats exhibited a 4.2-fold increase in cholesterol 7 alpha-hydroxylase-specific activity, a 4.5-fold increase in enzyme mass, a 10-fold increase in steady-state mRNA levels, and a 3.4-fold increase in transcriptional (nuclear "run-on") activity. Intraduodenal infusion of taurocholate at a rate of 36 mumol/100 g/h for 48 h in CBD rats caused a significant (p less than 0.05) decrease (64%) in cholesterol 7 alpha-hydroxylase-specific activity, mass (72%), steady-state mRNA levels (74%), and transcriptional activity (57%) as compared to CBD controls. Cholesterol feeding increased cholesterol 7 alpha-hydroxylase-specific activity (288%), poly(A) RNA levels (291%), and transcriptional activity (220%) as compared to control animals. These results provide convincing evidence that bile salts, either directly or indirectly, down-regulate in vivo transcription of the cholesterol 7 alpha-hydroxylase gene, which is probably the major mechanism regulating the levels of this enzyme. The results of this study also suggest that the promoter for cholesterol 7 alpha-hydroxylase may have both bile salt- and sterol-responsive elements.

[Pancreatic circulation in experimental biliary pancreatitis]. / Die Pankreasdurchblutung bei der experimentellen biliären Pankreatitis.

Measurements of pancreatic micro- and macrocirculation were performed to evaluate the pancreatitis-induced changes. Pigs were anesthetized and ventilated mechanically. Hypotension induced side-effects were avoided by adequate volume replacement. After laparatomy, splenectomy and gastroectomy the animals were enterotomized. Systemic hemodynamic parameters were monitored as well as pancreatic blood flow (Q), which was measured electromagnetically, and arterial and portal-venous blood gases. Pancreatic microcirculatory parameters were observed using fluorescence-videomicroscopy after i.v. administration of FITC dextran 150 and FITC labeled autologous erythrocytes. The pigs were randomly assigned to a control (n = 9) and a pancreatitis group (n = 10), the later being induced by the retrograde infusion of sodium-taurocholate. Systemic and pancreatic macrohemodynamic parameters remained constant in both groups, except for avdO2 and O2-consumption (O2-c) decreasing significantly in the pancreatitis group. At baseline 42% of all capillaries were perfused in both groups. In pancreatitis we detected focal areas with persistent stasis and areas which were continuously perfused. In these areas the portion of capillaries perfused by erythrocytes increased significantly to 67%. This was accompanied by an extravasation of FITC dextran. The finding of an unchanged Q beside reduced O2-c and avdO2 during pancreatitis is explained by the changes in pancreatic microcirculation. Focal stasis was observed beside areas showing typical signs of an acute inflammation: increased macromolecular permeability and capillary recruitment, e.g. oedema and hyperaemia.

Role of primary and secondary bile acids as feedback inhibitors of bile acid synthesis in the rat in vivo.

The effect of various primary and secondary bile acids on the rates of synthesis of all major bile acids was studied in the live rat with an extracorporal bile duct. Bile acid synthesis was determined using HPLC based on mass or by isotope dilution. Derepressed rates of bile acid synthesis (30-54 h) were inhibited by an infusion of taurocholic acid only at a supraphysiological dose of 500 mumol/kg per h, but not at 300 mumol/kg per h, which approximates the initial bile acid secretion (250 mumol/kg per h). When administered together with taurocholic acid (200 mumol/kg per h) only a high dose of taurochenodeoxycholic acid (100 mumol/kg per h) decreased taurocholic but not tauromuricholic or taurochenodeoxycholic acid synthesis. The only bile acid suppressing taurocholic acid (36-71%) and taurochenodeoxycholic acid (up to 33%) formation at an infusion rate close to the normal portal flux was deoxy- or taurodeoxycholic acid at 15-50 mumol/kg per h. It may be concluded that deoxycholic acid and possibly other secondary bile acids are much more potent inhibitors than primary bile acids.