Enhanced biliary excretion of canalicular membrane enzymes in estrogen-induced and obstructive cholestasis, and effects of different bile acids in the isolated perfused rat liver

Backgrounds/Aims: Canalicular membrane enzymes are normally released into bile by partially known processes. This study was undertaken to investigate whether hepatocellular cholestatis induced in rats by ethynylestradiol or obstructive cholestasis produced by complete biliary obstruction for 24 h is associated with an increased release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile, and to clarify how this process is affected by different bile acids.Methods: The studies were performed in the isolated perfused liver during infusion of sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate at increasing rates.Results: Maximum sodium taurocholate, taurochenodeoxycholate and tauroursodeoxycholate secretory rates were decreased in both cholestatic groups (complete biliary obstruction>ethynylestradiol) compared with controls. Maximum biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase were significantly increased in the ethynylestradiol group during infusion of sodium taurocholate and taurochenodeoxycholate, but not of tauroursodeoxycholate, and were increased in the complete biliary obstruction group during the infusion of sodium taurocholate and tauroursodeoxycholate but not of taurochenodeoxycholate. The biliary outputs of alkaline phosphatase and gamma-glutamyl transpeptidase showed a significant and direct linear relationship with sodium taurocholate and taurochenodeoxycholate secretory rates in both cholestatic groups. However, only in the complete biliary obstruction group did alkaline phosphatase and gamma-glutamyl transpeptidase excretion show a significant correlation with tauroursodeoxycholate secretory rates. The slope of the line, which indicated the mU of enzyme activity secreted per nmol of sodium taurocholate or taurochenodeoxycholate, was greater for gamma-glutamyl transpeptidase and alkaline phosphatase in both cholestatic groups (ethynylestradiol>complete biliary obstruction) than in the control group. Alkaline phosphatase activity in purified isolated canalicular and sinusoidal membranes was significantly increased in both cholestatic groups (complete biliary obstruction>ethynylestradiol), while gamma-glutamyl transpeptidase activity was unchanged compared with controls.Conclusion: The marked increase in sodium taurocholate and taurochenodeoxycholate-mediated release of alkaline phosphatase and gamma-glutamyl transpeptidase into bile in cholestatic rats suggests an increased lability of these intrinsic membrane proteins to the detergent effects of secreted bile acids. It remains to be elucidated whether this phenomenon, which was particularly intense in ethynylestradiol induced cholestasis, is important in the pathogenesis and perpetuation of bile secretory failure. In contrast, tauroursodeoxycholate administration did not result in enhanced biliary excretion of these membrane enzymes, in either the control group or the ethynylestradiol group, supporting the concept that this bile salt lacks the membrane toxicity of common bile acids.     

Influence of exocrine and endocrine pancreatic function on intestinal brush border enaymatic activities.

Digestive enzymatic activities (disaccharidases, alkaline phosphatase, peptide hydrolases) have been determined in the mucosa of 14 patients with chronic pancreatitis. All had an abnormal secretin-pancreozymin test. Four patients had insulin-dependent diabetes mellitus, four a pathological glucose tolerance test. Nine patients had steatorrhoea. Maltase, sucrase, and alkaline phosphatase activity was significantly elevated in patients with exocrine pancreatic insufficiency, whereas those of lactase, trehalase, and peptide hydrolase were normal. Patients with steatorrhoea had higher maltase and sucrase activity than those without steatorrhoea, whereas decreased glucose tolerance had no effect on brush border enzymatic activity. It is suggested thatdecreased exocrine rather than decreased endocrine pancreatic function is responsible for the increase in intestinal disaccharidase and alkaline phosphatase activity, possible by the influence of pacreatic enzymes on the turnover of brush border enzymes from the luminal side of the mucosal membranes or by direct hormonal stimulation though cholecystokinin.     

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 ofphosphatidylcholine species secreted into bile andthereby modulate bile metastability. The aim of thisstudy was to determine whether bile salts alterphosphatidylcholine species of the canalicular membrane, and ifthey do, to clarify whether the cytoprotective action ofhydrophilic bile salts is associated with modulation ofphosphatidylcholine composition in cell membrane bilayers. Bile salt-pool-depleted rats wereinfused intravenously with sodium taurocholate at aconstant rate (200 nmol/min/100 g body wt) for 2 hr,followed by infusion of either sodiumtauroursodeoxycholate, sodium tauroalphamuricholate, or sodiumtaurobetamuricholate (200 nmol/min/100 g) for 2 hr.Biliary outputs of cholesterol and phosphatidylcholineand phosphatidylcholine hydrophobicity in bile andsubcellular fractions were determined. The cytoprotectiveaction of hydrophilic bile salts was determined by therelease of canalicular membrane-localizing enzymes(alkaline phosphatase, leucine aminopeptidase) into bile. Tauroursodeoxycholate,taurobetamuricholate, and tauroalphamuricholatedecreased the release of these enzymes when compared tovalues under taurocholate infusion. Bilephosphatidylcholine hydrophobicity was also decreased by the bile salts, whereasthe cholesterol/phosphatidylcholine ratio was increased.In contrast, phosphatidylcholine hydrophobicity in thecanalicular membrane was increased by these three bile salts. In conclusion, hydrophilicbile salts promote biliary secretion of relativelyhydrophilic phosphatidylcholine secretion into bile, andconsequently phosphatidylcholine hydrophobicity in canalicular membranes increased. Such analteration in phosphatidylcholine species withincanalicular membrane enhances its lateral packingdensity with less fluidity, and this may account, inpart, for the cytoprotective action of hydrophilic bilesalts against hydrophobic bile salts.     

Inhibition of rat basal pancreatic secretion by intraduodenal bile

The effect on basal exocrine pancreatic secretion of diversion from and reintroduction into the duodenum of bile has been studied on conscious rats provided with pancreatic, biliary and duodenal fistulae. Diversion of bile from the intestine augmented protein output by 30%. After an eight-hour diversion recirculation of bile into the duodenum reduced pancreatic protein output by 30%; volume being not significantly modified. When either bile was diverted or the main bile duct was ligated, a similar inhibition of protein secretion was observed after intraduodenal injections of 20 mM solutions of taurocholate, taurochenodeoxycholate, chenodeoxycholate, and cholate, and of synthetic mixed micelles (bile salts, lecithin). Inhibitory action of bile salts on pancreatic secretion was seen equally whether or not the bile salts were in free or conjugated form or pancreatic juice returned to the intestine. We conclude that unlike man and the dog, bile as well as pancreatic juice inhibits the basal pancreatic exocrine secretion of the rat.     

Adaptive response of the enterohepatic circulation of bile acids to extrahepatic cholestasis

Experimental cholestasis induced by ligation of the common bile duct results in morphological and functional changes in the rat hepatocyte. The aim of this study was to evaluate the adaptive response of the transport process involved in the enterohepatic circulation of bile salts to obstructive cholestasis. Male Sprague-dawley rats with common bile duct ligation were killed after 48 and 72 hours. Portal and systemic blood and duodenal aspirates were collected. Taurocholate transport was measured in isolated ileal brush border (BBM) and liver basolateral membranes (BLM). Drastic reduction in intraluminal bile salt concentrations in ligated rats accompanied decreases in saturable taurocholate uptake by ileal BBMs. Kinetic analysis indicated that the decrease was attributable to reduction in transporter density and alteration in affinity for the substrate, both of which accentuated with increase in postligation time. In contrast, despite 20-fold higher portal venous bile salt concentrations, taurocholate uptake by hepatic sinusoidal membrane was lower in rats. Kinetic analysis and immunoblots developed using polyclonal antisera to the liver BLM bile acid transporter demonstrated a gradual decrease in transporter density with increase in postligation time accompanied by reduced taurocholate uptake by basolateral membrane. These results further support the concept that the ileal brush border membrane transporter is regulated by the availability of the substrate, whereas regulation of the bile salt transport across the liver BLM is independent of portal venous bile acid concentration. (Hepatology 1996 Mar;23(3):623-9)     

Conjugates of ursodeoxycholate protect against cholestasis and hepatocellular necrosis caused by more hydrophobic bile salts. In vivo studies in the rat

The protective effect of ursodeoxycholate conjugates against bile salt hepatotoxicity was studied in chronic bile fistula rats. Taurochenodeoxycholate or taurodeoxycholate, infused intraduodenally at 24 or 16 mumols/100 g rat per hour, respectively, caused cholestasis and severe hepatocellular necrosis within 8 hours. In contrast, tauroursodeoxycholate or taurocholate at 48 mumols/100 g rat per hour were choleretic. Tauroursodeoxycholate was not hepatotoxic, whereas taurocholate produced moderate hepatocellular necrosis. Simultaneous infusion of tauroursodeoxycholate to rats receiving taurochenoxycholate or taurodeoxycholate preserved bile flow and ameliorated hepatic injury in a dose-dependent manner. Tauroursodeoxycholate protected equally by intravenous and intraduodenal routes. Intravenous glycoursodeoxycholate also was protective. The hydrophobicity index of infused bile salts correlated well with their toxicity. Concurrent administration of ursodeoxycholate conjugates did not reduce biliary recovery of intraduodenally infused [24-14C]-taurocholate. Biliary alkaline phosphatase secretion was stimulated by infusion of taurocholate, taurodeoxycholate, or taurochenodeoxycholate; simultaneous infusion of ursodeoxycholate conjugates failed to prevent this increase. We conclude that ursodeoxycholate counteracts hepatoxicity of more hydrophobic bile salts via a direct effect at the level of the liver.     

Canalicular bile flow and bile salt secretion are maintained in rats with liver cirrhosis. Further evidence for the intact cell hypothesis

Different aspects of biliary physiology were studied in rat model of liver cirrhosis induced by CCl4/phenobarbitone. We measured bile flow, bile salt secretion, biliary secretion pressure and bile-to-plasma ratios of inert solutes under basal conditions and during infusion of taurocholate (0.4 and 0.8 mumol.min-1.100 g body wt.-1) in 11 cirrhotic and 10 control male Sprague-Dawley rats. Bile flow and biliary bile salt secretion did not differ between the two groups. Analyzing the relationship between bile salt secretion and bile flow, however, we found an increased slope (P less than 0.02) in the cirrhotic animals, suggesting a higher apparent osmotic activity of the bile salts secreted. Maximal biliary secretion pressure was maintained in cirrhotic animals (22.5 +/- 2.5 vs. 25.0 +/- 2.9 cmH2O) in the absence of exogenous bile salt. During taurocholate infusion it decreased to a lesser extent (P less than 0.001) in cirrhotic animals (13.5 +/- 3.4 vs. 19.3 +/- 3.8 cmH2O). Bile-to-plasma ratios of [3H]sucrose and [14C]ferrocyanide were markedly increased in cirrhotic rats. Biliary [14C]erythritol clearance was equal to bile flow in both groups. In the cirrhotic group, the [3H]sucrose bile/plasma ratio was positively correlated with spleen weight (r = 0.744, P less than 0.01), serum concentration of alkaline phosphatase (r = 0.583, P less than 0.05) and basal maximum biliary secretion pressure (r = 0.801, P less than 0.001). We conclude that chronic portal hypertension is associated with increased permeability of the blood/bile barrier. Nevertheless, bile flow and bile salt secretion are maintained in cirrhotic rats, giving support to the intact cell hypothesis for this important hepatocellular function.     

Effects of Ursodeoxycholate and Other Bile Salts on Levels of Rat Intestinal Alkaline Sphingomyelinase (A Potential Implication in Tumorigenesis)

Previous studies showed that bile salts had apromoting effect on colon cancer development and thiseffect was inhibited by ursodeoxycholate (UDC). Werecently found that both human colorectal adenomas and carcinomas were associated with a specificdecrease in alkaline sphingomyelinase activity. In thiswork, we compared the effects of ursodeoxycholate andother bile salts on the levels of rat intestinal alkaline sphingomyelinase both in theintestinal loops and after oral administration. Bilesalts at different concentrations were injected intointestinal loops and the dissociation of alkalinesphingomyelinase from the mucosa was assayed. We found that bilesalts, including taurocholate, taurodeoxycholate,glycocholate, glycochenodeoxycholate, and3-(3-cholamidopropyl dimethylammonio)-1-propanesulonate(CHAPS), dose dependently dissociated alkalinesphingomyelinase from the intestinal mucosa. UDC alonedid not dissociate the enzyme but significantlyinhibited the dissociation caused by other bile saltsand CHAPS. Feeding rats with 0.3% (w/w) taurocholate forfour days decreased peak activity of intestinal alkalinesphingomyelinase by 39% and total activity in theintestine by 20% and increased the output of the enzyme in the feces. In contrast, feeding 0.3%(w/w) UDC for four days increased the peak activity ofalkaline sphingomyelinase in the small intestine by 87%and the activity in the colon by 187% . The total activity of alkaline sphingomyelinase wasincreased by 80% and the output of the enzyme in thefeceswas only slightly increased by UDC administration.The changes in alkaline phosphatase after feeding taurocholate and UDC were much smaller. Ourresults indicate that UDC and other bile salts havedifferent effects on the levels of alkalinesphingomyelinase, which may be implicated in theirdifferent influences on cancer development reportedpreviously.     

Zymogram studies of human intestinal brush border and cytoplasmic peptidases.

Zymogram studies of peptide hydrolases from the human intestinal brush border and cytoplasmic fractions produced multiple bands--that is, up to seven--while the brush border membrane produced only a single band of enzyme activity. With all of the substrates tested except L-leucyl-L-leucyl-L-leucine, a band having anodic mobility identical with that produced by the brush border enzymes was produced by the cytoplasmic enzymes. With L-trileucine as a substrate, no overlapping band was produced. This band in the cytoplasmic fraction was heat sensitive, while that in the brush border fraction was not. Thus it would appear that there is a single human intestinal brush border peptide hydrolase capable of hydrolysing a variety of di- and tri-peptides. This peptide hydrolases of the brush border and the cytoplasmic fraction of human intestine are distinct.     

Increase of intestinal brush border hydrolases in mucosa of streptozotocin-diabetic rats

Summary Experimental diabetes mellitus in rats was induced by streptozotocin. Five days after administration of streptozotocin intestinal brush border hydrolases (maltase, sucrase, trehalase, lactase) and alkaline phosphatase were markedly elevated at all levels of the small intestine as measured in the total homogenate and in the isolated brush border preparation. Insulin treatment beginning 15 h after administration of streptozotocin was able to decrease the increased disaccharidase activity due to streptozotocin diabetes. In experimental diabetes mellitus of rats trransport as well as digestive functions of the intestinal mucosa are stimulated.This work has been presented at the meeting of the European Society for Clinical Investigation, Scheveningen, The Netherlands, April 1972 and has appeared in abstract form (1a)     

Posttranslational cleavage of rat intestinal lactase occurs at the luminal side of the brush border membrane

The intestinal sucrase-isomaltase precursor is cleaved at the brush border membrane by luminal proteases. Whether the lactase precursor also is cleaved by luminal proteases is uncertain. Lactase synthesis and processing was studied in 0- and 15-day-old rats after IP administration of [35S]methionine, and changes in precociously cortisone-induced sucrase-isomaltase were used as an internal control. Mucosal lactase and sucrase-isomaltase were separately immunoprecipitated and analyzed by autoradiography after electrophoresis. In both 0- and 15-day-old rats, mucosal lactase appeared as a 200K lactase precursor band at 30 minutes and as 200K and 225K lactase precursor bands at 60 minutes and was cleaved to form a 130K lactase band 120-240 minutes after labeling; sucrase-isomaltase similarly appeared as 210K and 220K bands at 30-60 minutes and was cleaved to form 140K I and 120K S subunits by 240 minutes in day 15 rats. To determine the role of luminal proteases, intestinal segments were isolated in situ and the luminal contents were flushed 30 minutes after labeling. Unflushed segments were used as controls. Only lactase precursor and sucrase-isomaltase precursor were present 240 minutes after labeling in flushed intestinal segments, but lactase precursor and sucrase-isomaltase precursor were cleaved in unflushed segments. Addition of trypsin or elastase into the lumen of flushed segments resulted in partial cleavage of lactase precursor but not of sucrase-isomaltase precursor. Luminal contents collected from the small intestine of day 15 rats 120 and 240 minutes after labeling showed 35S-labeled 130K and 80K polypeptides in lactase immunoprecipitates. It is concluded that intestinal lactase is synthesized as lactase precursor and transported to brush border membrane and cleaved by luminal proteases, and the amino end polypeptide cleaved from lactase precursor is released into the lumen.     

Secretin activation of the apical Na+-dependent bile acid transporter is associated with cholehepatic shunting in rats

The role of the cholangiocyte apical Na(+)-dependent bile acid transporter (ASBT) in bile formation is unknown. Bile acid absorption by bile ducts results in cholehepatic shunting, a pathway that amplifies the canalicular osmotic effects of bile acids. We tested in isolated cholangiocytes if secretin enhances ASBT translocation to the apical membrane from latent preexisting intracellular stores. In vivo, in bile duct-ligated rats, we tested if increased ASBT activity (induced by secretin pretreatment) results in cholehepatic shunting of bile acids. We determined the increment in taurocholate-dependent bile flow and biliary lipid secretion and taurocholate (TC) biliary transit time during high ASBT activity. Secretin stimulated colchicine-sensitive ASBT translocation to the cholangiocyte plasma membrane and (3)H-TC uptake in purified cholangiocytes. Consistent with increased ASBT promoting cholehepatic shunting, with secretin pretreatment, we found TC induced greater-than-expected biliary lipid secretion and bile flow and there was a prolongation of the TC biliary transit time. Colchicine ablated secretin pretreatment-dependent bile acid-induced choleresis, increased biliary lipid secretion, and the prolongation of the TC biliary transit. In conclusion, secretin stimulates cholehepatic shunting of conjugated bile acids and is associated with increased cholangiocyte apical membrane ASBT. Bile acid transport by cholangiocyte ASBT can contribute to hepatobiliary secretion in vivo.     

The effect of age on the biliary transport maximum (Tm) of bile salts in rats

Age-related differences in the biliary transport maxima (Tm) values for taurocholate (TC) and tauroursodeoxycholate (TUDC) were examined in female Fischer-344 rats as well as Wistar-derived rats of both sexes. The Tm values for TUDC were more than two times higher than corresponding values for TC in young (3-month-old) rats of both sexes. Tm values for both bile salts tended to decline with age, demonstrating a significant negative correlation between the Tm (micromol/min per g liver) and rat age (months) for all rat groups. The decline in Tm value was, however, dominant in the first year with little significant change after 1 year. The results in the present study coupled with our previous observations for Tm values of sulfobromophthalein (BSP) and conjugated BSP support our hypothesis that the transport capacity for bile canalicular membrane generally declines with age in rats.     

Downregulation of ileal bile acid absorption in bile-duct-ligated rats

BACKGROUND/AIMS: Accumulation of toxic bile acids in cholestasis contributes to liver injury and depends on their synthesis, secretion and intestinal absorption. In the present study, we investigated the effect of cholestasis on the active ileal absorption of bile acids in vivo and the adaptation of transporters involved in ileal bile acid absorption. METHODS: Male Wistar rats underwent ligation of the common bile duct or biliary diversion. Sham-operated rats served as controls. Active ileal bile acid absorption of taurocholate was measured by an intestinal perfusion technique. Transporter mRNA levels of the Na+/bile acid cotransporting protein (IBAT), ileal lipid binding protein (ILBP) and organic anion transporter subtype 3 (Oatp3) and protein expression of IBAT and ILBP were determined in the distal ileum. RESULTS: After bile duct ligation the intestinal absorption rates of taurocholate were lower (p<0.05) and after biliary diversion absorption rates were higher compared to sham-operated animals (p<0.05). The absorption rates were inversely correlated to serum bile acid concentrations. Levels of IBAT-, ILBP- and Oatp3- mRNA were not different between the groups. However, in cholestatic rats, the expression of the 99-kDa dimer of IBAT was decreased compared to controls (p<0.05), whereas the 46-kDa monomeric protein of IBAT and the expression of ILBP was unchanged. After biliary diversion a similar pattern of protein expression was observed, despite an increased absorption rate. CONCLUSIONS: Cholestasis leads to a decreased active ileal absorption of taurocholate. The changes in protein expression may not account for the different absorption rates. The intestinal absorption of bile acids seems to be regulated by their systemic concentration.     

Effect of sulfobromophthalein on biliary excretion of taurocholate and pravastatin in bile duct-ligated rat

Recent studies indicated that bile duct ligation down-regulates the expression of Na⁺/taurocholate cotransporting polypeptide and Na⁺-dependent taurocholate uptake by basolateral membrane vesicles in the rat. These findings suggest that hepatic taurocholate uptake in bile duct-ligated rats is mediated by the organic anion transporting polypeptide, a Na⁺-independent taurocholate uptake system which is common for sulfobromophthalein uptake. Therefore, the effect of sulfobromophthalein on biliary excretion of taurocholate and pravastatin in bile ductligated rats was studied. Although biliary excretion of pravastatin was markedly inhibited by sulfobromophthalein, biliary taurocholate excretion was not affected by sulfobromophthalein in bile duct-ligated rats. The excretory maximum of sulfobromophthalein in bile duct-ligated rats was reduced to one-fifth of control rats. These findings indicate that, in the bile duct-ligated rats, taurocholate uptake is mediated not by the multispecific organic anion transporter, but by other uptake system(s).     

Deoxycholate depresses small-intestinal enzyme activity.

Feeding sodium deoxycholate orally to rats for four days caused depression of the activity of the small intestinal enzymes lactase, sucrase, maltase, alkaline phosphatase, and N-acetyl-beta-glucosaminidase. The first four are brush border enzymes, the last a lysosomal enzyme. Alkaline phosphatase activity recovered very rapidly and rebounded to above the normal level within 24 hours. The activity of the three disaccharidases returned to normal within seven days while no recovery was observed within 96 hours of the activity of the lysosomal enzyme, N-acetyl-beta-glucosaminidase, after removing the bile salt from the diet.     

Functional reconstitution of the canalicular bile salt transport system of rat liver.

Recent studies have suggested that the canalicular bile salt transport system of rat liver corresponds to a 100-kDa membrane glycoprotein. In the present study we attempted to functionally reconstitute the 100-kDa protein into artificial proteoliposomes. Canalicular membrane proteins were solubilized with octyl glucoside in the presence of asolectin phospholipids. The extracts were treated with preimmune serum or the 100-kDa protein selectively immunoprecipitated with a polyclonal antiserum. Proteins remaining in the supernatant were then incorporated into proteoliposomes by gel-filtration chromatography. Canalicular proteoliposomes containing the 100-kDa protein exhibited transstimulatable taurocholate uptake that could be inhibited by 4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). In contrast, no DIDS-sensitive transstimulatable taurocholate uptake was found in 100-kDa protein-free canalicular proteoliposomes. However, when the immunoprecipitated 100-kDa protein was dissociated from the antibodies and exclusively incorporated into liposomes, reconstitution of DIDS-sensitive transstimulatable and electrogenic taurocholate anion transport was again positive. Although incorporation of solubilized basolateral membrane proteins into liposomes also resulted in a prompt reconstitution of Na+ gradient-driven taurocholate uptake, the anti-100-kDa antibodies had no effects on the reconstituted transport activity of basolateral proteins. Thus, the findings establish that the previously characterized canalicular-specific 100-kDa protein is directly involved in the transcanalicular secretion of bile salts.     

Hydrophilic bile salts have a cytoprotective effect against cyclosporine A-induced cholestasis through enhanced canalicular membrane fluidity and transporter activity

Cyclosporine A (CsA) reduces liver canalicular membrane (CM) fluidity to cause a disproportionate reduction of biliary lipid secretion (the uncoupling phenomenon) without affecting adenosine triphosphate-dependent (ABC) transporters except for Mdr1. This study investigated whether hydrophilic bile salts inhibit CsA-induced cholestasis, focusing on CM fluidity and ABC transporter expression. Male Sprague–Dawley rats were infused with taurocholate (TC) (200 nmol/min/100 g body weight) for 2 h, flowed by infusion with tauroursodeoxycholate (TUDC), tauroalphamuricholate, or taurobetamuricholate (100 nmol/min/100 g body weight plus TC at 100 nmol/min/100 g body weight). Thereafter, CsA (20 mg/kg body weight) was injected as a bolus and bile was collected for 2 h. Canalicular membrane vesicles were prepared for analysis of cholesterol (CH), phospholipid (PL), CM fluidity, and expression of ABC transporters (Mdr1, Bsep, Mdr2, and Mrp2). CsA administration reduced biliary lipid secretion along with a disproportionately smaller decline of bile salt secretion. Hydrophilic bile salts significantly inhibited cholestasis after CsA injection by increasing CM fluidity and by increasing the expression of Mrp2 and Bsep, whereas Mdr1 and Mdr2 were unaltered. (1) Hydrophilic bile salts inhibit CsA-induced cholestasis, presumably by increasing CM fluidity, and this action was greatest with TUDC. (2) The fact that ABC transporters, except for Mdr1 and Mdr2, were overexpressed in the CM after infusion of these bile salts suggests that cytoprotective bile salts functions may increase transporter mass as well as enhancing transporter activity.     

Physiological factors controlling release of enterokinase from rat enterocytes

The quantitative release of enterokinase from isolated rat enterocytes following treatment with taurocholate-taurodeoxycholate, papain, chymotrypsin, elastase, carbamylcholine, and cholecystokinin-octapeptide was examined. Alkaline phosphatase and lactate dehydrogenase activities were evaluated simultaneously to check for specificity. Bile salts promoted a concentration-dependent release of all enzymes. Concomitantly, bile salts also led to cell destruction in proportion to the amount of enzymes released. Proteases caused the release of enterokinase and alkaline phosphatase with no concomitant increase of lactate dehydrogenase or cell lysis. At equal concentrations, papain released more enzymes than chymotrypsin and elastase. Chymotrypsin and elastase, however, led to higher ratios of enterokinase to alkaline phosphatase found in the media and suggested a selective release of enterokinase (EK) over that of alkaline phosphatase. Bile salts and pancreatic proteases together seem to have an additive effect of the release of EK. Carbamylcholine and cholecystokinin-octapeptide had no effect on enzyme release. These results suggested that pancreatic proteases are involved in the release of enterokinase by a selective action. Bile salts may also play a role through a nonselective detergent effect.     

Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat

The effects of biliary diversion on pancreatic enzyme activities of intestinal contents was studied in conscious rats prepared with biliary and pancreatic fistulae. Diversion of bile from the intestine for 1 day caused on 80% decrease in trypsin and chymotrypsin activities of intestinal contents, in spite of increased (230%) pancreatic trypsin and chymotrypsin secretion. Bile diversion in fed rats caused a smaller decrease (58%) in trypsin and chymotrypsin activities of intestinal contents. Sodium taurocholate (100 mumol/hr intraduodenally) partially reversed the changes in pancreatic secretion and intestinal contents' activities of trypsin and chymotrypsin caused by bile diversion. The results indicated that bile was important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestine. The mechanism for this was studied by comparing the rate of disappearance of trypsin activity in vivo and in vitro. Bovine trypsin, with or without sodium taurocholate, was infused intraduodenally into conscious rats deprived of bile-pancreatic juice and the recovery of trypsin activity from the small intestine determined. Taurocholate increased recovery of trypsin from the small intestine more than threefold, but inactivation of bovine trypsin in vitro was not retarded by sodium taurocholate. The results indicate that bile in the small intestine controls the rate of disappearance of intraluminal trypsin and chymotrypsin activities, probably by inhibiting their autodigestion in vivo. We previously reported that bile duct ligation in rats caused decreased trypsin and chymotrypsin activities in the small intestine, but increased pancreatic enzyme secretion. We concluded that trypsin and chymotrypsin underwent accelerated inactivation in the small intestine in the absence of bile. The present study was designed to explore the mechanism for the effects of bile deprivation on intraluminal proteolytic enzyme activities in the rat.