Biliary excretion of inorganic electrolytes: its role in hepatic bile formation

To define the role of inorganic electrolyte secretion in hepatic bile formation, the effects of secretin, glucagon, and differently structured bile acids on bile flow and composition were studied in the dog, guinea pig, and rat. In the dog and guinea pig, secretin (2.5-10 clinical units X kg-1 X 30 min-1) increased bile flow and bicarbonate concentration in bile, a finding consistent with the hypothesis that the hormone stimulates a bicarbonate-dependent secretion possibly at the level of the bile ductule-duct. In the rat, secretin (5-15 CU X kg-1 X 30 min-1) failed to increase bile secretion. Glucagon (1.25-300 micrograms X kg-1 X 30 min-1) increased bile flow in all the three species, and produced no changes in biliary bicarbonate concentrations in the dog and rat. In the guinea pig, however, glucagon choleresis was associated with an increase in bicarbonate concentration in bile, similar to that observed with secretin. The choleretic activities of various bile acids (taurocholate, chenodeoxycholate, glycochenodeoxycholate, tauroursodeoxycholate, and ursodeoxycholic acid, infused at 30-360 mumol X kg-1 X 30 min-1) were similar in the rat (6.9-7.2 microL/mumol), but different in the guinea pig (11-31 microL/mumol). In the latter species, the more hydrophobic the bile acid, the greater was its choleretic activity. In all instances, bile acid choleresis was associated with a decline in the biliary concentrations of chloride, but with no major change in bicarbonate levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secretin stimulates exocytosis in isolated bile duct epithelial cells by a cyclic AMP-mediated mechanism.

Intrahepatic bile duct epithelial cells, or cholangiocytes, contribute to bile secretion in response to hormones, including secretin. However, the mechanism by which secretin stimulates ductular bile flow is unknown. Since recent data in nonhepatic epithelia have suggested a role for exocytosis in fluid secretion, we tested the hypothesis that secretin stimulates exocytosis by isolated cholangiocytes. Cholangiocytes were isolated from normal rat liver by a newly described method employing enzymatic digestion and mechanical disruption followed by immunomagnetic separation using specific monoclonal antibodies, and exocytosis was measured using a fluorescence unquenching assay employing acridine orange. Secretin caused a dose-dependent (10(-12)-10(-7) M) increase in acridine orange fluorescence by acridine orange-loaded cholangiocytes with a peak response at 10 min; the half-maximal concentration of secretin was 7 x 10(-9) M. The secretin effect was inhibited by preincubation of cholangiocytes with colchicine (30% inhibition, p less than 0.05) or trypsin (90% inhibition, p less than 0.001); no inhibition was seen with lumicolchicine and heat-inactivated trypsin. Cholecystokinin, insulin, and somatostatin had no effect on fluorescence of acridine orange-loaded cholangiocytes; secretin had no effect on fluorescence of acridine orange-loaded hepatocytes or hepatic endothelial cells. Exposure of isolated cholangiocytes to secretin at doses that stimulated exocytosis caused a dose-dependent increase in cyclic AMP levels (218% maximal increase, p less than 0.05); moreover, an analogue of cyclic AMP stimulated exocytosis by cholangiocytes. Secretin had no effect on intracellular calcium concentration using Fura-2-loaded cholangiocytes assessed by digitized video microscopy. Our results demonstrate, for the first time, that secretin stimulates exocytosis by rat cholangiocytes. The effect is cell- and hormone-specific, dependent on intact microtubules, on a protein(s) on the external surface of cholangiocytes, and on changes in cellular levels of cyclic AMP. The results are consistent with the hypothesis that secretin-induced changes in bile flow may involve an exocytic process.     

Bile secretion in the fistulated pig: effect of the method used for bile reinfusion

The aim of this work was to investigate the effects on bile secretion of flow rate and site of reinfusion of the collected bile to the animal. Thirty-two pigs weighing 50 +/- 3 kg at the beginning of the experiment were fitted with a reentrant fistula in the lower common bile duct and in the upper duodenum. Bile collected from the bile duct was reinfused in four different ways (four groups of 8 animals each): into the duodenum or the lower common bile duct at a constant flow rate using a peristaltic pump, or into the duodenum or the lower common bile duct at a rate mimicking the flow rate of the secretion using an automatic apparatus. Reinfusing the bile into the lower common bile duct at a rate mimicking the secretion rate provided a daily bile acid production about 21% higher than the level recorded with the other three methods. This was mainly due to a higher bile acid concentration since the bile flow was only slightly affected by the treatment.     

Effects of pancreatic polypeptide on biliary flow and bile acid secretion stimulated by secretin and cholecystokinin in the conscious pig

Fourteen castrated male Large White pigs, weighing 42.5 +/- 1.0 kg, were fitted with biliary and duodenal fistulae for biliary secretion studies. Furthermore, catheters were placed in a carotid artery for blood sampling and in a jugular vein for peptide infusion. Bile was automatically restituted to the animals and continuously sampled for analysis on experimental days. Following an 8 day recovery period, infusion studies were performed after an overnight fast. After a 30 min basal period, sustained biliary flow and bile acid output were obtained and maintained throughout the assay with secretin (36 pmol/kg/h) and CCK-8 (600 pmol/kg/h) infusion. Then, 200, 400, 600, 800 or 1200 pmol/kg/h of porcine pancreatic polypeptide (PP) were infused for 60 min. Secretin plus CCK infusion was continued for 1 h after PP infusion was stopped. Each dose of PP was given on a separate day. Biliary flow was not affected by PP except for the dose of 400 pmol/kg/h. On the contrary, bile acid concentration and output decreased with the lowest dose of PP (200 pmol/kg/h). As soon as the first dose of PP was infused, bile acid concentration and output fell to about 60% of values obtained with secretin plus CCK. Plasma levels of PP were below or similar to postprandial values for 200, 400 and 600 pmol/kg/h and they were significantly larger with 800 and 1200 pmol/kg/h. Bile acid concentration and output did not return to values obtained with secretin plus CCK infusion after cessation of PP infusion. In conclusion, porcine PP given in physiological doses to the pig decreases bile acid output whereas biliary flow remains unaffected.     

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.     

Upregulation of secretin receptors on cholangiocytes after bile duct ligation

Secretin not only increases ductular bile secretion in vivo in rats after bile duct ligation (BDL) [1], but also increases cAMP levels and stimulates exocytosis in isolated cholangiocytes [2]. Although we have previously reported that secretin receptor mRNA was upregulated in cholangiocytes after BDL [3], the cholangiocyte secretin receptor has not been functionally characterized or quantified after BDL. In this work, we used a novel, photolabile and biologically active analogue of secretin to quantify and characterize secretin receptors on cholangiocytes isolated from normal and BDL rats. The cholangiocyte secretin receptor bound radioligand with high affinity and in a rapid, reversible, and temperature-dependent manner. While receptors on cholangiocytes from normal and BDL rats were functionally and biochemically identical, receptor density on cholangiocytes was increased 5-fold following BDL. The combination of increased cell number with increased functional secretin receptors per cell is due to the fact that cholangiocyte hyperplasia represents a reactive response to a cholestatic condition and this effort on the part of the organism to maintain bile secretion, explains the increased hormone-responsive choleresis observed after BDL and may reflect an adaptive response of the organism to cholestasis.     

Role of the neuropeptide,bombesin, in bile secretion.

Since ancient times, bile secretion has been considered vital for maintaining health. One of the main functions of bile secretion is gastric acid neutralization with biliary bicarbonate during a meal or Pavlovian response. Although the liver has many extrinsic and intrinsic nerve innervations, the functional role of these nerves in biliary physiology is poorly understood. To understand the role of neural regulation in bile secretion, our recent studies on the effect of bombesin, a neuropeptide, on bile secretion and its underlying mechanisms will be reviewed. Using isolated perfused rat livers (IPRL) from both normal and 2 week bile duct ligated rats, as well as hepatocyte couplets and isolated bile duct units (IBDU) from normal rat livers, bombesin was shown to stimulate biliary bicarbonate and fluid secretion from bile ducts. Detailed pH studies indicated that bombesin stimulated the activity of Cl-/HCO3- exchanger, which was counterbalanced by a secondary activation of electrogenic Na+/HCO3- symport. Quantitative videomicroscopic studies showed that bombesin-stimulated fluid secretion in IBDU was dependent on Cl- and HCO3- in the media, anion exchanger(s), Cl- and K+ channels, and carbonic anhydrase, but not on the microtubular system. Furthermore, this bombesin response is inhibited by somatostatin but not substance P. Finally, studies of secondary messengers in isolated cholangiocytes and IBDU indicated that bombesin had no effect on intracellular cAMP, cGMP, or Ca++ levels in cholangiocytes. These results provide evidence that neuropeptides such as bombesin can directly stimulate fluid and bicarbonate secretion from cholangiocytes by activating luminal Cl-/HCO3- exchange, but by different mechanisms from those established for secretin. These findings, in turn, suggest that neuropeptides may play an important regulatory role in biliary transport and secretion. Thus, this neuropeptidergic regulation of bile secretion may provide a plausible mechanism for the bicarbonate-rich choleresis seen with meals or Pavlovian response.     

Influence of bile salts on the endogenous excretion of bile pigments

Effect of the infusion of glycodeoxycholate (GDC), taurocholate (TC) and dehydrocholate (DHC) on bile flow and on bile salt, biliary lipid and bile pigment secretion, has been studied in pentobarbital-anesthetized rabbits. GDC increased bile flow the most, while DHC increased it more than TC. The different choleretic actions of these bile salts cannot be explained by means of variations in their capacity to form micelles. Only GDC and TC were able to stimulate biliary lipid secretion, which suggests that both bile salts increase the formation of mixed micelles. GDC and TC to a lesser extent increased bile pigment excretion, DHC being without effect. These results favour the hypothesis that micellar binding could be an important factor responsible for the effect of bile acids on bile pigment excretion and should not be completely ruled out.     

Hepatic bile and pancreatic exocrine secretions evoked by gastrointestinal peptides in sheep

The secretory response of hepatic bile and exocrine pancreas to gastrointestinal peptides has been studied in chronically cannulated sheep. Pancreatic juice flow and protein output were evoked dose dependently by intraportal injection of secretin, CCK-8, caerulein, VIP and neurotensin. However, biliary secretion was evoked by only secretin. Biliary and pancreatic exocrine secretions were enhanced by delivered gastric juice into the duodenum as followed by the increased plasma concentration of immunoreactive secretin (IRS). Results suggest that secretin is the major peptide that regulates pancreatic exocrine secretion and hepatic bile production in the sheep.     

Comparison of helodermin, VIP and PHI in pancreatic secretion and blood flow in dogs

Helodermin, VIP and PHI, which share a high degree of homology with secretin, have been identified in the gut but their physiological role is unknown. In this study 3 series of tests were carried out to determine the actions of helodermin, VIP and PHI on pancreatic secretion in 6 conscious dogs and amylase release from the dispersed canine pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects in 24 anesthetized dogs. Helodermin, VIP and PHI infused i.v. in graded doses (12.5-200 pmol/kg.h) resulted in a dose-dependent increase in pancreatic HCO3 secretion reaching, respectively, 100%, 7% and 2% of secretin maximum. When combined with constant dose infusion of CCK-8 (100 pmol/kg.h), helodermin but not VIP or PHI augmented dose-dependently the HCO3 secretion. When added in various concentrations (10(-10)-10(-5)M) to the incubation medium of dispersed pancreatic acini only helodermin but not VIP or PHI increased dose-dependently amylase release reaching about 50% of CCK-8 maximum. In anesthetized dogs, the pancreatic blood flow (PBF) measured by electromagnetic blood flowmetry showed an immediate and dose-dependent increase following the injections of various doses of helodermin, VIP, PHI and secretin, the peak blood flow preceding by about 1 min the peak secretory stimulation. This study shows that helodermin resembles secretin in its potent pancreatic HCO3 stimulation but differs from VIP or PHI which are poor secretagogues but potent vasodilators. We conclude that if tested peptides are released in the gut, helodermin, like secretin, may be involved in the hormonal stimulation of exocrine pancreas, whereas VIP and PHI may serve mainly as vasodilators in the pancreatic circulation.     

Alpha-adrenergic influences on exocrine pancreatic secretion in the rabbit

Action of phenylephrine (35 micrograms/Kg/min) alone or previously blocked by phentolamine (100 micrograms/Kg/min) on exocrine pancreatic secretion of anaesthetized rabbits has been studied, in basal state or under stimulation by secretin (1 C.U./Kg/h) or by the octapeptide of cholecystokinin (OP-CCK) (0.15 Ivy dog units/Kg/h). Phenylephrine increased arterial pressure. This effect was blocked by phentolamine. However no variations were seen in pancreatic blood flow in any of the experimental conditions assayed. Phenylephrine produced a secretin-like effect on hydroelectrolytic secretion in basal conditions. This action was maintained after the infusion of secretin but not after OP-CCK. This effect was not blocked by phentolamine. Phenylephrine increased protein secretion in the basal state, an action that was blocked by phentolamine. After secretin or OP-CCK stimulation phenylephrine did not increase protein secretion. It is concluded that phentolamine blocks the effects of phenylephrine on acinar cells but not on ductular cells.     

Isolation of functional polarized bile duct units from mouse liver

The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on Cl- and HCO3-in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models.     

Three-day continuous drainage of pancreatic juice in the cortisone-treated conscious rat: Analysis of enzyme activities and ultrastructural changes

Summary We have investigated the short-term effects of hydrocortisone (60 mg/kg per day) and placebo on basal and stimulated pancreatic secretion in the conscious rat. Volume and enzyme secretion were determined; fine structural changes were examined simultaneously.The pancreatic and bile ducts were cannulated separately; pancreatic juice was drained via an isolated fistula, and bile was recirculated into the duodenum. The application of hydrocortisone led to an almost complete inhibition of the secretory response of the exocrine pancreas when stimulated with 0.25 U secretin in combination with 5 × 10^-8 g caerulein per h. It strongly affected the secretion rates of volume, protein, lipase, chymotrypsin, trypsin and carboxypeptidase, whereas the secretion rate of alpha-amylase continued to show a slight increase after stimulation.After stimulation with secretin and caerulein, the hydrocortisone-treated animals showed a higher density of zymogen granules in the acinar cell and an increase in the number of autophagic vacuoles in comparison to the equally stimulated placebo-treated rats.It is concluded that the short-term inhibition of pancreatic secretion by hydrocortisone occurs largely as a result of an inhibition of cellular enzyme discharge.Supported by the Deutsche Forschungsgemeinschaft, Ga 279     

Roles of 5-HT receptors in the release and action of secretin on pancreatic secretion in rats

5-Hydroxytryptamine (serotonin, 5-HT) is a hormone and neurotransmitter regulating gastrointestinal functions. 5-HT receptors are widely distributed in gastrointestinal mucosa and the enteric nervous system. Duodenal acidification stimulates not only the release of both 5-HT and secretin but also pancreatic exocrine secretion. We investigated the effect of 5-HT receptor antagonists on the release of secretin and pancreatic secretion of water and bicarbonate induced by duodenal acidification in anesthetized rats. Both the 5-HT(2) receptor antagonist ketanserin and the 5-HT(3) receptor antagonist ondansetron at 1-100 microg/kg dose-dependently inhibited acid-induced increases in plasma secretin concentration and pancreatic exocrine secretion. Neither the 5-HT(1) receptor antagonists pindolol and 5-HTP-DP nor the 5-HT(4) receptor antagonist SDZ-205,557 affected acid-evoked release of secretin or pancreatic secretion. None of the 5-HT receptor antagonists affected basal pancreatic secretion or plasma secretin concentration. Ketanserin or ondansetron at 10 microg/kg or a combination of both suppressed the pancreatic secretion in response to intravenous secretin at 2.5 and 5 pmol x kg(-1) x h(-1) by 55-75%, but not at 10 pmol x kg(-1) x h(-1). Atropine (50 microg/kg) significantly attenuated the inhibitory effect of ketanserin on pancreatic secretion but not on the release of secretin. These observations suggest that 5-HT(2) and 5-HT(3) receptors mediate duodenal acidification-induced release of secretin and pancreatic secretion of fluid and bicarbonate. Also, regulation of pancreatic exocrine secretion through 5-HT(2) receptors may involve a cholinergic pathway in the rat.     

Bile secretion in the chicken: effects of secretin, cholecystokinin- pancreozymin and duodenal mucosal extract

The effects of i.v. administration of secretin, CCK-PZ, acid extracts from the duodenal mucosa and the duodenal acidification of the intestine on bile secretion were studied in anaesthetized chickens. Secretin and acid extracts from the duodenal mucosa, which increase bile flow, caused comparable modifications in bile composition; infusion of HCl to the duodenum only induced slight modifications. CCK-PZ caused a pronounced cholecystokinetic effect and, to a lesser degree, it also showed choleretic effects. The results suggest that in the hormonal regulation of bile secretion in the chicken CCK-PZ is more important than secretin and furthermore that the choleretic activity of the latter must be carried out by other secretin-like peptides.     

Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes

We sought to develop a cholangiocyte cell culture system that has preservation of receptors, transporters, and channels involved in secretin-induced secretion. Isolated bile duct fragments, obtained by enzyme perfusion of normal rat liver, were seeded on collagen and maintained in culture up to 18 wk. Cholangiocyte purity was assessed by staining for gamma-glutamyl transpeptidase (gamma-GT) and cytokeratin-19 (CK-19). We determined gene expression for secretin receptor (SR), cystic fibrosis transmembrane conductance regulator, Cl(-)/HCO(3)(-) exchanger, secretin-stimulated cAMP synthesis, Cl(-)/HCO(3) exchanger activity, secretin-stimulated Cl(-) efflux, and apical membrane-directed secretion in polarized cells grown on tissue culture inserts. Cultured cholangiocytes were all gamma-GT and CK-19 positive. The cells expressed SR and Cl(-)/HCO(3)(-) exchanger, and secretin-stimulated cAMP synthesis, Cl(-)/HCO(3)(-) exchanger activity, and Cl(-) efflux were similar to freshly isolated cholangiocytes. Forskolin (10(-4) M) induced fluid accumulation in the apical chamber of tissue culture inserts. In conclusion, we have developed a novel cholangiocyte line that has persistent HCO(3)(-), Cl(-), and fluid transport functions. This cell system should be useful to investigators who study cholangiocyte secretion.     

Migration of gall stones.

The factors influencing the migration of gall stones are ill understood. Altogether 331 patients undergoing cholecystectomy were studied prospectively. The diameters of the cystic and common bile ducts and of stones in the gall bladder and bile ducts were measured. Increasing pressure was applied to the freshly excised gall bladder in an attempt to evacuate stones through the cystic duct. Stones passed in 33 (60.0%) of patients with choledocholithiasis, 45 (67.2%) of patients with pancreatitis, and 7 (3.2%) of patients without either pancreatitis or choledocholithiasis. Stones migrated in 6 (3.0%) who had a normal cystic duct diameter (less than or equal to 4 mm) and in 46 (32.5%) with a duct over 4 mm diameter. Common bile duct stones were often larger than the diameter of the cystic duct and when reintroduced into the gall bladder would not migrate. The passage of debris (less than or equal to 1 mm) through the cystic duct bore no relation to the presence or absence of choledocholithiasis or a dilated cystic duct. Small stones (1-4 mm diameter) must migrate to initiate and facilitate further migration; some must increase in size in the common bile duct. Increased biliary pressure consequently dilates the duct system retrogradely, allowing larger stones to follow. Patients at risk of stone migration and thereby pancreatitis and jaundice have large ducts that can be detected by ultrasound assessment.     

Morphology of the prairie dog gallbladder: normal characteristics and changes during early lithogenesis

Studies were undertaken to describe the normal structure of the prairie dog gallbladder and adjacent cystic duct, and then to determine sequential changes that occurred as abnormalities in bile composition developed during high cholesterol feeding. Control animals were fed a diet with trace cholesterol, while experimental animals were fed a diet enriched with 1.2% cholesterol for 1, 2, 3, or 4 weeks. Light microscopy and scanning and transmission electron microscopy were used to characterize morphologic changes at each time interval. Biliary lipid composition was altered in all experimental groups, evidenced by significant decreases in bile-acid-to-cholesterol ratios. Cholesterol crystals appeared in experimental bile at 1 and 2 weeks, while stones formed at 3 and 4 weeks. The cystic duct and neck of the gallbladder occasionally displayed goblet cells. Little mucus was demonstrable in principal cells of the gallbladder, but much more in those lining the cystic duct. After 2 weeks of lithogenic diet, there was an increase in mucus content and secretion from all areas, as well as an influx of polymorphonuclear and mononuclear leukocytes. Accumulation of plasma cells in the lamina propria was an especially prominent feature of experimental tissues. These results suggest that 1) there is regional heterogeneity in the mucus content of the gallbladder and cystic duct of the prairie dog, and 2) both regions respond to lithogenesis with mucus hypersecretion and acute and chronic inflammatory changes prior to the appearance of cholesterol gallstones.     

Enterohepatic circulation in hamsters with an extracorporeal bile duct.

The present study describes a novel technique for investigations of the enterohepatic circulation in the hamster with an extracorporeal bile duct that allows long-term bile collection in the free-moving animal. The animals recovered for 7 days after the operation before the external loop was cut and bile was collected over a period of 78 h. Under these optimal conditions, initial bile flow (651 +/- 89 microliters per 100 g.h-1) and the secretion rates of biliary lipids were several-fold higher than reported in an earlier study using the acute fistula hamster. Biliary cholesterol secretion amounted to 369 +/- 32 nmol per 100 g.h-1, phospholipid secretion was 2.6 +/- 0.3 mumol per 100 g.h-1, and total bile acid secretion was 31.9 +/- 2.2 mumol per 100 g.h-1. A clearcut diurnal rhythm was demonstrated for bile flow and all biliary constituents. After 9 h the depletion of the bile acid pool was complete and cholic acid synthesis derepressed 1.4-fold from a basal rate of 818 nmol per 100 g.h-1, whereas the derepression of chenodeoxycholic acid synthesis was even less pronounced. Biliary cholesterol output increased 2.2-fold, but the phospholipid secretion was constant during the full experiment. It may be concluded that the technique of an extracorporeal bile duct in the free-moving animal allows studies of bile secretion under optimal conditions. Most likely the bile secretion rates given above approach the physiological rates in the hamster.