Bicarbonate Output Research Articles

Non-steroidal FXR agonist cilofexor improves cholestatic liver injury in the Mdr2-/- mouse model of sclerosing cholangitis

The nuclear receptor farnesoid X receptor (FXR) is a key regulator of hepatic bile acid (BA) and lipid metabolism, inflammation and fibrosis. Here, we aimed to explore the potential of cilofexor (GS-9674), a non-steroidal FXR agonist, as a therapeutic approach for counteracting features of cholestatic liver injury by evaluating its efficacy and mechanisms in the Mdr2/Abcb4 knockout (-/-) mouse model of sclerosing cholangitis. FVB/N wild-type and Mdr2-/- or BALB/c wild-type and Mdr2-/- mice were treated with 0, 10, 30 or 90mg/kg cilofexor by gavage every 24h for 10 weeks. Serum biochemistry, gene expression profile, hydroxyproline content, and picrosirius red and F4/80 immunostaining, were investigated. Bile flow, biliary bicarbonate and BA output, and hepatic BA profile, were assessed. Cilofexor treatment improved serum levels of aspartate aminotransferase, alkaline phosphatase as well as BAs in Mdr2-/- animals. Hepatic fibrosis was improved, as reflected by the reduced picrosirius red-positive area and hydroxyproline content in liver sections of cilofexor-treated Mdr2-/- mice. Intrahepatic BA concentrations were lowered in cilofexor-treated Mdr2-/- mice, while hepatobiliary bile flow and bicarbonate output were increased. Collectively the current data show that cilofexor treatment improves cholestatic liver injury and decreases hepatic fibrosis in the Mdr2-/- mouse model of sclerosing cholangitis. Treatment with cilofexor, a non-steroidal farnesoid X receptor (FXR) agonist, improved histological features of sclerosing cholangitis, cholestasis and hepatic fibrosis in the Mdr2-/- mouse model. These findings indicate, that pharmacological stimulation of intestinal FXR-mediated gut-liver signaling, via fibroblast growth factor 15 (thereby reducing bile acid synthesis), may be sufficient to attenuate cholestatic liver injury in the Mdr2-/- mouse model of sclerosing cholangitis, thus arguing for potential therapeutic properties of cilofexor in cholestatic liver diseases.

Reduced Hepatocyte Bicarbonate Output Accelerates Periductal Inflammation and Fibrosis in mdr2-/- Mouse Liver

Background/Aims: Mice deficient for the canalicular phospholipid transporter MDR2 (ABCB4) develop sclerosing cholangitis due to high biliary concentrations of monomeric bile acids. This study determines whether a selective reduction in biliary bicarbonate output, secondary to the deletion of the hepatocyte-expressed carbonic anhydrase CAXIV (Car14) aggravates the bile acid-induced damage observed in the mdr2-/- mouse model. Methods: Bile flow was measured gravimetrically and HCO3- output by microtitration before and during stimulation with intravenously applied tauroursodesoxycholic acid (TUDCA) in car14-/-mdr2-/- (abcb4-/-), car14-/-/mdr2+/+, car14+/+/mdr2-/- and wt mice. Cholangiocyte proliferation, hepatic inflammation and fibrosis was studied by gene and/or protein expression for proinflammatory and profibrotic cytokines, cholangiocyte proliferation markers, and by (immuno) histochemical assessment. The impact of Car14 deficiency was also assessed in a xenobiotic cholangitis model. Results: TUDCA stimulated HCO3-output was significantly increased in 6 week old mdr2-/- mice, and significantly decreased in both car14-/- as well as car14-/-/mdr2-/-mice, compared to wt, while bile flow was unaltered. Both bile flow and HCO3- output were significantly decreased in 11 week old mdr2-/-, and more so in car14-/-/mdr2-/- mice. Loss of Car14 significantly increased inflammatory liver injury and cholangiocyte proliferation, and aggravated liver fibrosis in car14-/-/mdr2-/- mice compared to mdr2-/- mice. In contrast, the absence of Car14 did not affect the hepatic functional and morphological alterations in 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) fed mice. Conclusions: Car14 deletion reduced biliary HCO3- output and aggravated the functional, inflammatory and morphological alterations in the liver of mdr2-/-mice. These results demonstrate the importance of sufficient hepatocellular bicarbonate output in the protection of the hepatobiliary epithelium against toxic bile acids.

Neurotensin and xenin stimulates pancreatic exocrine secretion through the peripheral cholinergic nerves in conscious sheep

The present study aimed to clarify the effects of neurotensin and xenin on pancreatic exocrine secretion in conscious sheep and their mechanism of actions. The animals were equipped with two silastic cannulae in the common bile duct to separately collect pancreatic fluid and bile, and a silastic cannula in the proximal duodenum to continuously return the mixed fluids. NT and xenin were intravenously injected at range of 0.01–3.0 nmol/kg during the phase I of duodenal migrating motor complex. A single intravenous NT injection significantly and dose-dependently increased pancreatic fluid, protein, and bicarbonate outputs. The effect of NT at 1 nmol/kg was completely inhibited by a background intravenous infusion of atropine methyl nitrate at a dose of 10 nmol/kg/min, however, the effect was not altered by a prior injection of the neurotensin receptor subtype (NTR)-1 antagonist SR 48692 at 60 nmol/kg. Moreover, a single intravenous xenin-25 injection significantly and dose-dependently increased pancreatic fluid and protein output, whereas the effect of xenin-25 did not clearly show dose-dependence. The prior SR 48692 injection at 30 nmol/kg did not significantly alter the effects of xenin-25 at 0.3 nmol/kg, while the atropine infusion significantly inhibited the increase in fluid secretion. Under the atropine infusion, xenin-25 at 0.3 nmol/kg did not increase protein and bicarbonate outputs, whereas the inhibitory effect of the atropine was not significant compared to that of the single injection of xenin-25. A single intravenous injection of NTR-2 agonist levocabastine at 0.1–3 nmol/kg did not alter pancreatic exocrine secretion. These results suggest that both NT and xenin-25 effectively stimulates pancreatic exocrine secretion through the peripheral cholinergic system in sheep and that NTR-2 is not involved in the regulation of pancreatic exocrine secretion, however, we did not precisely determine the role of NTR-1 in the actions of both the peptides on pancreatic exocrine secretion.

Loss of luminal carbonic anhydrase XIV results in decreased biliary bicarbonate output, liver fibrosis, and cholangiocyte proliferation in mice

Carbonic anhydrase XIV (Car14) is highly expressed in the hepatocyte, with predominance in the canalicular membrane and its active site in the extracellular milieu. The aim of this study is to determine the physiological relevance of Car14 for biliary fluid and acid/base output, as well as its role in the maintenance of hepatocellular and cholangiocyte integrity. The common bile duct of anesthetized car14−/− and car14+/+ mice was cannulated and hepatic HCO3− output was measured by microtitration and bile flow gravimetrically before and during stimulation with intravenously applied tauroursodeoxycholic acid (TUDCA). Morphological alterations and hepatic damage were assessed histologically and immunohistochemically in liver tissue from 3- to 52-week-old car14−/− and car14+/+ mice, and gene and/or protein expression was measured for pro-inflammatory cytokines, fibrosis, and cholangiocyte markers. Biliary basal and more so TUDCA-stimulated HCO3− output were significantly reduced in car14−/− mice of all age groups, whereas bile flow and hepatic and ductular morphology were normal at young age. Car14−/− mice developed fibrotic and proliferative changes in the small bile ducts at advanced age, which was accompanied by a reduction in bile flow, and an upregulation of hepatic cytokeratin 19 mRNA and protein expression. Membrane-bound Car14 is essential for biliary HCO3− output, and its loss results in gradual development of small bile duct disease and hepatic fibrosis. Bile flow is not compromised in young adulthood, suggesting that Car14-deficient mice may be a model to study the protective role of biliary canalicular HCO3− against luminal noxi to the cholangiocyte.

Reporting a phase II, open-label study assessing the safety and efficacy of MS1819-SD, a recombinant lipase, for the treatment of exocrine pancreatic insufficiency

Background: Chronic pancreatitis is the most common cause of exocrine pancreatic insufficiency (EPI) in adults. EPI is a deficiency in exocrine pancreatic enzymes resulting in an inability to maintain normal digestion. This inadequate digestion, especially fat malabsorption, occurs when intraduodenal levels of lipase fall below 5–10% of normal enzyme output. Furthermore, in EPI due to chronic pancreatitis, decreased bicarbonate output leads to impairment of micelle formation of fats. Fat maldigestion is compounded by decreased pancreatic secretion of lipase and colipase, further dampening hydrolysis of intraluminal fat. Methods: A multicenter, Phase II, open-label, non-randomized study was conducted to investigate the safety of escalating doses (280 mg/day, 560 mg/day, 1120 mg/day and 2240 mg/day) of MS1819 Spray Dried (MS1819-SD, a lipase produced by the LIP2 gene of Yarrowia lipolytica using recombinant DNA technology) in patients with chronic pancreatitis (NCT03481803) and decreased fecal pancreatic elastase-1 (FE-1). Patients ‘washed-out’ their standard of care treatment for EPI establishing a baseline. Each patient subsequently received escalating doses of MS1819-SD in 2-week increments. The total treatment phase ranged from 48 to 60 days. Safety was assessed by adverse event (AE) incidence, including clinical or laboratory abnormalities, and efficacy was determined using Coefficient of Fat Absorption (CFA) change from baseline. Systemic exposure and immunogenicity potential of MS1819 were investigated in patients’ serum. Results: A total of 11 patients (9 males, 2 females) were enrolled in France, Australia and New Zealand. The study mean age was 59.2 years (11.19 standard deviation [SD]) and mean weight was 70.6 kg (12.84 SD). Disease etiology was alcoholism in 4 patients, idiopathic in 3 patients, genetic in 2 patients, auto-immune in 1 patient and gallstone induced pancreatitis for 1 patient. In the highest dose group (2240 mg/day) 4/10 patients reported an AE. One serious AE occurred at this dose but it was not considered to be treatment related (pre-planned removal of stent). A drug-related AE was reported at the 1120 mg dose (slight hepatic cytolysis, mild in nature). No circulating MS1819 was detected hence the mechanism of this adverse event cannot be explained or related to systemic exposure. MS1819 immunogenicity was observed in some patients with post-treatment anti-drug antibodies (ADAs) detected. However, some pre-existing ADAs were also detected and the incidence of ADAs was mild (3/10 patients) with moderate ADA titers. Increases in CFA were reported with all doses of MS1819-SD, these increases were particularly high in patients with baseline CFA <40%. Comparing CFA with FE-1 (24 patients with both recorded at screening) a positive spearman rank correlation of 0.64 (p < 0.001) was obtained. Although the study was not powered to detect a difference in efficacy parameters, the least square mean increase in CFA was 21.8% with MS1819-SD 2240 mg (p = 0.002; per protocol analysis). Conclusion: In general, a favorable safety profile was reported at all doses of MS1819-SD included in the study. Statistically significant and clinically meaningful increases in CFA compared to baseline were recorded. Although the efficacy analysis was pre-planned, the study was not powered to demonstrate differences in the groups and only a small number of patients were included. Favorable trends were also observed on other evaluated endpoints, including the Bristol stool scale, number of daily evacuations and stool weight. Additional studies are warranted to further evaluate the use of MS1819-SD to treat EPI in patients with chronic pancreatitis or cystic fibrosis.

Rifampicin Induces Bicarbonate-Rich Choleresis in Rats: Involvement of Anion Exchanger 2.

Previous studies have shown that rifampicin induced choleresis, the mechanisms of which have not been described. The aim of this study was to investigate the mechanisms underlying in vivo rifampicin-induced choleresis. In one experimental set, rats were treated chronically with rifampicin on days 1, 3 and 7. Serum and biliary parameters were assayed, and mRNA and protein levels, as well as the locations of the hepatic export transporters were analyzed by real-time PCR, western blot and immunofluorescence. Ductular mass was evaluated immunohistochemically. In another experimental set, rats received an acute infusion of rifampicin. The amount of rifampicin in bile was detected using HPLC. Biliary parameters were monitored following intrabiliary retrograde fluxes of the Cl(-)/HCO3 (-) exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) in the infused rats. Biliary bicarbonate output increased in parallel to the augmented bile flow in response to rifampicin, and this effect was abolished with intrabiliary administration of DIDS, but not NPPB. The biliary secretion of rifampicin with increases in bile flow and biliary rifampicin in response to different infused doses of the antibiotic show no significant correlations. After rifampicin treatment, the expression level of anion exchanger 2 (AE2) increased, while the location of hepatic transporters did not change. However, RIF treatment did not increase ductular mass significantly. These results indicate that the increase in bile flow induced by rifampicin is mainly due to increased HCO3 (-) excretion mediated by increased AE2 protein expression and activity.

Helicobacter pylori and pancreatic diseases.

A possible role for Helicobacter pylori (H. pylori) infection in pancreatic diseases remains controversial. H. pylori infection with antral predomination leading to an increase in pancreatic bicarbonate output and inducing ductal epithelial cell proliferation could contribute to the development of pancreatic cancer via complex interactions with the ABO genotype, dietary and smoking habits and N-nitrosamine exposure of the host. Although the individual study data available so far is inconsistent, several meta-analyses have reported an increased risk for pancreatic cancer among H. pylori seropositive individuals. It has been suggested that H. pylori causes autoimmune pancreatitis due to molecular mimicry between H. pylori α-carbonic anhydrase (α-CA) and human CA type II, and between H. pylori plasminogen-binding protein and human ubiquitin-protein ligase E3 component n-recognin 2, enzymes that are highly expressed in the pancreatic ductal and acinar cells, respectively. Future studies involving large numbers of cases are needed in order to examine the role of H. pylori in autoimmune pancreatitis more fully. Considering the worldwide pancreatic cancer burden, as well as the association between autoimmune pancreatitis and other autoimmune conditions, a complete elucidation of the role played by H. pylori in the genesis of such conditions could have a substantial impact on healthcare.

Duodenal application of Li+ in a submaximal therapeutic dose inhibits exocrine pancreatic secretion and modulates gastro-duodenal myoelectrical activity in a conscious pig model

This study tested whether duodenal application of lithium inhibits gastroduodenal motility, and whether it suppresses secretion from the exocrine pancreas. Five suckling pigs, 16-18 days old, were surgically fitted with 3 serosal electrodes on the wall of the gastric antrum and the duodenum for electromyography of smooth muscles, and with a pancreatic duct catheter and a duodenal T-cannula for collection and re-entrant flow of pancreatic juice. After the recovery period, on alternative days, each animal was tested once with an intraduodenal infusion of Li+ (100 mmol·L(-1) C3H5LiO3, 10 mL·kg(-1)·h(-1)) for 1 h, and once with an intraduodenal infusion of NaCl (154 mM, 10 ml·kg(-1)·h(-1)), also for 1 h, with the first treatment, i.e., Li+ or NaCl, randomly assigned. Individual pigs served as their own controls, with data recorded prior to a treatment being used as the baseline. Li+ increased the duration of quiescence (P < 0.05) and activity phase (P < 0.05) in the antrum, thus increasing (P < 0.05) the duration of antral myoelectrical cycles. Li+ shortened (P < 0.05) phase I, but it did not affect phase II or phase III or the MMC in the duodenum. Li+ inhibited pancreatic juice outflow as well as pancreatic enzyme and bicarbonate output (P < 0.05 for all pancreatic parameters).

The Significance of the Volume of Pancreatic Juice Measured at Secretin Stimulation Testing

(1) To quantitate volume production during secretin stimulation testing in patients suspected of having chronic pancreatitis (CP); (2) to compare volume production to clinical criteria for the diagnosis of CP. A total of 224 patients referred for suspected CP were reviewed retrospectively for clinical information supporting the diagnosis of CP. The patients were divided into 2 groups: those with peak bicarbonate (PB) of less than 80 mEq/L and those with PB of 80 mEq/L or greater (ie, CP and no CP). For a separate comparison, the patients were also placed into similar 2 groups based on clinical criteria. The volume, total bicarbonate output, volume per kilogram, and PB of pancreatic juice after secretin stimulation in patients thought to have CP were compared to those thought not to have CP. Volume was lower in the patients with PB of less than 80 mEq/L (206 ± 114 and 269 ± 106 mL) and lower in patients who met clinical criteria for CP (203 ± 109 and 271 ± 108 mL), P < 0.001 for both, but there was significant overlap (volume alone did not accurately discriminate CP from no CP). During secretin stimulation testing, bicarbonate parameters likely are better predictors of CP than volume parameters. Changes in the production of the volume of pancreatic juice during secretin stimulation likely reflect relatively late changes in pancreatic function.

Abstract ED02-03: Gene, environment, and risk factor interaction in pancreatic cancer

Abstract ED02-03: Gene, environment, and risk factor interaction in pancreatic cancer

71 Functional Significance of Endoscopic Ultrasound (EUS) Chronic Pancreatitis (CP) Criteria: Comparison with Secretin and Cholecystokinin (CCK) Pancreatic Function Testing (Pft)

(PB), Volume (V), Volume/kg (flow), and Total Bicarbonate Output (TBO) on each patient. Questions: During SST, do V, flow, and TBO of pancreatic juice adequately predict CP as well as the PB? What level of V, flow, or TBO reliably discriminates CP from non-CP? Results: 43.2 % of patients had CP by peak bicarbonate criteria (PB<80mEq/L). Of those, the sensitivity for CP using flow was only 14%. The percent of non-CP patients, ie false positives, with low flow was still 2.7%. The sensitivity for CP using TBO was only 47.4%. TBO was low in 5.3% of non-CP (specificity of 94.7%). Using the Wilcoxon signed rank sum test, the following were observed: (1) Mean V: non-CP (281ml ± 107) vs CP patients (207ml ± 115) p<0.0001. (2) Mean flow: non-CP (4.0ml/kg ± 1.3) vs CP patients (2.8ml/ kg ± 1.3) p<0.0001. (3)Mean TBO: non-CP (23 mEq/hr ± 8.7) vs CP patients (11 mEq/hr ± 7.1) p<0.0001. Using the Spearman linear correlation test, considering all patients, the following observations were made (coefficients in parentheses and all p-values <0.0001): (1) V correlates with PB (0.3537) (2) Flow and especially TBO were more strongly correlated with PB (0.4070 and 0.6571, respectively). Receiver operating curves were made for V, flow, and TBO to see if each could predict the presence of CP. The AUC's were 0.721, 0.769, and 0.867, respectively. Conclusions: Volume and flow correlate linearly with peak bicarbonate concentration during SST but not as well as total bicarbonate output, and a large drop in volume, flow, or total bicarbonate output is required to bring a patient into the PB<80 mEq/L category. Volume and flow do predict the presence of advanced CP but are very insensitive and bicarbonate output is only a somewhat better predictor. Based on data from the secretin stimulation test, S-MRCP's use of volume will miss a significant portion of patients with early CP, particularly those with small duct disease.

A prospective crossover study comparing secretin-stimulated endoscopic and Dreiling tube pancreatic function testing in patients evaluated for chronic pancreatitis

Direct pancreatic function tests (PFT) are conventionally performed with use of double-lumen "Dreiling" collection tubes. We have developed an endoscopic collection method (ePFT) that eases the performance of these tests. Our aim was to compare the bicarbonate results obtained from the secretin ePFT and Dreiling PFT methods in patients evaluated for chronic pancreatitis. A prospective crossover design was used to compare the PFT methods. Tertiary care referral center. Twenty-four patients undergoing an evaluation for chronic pancreatitis underwent the secretin-stimulated ePFT and Dreiling PFT methods on separate days. Duodenal fluid bicarbonate concentrations and estimated bicarbonate outputs were compared. The mean difference in peak bicarbonate concentration (Dreiling PFT minus ePFT) was 7 mEq/L (SD 20) and not statistically significant (P = .11). A good correlation in peak bicarbonate concentrations (r = 0.74, 95% CI, 0.48-0.88) and estimated bicarbonate output (r = 0.78, 95% CI, 0.54-0.90) was observed between the two PFT methods. The sensitivities and specificities of the secretin ePFT and Dreiling PFT could not be compared because of the lack of a histologic gold standard. The secretin ePFT yields results similar to those of the Dreiling PFT in patients evaluated for chronic pancreatitis.

C-type natriuretic peptide stimulates pancreatic exocrine secretion in the rat: Role of vagal afferent and efferent pathways

We previously reported that C-type natriuretic peptide (CNP) increases amylase release in isolated pancreatic acini through natriuretic peptide receptor C activation and enhances pancreatic exocrine secretion via vagal pathways when applied to the brain. In the present study we sought to establish whether CNP was involved in the peripheral regulation of pancreatic secretion. Anesthetized rats were prepared with pancreatic duct cannulation, pyloric ligation and bile diversion into the duodenum. CNP dose-dependently enhanced pancreatic flow, chloride and protein excretion but did not modify bicarbonate output. A selective natriuretic peptide receptor C agonist enhanced pancreatic flow and mimicked CNP-evoked protein output but failed to modify chloride secretion. Truncal vagotomy, perivagal application of capsaicin and hexamethonium reduced CNP-evoked pancreatic flow and abolished chloride excretion but did not affect protein output. Furthermore, pre-treatment with atropine reduced both CNP-stimulated pancreatic flow and chloride excretion but failed to modify protein excretion. Partial muscarinic blockade of CNP-evoked chloride output suggested that mediators other than acetylcholine were involved. However, CNP response was unaltered by cholecystokinin and vasoactive intestinal peptide receptor blockade or by nitric oxide synthase inhibition. In conclusion, CNP-stimulated pancreatic flow through the activation of the natriuretic peptide receptor C and the vago-vagal reflex but it increased protein output only by natriuretic peptide receptor C activation and chloride excretion by vago-vagal reflexes. Present results suggest that CNP may play a role as a local regulator of the exocrine pancreas.

Pancreatic secretion after selective and truncal vagotomy in the dog.

Abstract External pancreatic secretion was studied in 4 dogs: 2 with a Thomas cannula and 2 with a new cannula in which a duodenal pouch acts as an extension of the pancreatic duct. Peak bicarbonate outputs after a meal were about a third of the maximal secretory capacity of the pancreas in response to single intravenous injections of pure secretin, whereas peak protein concentrations and outputs after a meal were of the order of the maximum response to injections of secretin and pancreozymin. There were no consistent changes in hormonestimulated pancreatic secretion after selective vagotomy. Meal-stimulated pancreatic secretion was markedly reduced after truncal vagotomy (81 per cent reduction in peak bicarbonate output, 65 per cent in peak protein output). Secretin-stimulated bicarbonate output was markedly decreased 3 weeks after truncal vagotomy, but then gradually increased to control values, and in 1 dog to twice the control. Pancreozymin-stimulated protein output was increased after truncal vagotomy, reaching values two to three times the control. Local anaesthetics applied topically to the periampullary duodenal mucosa markedly inhibited secretin-stimulated bicarbonate output even after truncal vagotomy, but there was only slight reduction after subcutaneous lignocaine. The reduction in food-stimulated pancreatic secretion after truncal vagotomy is unlikely to be caused by pancreatic denervation, since the postvagotomy pancreatic response to direct stimulation with exogenous hormones is normal or supranormal, and is probably due to decreased release of gastrointestinal hormones after a meal. Local reflex arcs may influence the pancreas and may explain (by denervation supersensitivity) the post-vagotomy increase in pancreatic secretion as well as the effects of topical local anaesthetics. The role of neural factors in the regulation of external pancreatic secretion has long been disputed. This subject is now of particular clinical interest in relation to the causes of post-vagotomy diarrhoea and steatorrhoea (for a review of the literature, see Cox, 1969). With the development of a new cannula for studying external pancreatic secretion in the unanaesthetized dog (Thambugala, 1971) the opportunity was taken to study the effects of selective and truncal vagotomy on the pancreatic secretory responses to food and pure secretin and pancreozymin.

The effect of acute ethanol administration on the exocrine pancreatic secretion of the pig.

The acute effect of intravenous and intragastric alcohol on exocrine pancreatic secretion was studied in pigs with chronic pancreatic and gastric fistulas. A new H-type pancreatic cannula was designed for the study. With intravenous alcohol and the gastric fistula open, increased gastric acid secretion occurred but pancreatic enzyme output was inhibited. When acid was allowed to inter the duodenum by closing the gastric fistula, intravenous alcohol produced a secretin-like effect of increased pancreatic volume and bicarbonate secretion. Intragastric alcohol caused an increase in volume, bicarbonate and enzyme output. This was probably due initially to direct gastroduodenal stimulation as the same response occurred following intragastric mannitol of similar volume, pH and osmolality. The subsequently sustained increase in volume and bicarbonate was possibly a secondary secretin-like response following absorption of alcohol. It is concluded that the pig pancreas responds to alcohol as has been described in other animals. The use of a single model has allowed clarification of various phases of response invoking the effects of secretin, gastric and glucagon.

The Effect of Moderate Sedation on Exocrine Pancreas Function in Normal Healthy Subjects: A Prospective, Randomized, Cross-Over Trial Using the Synthetic Porcine Secretin Stimulated Endoscopic Pancreatic Function Test (ePFT)

We have developed a purely endoscopic collection method for the assessment of pancreatic secretory function (ePFT). The pancreatic secretory effects of sedation medications utilized during endoscopic procedures are not completely known. To study the effect of moderate sedation on the exocrine pancreas gland in a prospective, randomized trial. Healthy volunteers were randomized by computers to one of two treatments (A-no sedation, B-sedation) in period 1 and crossed-over to the other treatment in period 2 with a minimal washout interval of 7 days. Sedation dosage was standardized for each patient based on age, gender and weight from a previously published dosing nomogram. Synthetic porcine secretin (ChiRhoClin, Inc., Burtonsville, Maryland) was used as the pancreatic stimulant. Duodenal fluid samples were aspirated via the endoscope every 5 min for 1 h and sent on ice to our hospital laboratory for the measurement of pancreatic secretory electrolyte concentrations by autoanalyzer. A total of 17 healthy volunteers were enrolled. Sixteen subjects (8 males and 8 females) completed the randomized prospective trial. Median intravenous meperidine and midazolam sedation dose was 62.5 mg and 2.5 mg, respectively. Maximum pancreatic juice flow occurred during the early phase of secretion and maximum bicarbonate concentration occurred during the late phase of secretion. Analysis of the electrolyte composition of the endoscopically collected duodenal drainage fluid revealed a constant cation concentration for both sodium and potassium over the 1 h collection period. The anions, chloride and bicarbonate, exhibited a reciprocal relationship identical to that seen in traditional gastroduodenal tube collection studies. There was no statistical difference observed between the sedation and no sedation groups. The estimated total bicarbonate output (area under curve, AUC) for the sedated and non-sedated groups were 5,017 meq + 724 (range 3,663-6,173) and 5,364 meq +/- 583 (range 4,323-6563) respectively (p= 0.0656). The mean peak bicarbonate concentrations for sedated (n = 8) versus non-sedated (n = 8) groups were 103 +/- 11 meq/L (range 78-125) and 106 +/- 11 meq/L (range 87-138), respectively (p= 0.1346). There was excellent correlation of peak bicarbonate concentrations when sedation and no sedation groups were compared (r= 0.744, p < 0.05; Spearman rank correlation). There were no episodes of pancreatitis. (a) Moderate sedation used for upper endoscopy does not effect the clinical diagnostic parameters (peak bicarbonate concentration or total bicarbonate output) utilized to diagnose pancreatic insufficiency. (b) Analysis of duodenal drainage fluid collected endoscopically after synthetic secretin stimulation produces an identical pancreatic secretory curve described with traditional gastroduodenal tube collection methods.

Heat-stable enterotoxin ofEscherichia colistimulates a non-CFTR-mediated duodenal bicarbonate secretory pathway

The cystic fibrosis (CF) transmembrane conductance regulator (CFTR) is an important pathway for duodenal mucosal bicarbonate secretion. Duodenal biopsies from CF patients secrete bicarbonate in response to heat-stable enterotoxin from Escherichia coli (STa) but not cAMP. To explore the mechanism of STa-induced bicarbonate secretion in CF more fully, we examined the role of CFTR in STa-stimulated duodenal bicarbonate secretion in mice. In vivo, the duodenum of CFTR (-/-) or control mice was perfused with forskolin (10(-4) M), STa (10(-7) M), uroguanylin (10(-7) M), 8-bromoguanosine 3',5'-cGMP (8-Br-cGMP) (10(-3) M), genistein (10(-6) M) plus STa, or herbimycin A (10(-6) M) plus STa. In vitro, duodenal mucosae were voltage-clamped in Ussing chambers, and bicarbonate secretion was measured by pH-stat. The effect of genistein, DIDS (10(-4) M), and chloride removal was also studied in vitro. Control, but not CF, mice produced a significant increase in duodenal bicarbonate secretion after perfusion with forskolin, uroguanylin, or 8-Br-cGMP. However, both control and CF animals responded to STa with significant increases in bicarbonate output. Genistein and herbimycin A abolished this response in CF mice but not in controls. In vitro, STa-stimulated bicarbonate secretion in CF tissues was inhibited by genistein, DIDS, and chloride-free conditions, whereas bicarbonate secretion persisted in control mice. In the CF duodenum, STa can stimulate bicarbonate secretion via tyrosine kinase activity resulting in apical Cl(-)/HCO(3)(-) exchange. Further studies elucidating the intracellular mechanisms responsible for such non-CFTR mediated bicarbonate secretion may lead to important therapies for CF.

Long-term bile diversion enhances basal and duodenal oleate-stimulated pancreatic exocrine secretion in dogs.

There have been no previous reports whether long-term bile diversion enhances pancreatic exocrine secretion. The aim of this study was to elucidate the effect of long-term bile diversion on pancreatic exocrine secretion. Four mongrel dogs were prepared for chronic gastric and pancreatic fistulas and received intraduodenal sodium oleate infusion (controls). These dogs, then underwent diversion of bile from the intestines by ligating the common bile duct and interposing a segment of jejunum between the gallbladder and the urinary bladder (total biliary diversion [TBD]). After three weeks, the dogs received an identical sodium oleate infusion. TBD augmented basal pancreatic exocrine secretion compared with controls (4.4-fold increase in basal flow volume; 9.0-fold increase in bicarbonate output; and 3.3-fold increase in protein output). Likewise, TBD augmented oleate-stimulated exocrine secretion (2.0-fold increase in cumulative flow volume; 2.6-fold increase in bicarbonate output; and 1.4-fold increase in protein output). TBD also augmented basal and oleate-stimulated plasma cholecystokinin levels. Administration of a Cholecystokinin-A receptor antagonist (loxiglumide) after TBD reduced the flow volume and bicarbonate output to the control levels, and the protein output to less than a half of the control level. Long-term bile diversion enhances basal and oleate-stimulated pancreatic exocrine secretion, at least partly via increased cholecystokinin secretion.

Effect of Vasoactive Intestinal Peptide and Pituitary Adenylate Cyclase-Activating Polypeptide on Pancreatic, Hepatic and Duodenal Mucosal Bicarbonate Secretion in the Pig

Background and Method: The potency of pituitary adenylate cyclase-activating polypeptide (PACAP) and the related vasoactive intestinal peptide (VIP) on pancreaticobiliary and duodenal mucosal bicarbonate secretion (DMBS) was studied in anaesthetized pigs. VIP, PACAP27 and PACAP38 were infused at doses of 250, 500, 1,000, 2,000 and 6,000 pmol/kg·h. Results: Infusion of VIP in these concentrations increased the portal plasma concentrations of VIP from 9 to 102, 220, 600, 850 and 3,795 pM, respectively. At doses of 1,000 and 6,000 pmol/kg·h all three peptides significantly stimulated pancreatic bicarbonate secretion, VIP from 0.0 to 0.3 and 7.6 mmol/h, PACAP27 from 0.0 to 2.3 and 8.2 mmol/h, and PACAP38 from 0.0 to 0.2 and 7.4 mmol/h, respectively. Only during infusion of 6,000 pmol/kg·h did the three peptides elicit a significant increase in hepatic bicarbonate output. At this dose VIP increased hepatic bicarbonate output from 0.4 to 3.8 mmol/h, PACAP27 from 0.3 to 3.6 mmol/h, and PACAP38 from 0.1 to 1.4 mmol/h, respectively. Infusion of VIP (500, 1,000, 2,000 and 6,000 pmol/kg·h) dose-dependently and significantly enlarged DMBS from 0.09 to 0.19, 0.41, 0.54 and 0.53 mmol/h, respectively. A significant effect of PACAP27 and PACAP38 on DMBS was only obtained at a dose of 6,000 pmol/kg·h, and at this dose VIP-induced DMBS was 4- and 8-fold higher than that induced by PACAP27 and PACAP38, respectively. Conclusions: VIP, PACAP27 and PACAP38 all have a pharmacological effect on both DMBS and pancreaticobiliary bicarbonate secretion. When comparing these results with the distribution of VIP/PACAP neurons and relevant receptors, it seems likely that VIP and PACAP have a physiological importance as neurotransmitters in the pancreas and duodenum, but not in the liver. The differences in the effect of VIP and PACAP on DMBS observed in this study indicate that there are variations between pancreatic and duodenal PACAP/VIP receptor subtypes, or that the duodenal inactivation of the peptides is different from the inactivation in the pancreas.

Insulin inhibits secretin-induced pancreatic bicarbonate output via cholinergic mechanisms.

Exogenous insulin inhibits secretin-stimulated pancreatic bicarbonate output via a dose-dependent mechanism; this effect is prevented by pancreatic denervation. To investigate possible cholinergic mediation, we examined the effect of bethanechol on secretin-stimulated pancreatic secretion during euglycemic, hyperinsulinemic clamp. In four dogs with chronic pancreatic fistulas, euglycemic, hyperinsulinemic clamp (1.25 mU/kg/min) was begun after a 30-minute basal period; computer-assisted glucose administration maintained euglycemia. Control studies were performed with volume-matched and rate-matched vehicle infusion. After 1 hour, secretin infusion was begun at a dosage of 16 ng/kg/h; the dose was doubled every 30 minutes. The studies were then repeated during background bethanechol infusion (90 microg/kg/h) begun 30 minutes after clamp initiation. Pancreatic juice was analyzed for bicarbonate and protein; serum samples were analyzed for glucose and insulin. Exocrine outputs and serum glucose and insulin levels did not differ in the basal period. Insulin levels were significantly elevated during the euglycemic, hyperinsulinemic clamp (62 microU/mL versus 12 microU/mL; p < 0.01); glucose levels did not differ. As before, secretin-induced bicarbonate output was inhibited by euglycemic, hyperinsulinemic clamp. The inhibitory effect of insulin was reversed by bethanechol. Despite the euglycemic, hyperinsulinemic clamp, secretin-induced bicarbonate output was potentiated by bethanechol at higher secretin doses (p < 0.05). These data confirm that cholinergic mechanisms mediate insulin's inhibition of secretin-induced pancreatic bicarbonate output.