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Background & Aims: Growth of exocrine pancreas is regulated by gastrointestinal hormones, notably cholecystokinin (CCK). CCK-driven pancreatic growth requires calcineurin (CN), which activates Nuclear Factor of Activated T cells (NFATs), but the genetic underpinnings and feedback mechanisms that regulate this response are not known., Methods: Pancreatic growth was stimulated by protease inhibitor (PI)-containing chow, which induces secretion of endogenous CCK. Expression profiling of PI stimulation was performed on Affymetrix 430A chips, and CN was inhibited via FK506. Exocrine pancreas-specific overexpression of CN inhibitor Regulator of Calcineurin 1 (Rcan1) was achieved by breeding elastase-Cre(estrogen receptor [ER]) transgenics with "flox-on" Rcan1 mice., Results: CN inhibitor FK506 blocked expression of 38 genes, as confirmed by quantitative polymerase chain reaction. The CN-dependent genes were linked to growth-related processes, whereas their promoters were enriched in NFAT and NFAT/AP1 sites. Multiple NFAT targets, including Rcan1, Rgs2, HB-EGF, Lif, and Gem, were validated by chromatin immunoprecipitation. One of these, a CN feedback inhibitor Rcan1, was induced >50 fold during 1-8 hours course of pancreatic growth and strongly inhibited (>99%) by FK506. To examine its role in pancreatic growth, we overexpressed Rcan1 in an inducible, acinar-specific fashion. Rcan1 overexpression inhibited CN-NFAT signaling, as shown using an NFAT-luciferase reporter and quantitative polymerase chain reaction. Most importantly, the increase in exocrine pancreas size, protein/DNA content, and acinar proliferation were all blocked in Rcan1 overexpressing mice., Conclusions: We profile adaptive pancreatic growth, identify Rcan1 as an important new feedback regulator, and firmly establish that CN-NFAT signaling is required for this response., (Copyright (c) 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Proteolytic release and activation of latent transforming growth factor beta (TGF-beta) by the hepatic stellate cells (HSCs) are key events for pathogenesis of hepatic fibrosis, and protease inhibitors suppress TGF-beta generation by cultured HSCs, suggesting their potential use as antifibrogenic agents. We explored this idea using camostat mesilate, a serine protease inhibitor, to determine its effects and mechanisms of action in vivo., Methods: Camostat mesilate was either added to cultured rat HSCs or administered orally to rats during porcine serum treatment, followed by overexpression of urokinase. We measured cellular and hepatic levels of plasmin, TGF-beta, TGF-beta activity, activated HSC markers (increased cell number, morphologic change, and expression of both alpha-smooth muscle actin and collagen(alpha2)[I]), and fibrosis (Azan-staining and quantification of hydroxyproline content)., Results: Camostat mesilate (500 micromol/L) inhibited generation of TGF-beta by suppressing plasmin activity and reduced the activity of TGF-beta, which blocked in vitro activation of HSCs. In the in vivo model, camostat mesilate (1-2 mg/g of diet) markedly attenuated an increase in hepatic plasmin and TGF-beta levels, HSC activation, and hepatic fibrosis without apparent systemic or local side effects, all of which were reverted by restoration of hepatic plasmin activity., Conclusions: Camostat mesilate prevents porcine serum-induced rat hepatic fibrosis via a profound reduction in TGF-beta generation.

Background & Aims: Cholecystokinin (CCK) is known to stimulate the synthesis of digestive enzymes in the pancreas at the translational level. We investigated in vivo the biochemical regulation of initiation factors important for the stimulation of translation of digestive enzyme protein in rat pancreas by CCK., Methods: Intraperitoneal injection of CCK or intragastric administration of a trypsin inhibitor to elicit endogenous CCK release was followed by removal and preparation of pancreas for protein evaluation. Isoelectric focusing was used to evaluate the phosphorylation of the initiation factor eIF4E, and Western blotting and immunoprecipitation followed by Western blotting were used to study the phosphorylation state and amount of other interacting factors., Results: CCK treatment induced a time- and dose-dependent phosphorylation of pancreatic eIF4E and its binding protein (PHAS-I). Because the release of eIF4E from its binding protein as a result of phosphorylation is followed by formation of a messenger RNA cap-binding complex that includes the initiation factor eIF4G, we evaluated the association of eIF4G with released eIF4E and showed that it was increased by CCK. These events occurred over a range of CCK doses from 0.2 to 5 microg/kg. We also evaluated the effect of endogenous CCK by administering a synthetic trypsin inhibitor, camostat (100 mg/kg). Camostat treatment markedly increased the phosphorylation of both PHAS-I and eIF4E and the formation of eIF4E-eIF4G complex. Thus, both exogenous and endogenous CCK activate translational initiation factors in vivo., Conclusions: Activation of translational machinery necessary for initiation of protein synthesis likely contributes to the normal postprandial synthesis of pancreatic digestive enzymes.

Background & Aims: Feedback regulation of pancreatic enzyme secretion is well established in animals, and their pancreases are able to adapt to intraduodenal inhibition of pancreatic enzymes by proteinase inhibitors such as Camostate (FOY-305; Schwarz GmbH, Monheim, Germany). In this study, we addressed whether similar adaptive changes occur in the human pancreas after 4 weeks of 2 g/day Camostate application., Methods: Before, at the end of, and 2 weeks after 4-week Camostate treatment (four times 500 mg daily), pancreatic changes were analyzed with the use of a secretin-cerulein test, a test-meal stimulation, cholecystokinin plasma measurement, and standardized ultrasonographic investigations of the pancreas., Results: Duodenal trypsin output after secretion stimulation was significantly increased (+44%; P < 0.01) and duodenal bicarbonate output decreased 22% (P < 0.05) after 4 weeks of Camostate application. The size of the pancreatic head (vertical) increased 8% (P < 0.05) at week 4 and decreased to pretreatment values 2 weeks after treatment (week 6). The other three diameters measured (head oblique, body, and tail) showed a similar pattern. Stimulated cholecystokinin plasma levels 15 minutes after application of a standard test meal increased 62% (P < 0.05)., Conclusions: The human pancreas adapts to oral application of the proteinase inhibitor Camostate. These findings support the theory that feedback control of the exocrine pancreas operates in humans.

Background & Aims: The role of small intestinal proteolytic activity in the regulation of upper gastrointestinal function in humans is poorly understood. The aim of this study was to determine the importance of proteolytic activity for protein- or amino acid-induced cholecystokinin release and pancreaticobiliary secretion., Methods: In 9 healthy subjects, saline was perfused intraduodenally for 3 hours either with or without the synthetic protease inhibitor camostate. During the last hour, albumin or amino acids in the same molecular composition as albumin were also perfused., Results: Perfusion with camostate, in concentrations that abolished intraduodenal proteolytic activity, had no effect on unstimulated plasma cholecystokinin concentrations or gallbladder emptying, but markedly (P < 0.05) increased unstimulated pancreatic enzyme output. Perfusion with protein distinctly stimulated cholecystokinin release, gallbladder emptying, and pancreatic enzyme output (P < 0.05). Perfusion with camostate resulted in significantly lower protein-stimulated plasma cholecystokinin, gallbladder, and pancreatic enzyme responses (P < 0.05). Perfusion with amino acids also stimulated plasma cholecystokinin, gallbladder emptying, and pancreatic enzyme output (P < 0.05). Camostate did not inhibit these values., Conclusions: This study shows that appropriate digestion of protein is required to stimulate plasma cholecystokinin release, gallbladder emptying, and pancreatic enzyme secretion in humans.

Background/aims: Bombesin-stimulated plasma cholecystokinin levels decrease after an initial increase despite continuous infusion of bombesin. The aim of this study was to determine if a feedback mechanism, mediated by bile salts or proteolytic enzymes, is responsible for this decline., Methods: Bombesin (1.0 ng.kg-1.min-1) was infused into volunteers for 180 minutes on separate occasions. Cholestyramine, colestipol, camostate, or saline were perfused intraduodenally during the second hour of the tests. Cholestyramine was also administered without infusion of bombesin., Results: Colestipol and cholestyramine, dependent on their bile salt-binding capacity, markedly enhanced (P < 0.05) bombesin-stimulated plasma cholecystokinin from 2.1 +/- 0.5 pmol/L to 6.4 +/- 2.2 pmol/L and 12.1 +/- 3.3 pmol/L (P < 0.05 vs. colestipol), respectively, and further decreased gallbladder volume (P < 0.05) from 9.4 +/- 1.6 mL to 2.0 +/- 0.4 mL and 2.2 +/- 0.5 mL, respectively. The protease inhibitor camostate had no effect. Bile salt precipitation also enhanced plasma pancreatic polypeptide responses (P < 0.01) but did not alter gastrin responses. Plasma cholecystokinin responses to cholestyramine without bombesin infusion varied considerably, but increments were highly correlated to decreases in gallbladder volume (r = 0.91; P < 0.005)., Conclusions: Bile salt sequestration but not protease inactivation enhances plasma cholecystokinin and gallbladder responses to bombesin infusion in humans.

We investigated a hormonal mechanism in a trypsin inhibitor-induced pancreatic hypersecretion in rats. Intraduodenal administration of a synthetic trypsin inhibitor, camostat, resulted in significant increases in plasma concentration of both secretin and cholecystokinin in a dose-related manner that paralleled a significant increase in exocrine pancreatic secretion. To eliminate the effect of circulating secretin in rats, a rabbit antisecretin serum was given IV that resulted in a 77% reduction in bicarbonate secretion stimulated by intraduodenal camostat. A cholecystokinin receptor antagonist, MK-329, also inhibited significantly the camostat-induced increase in pancreatic secretion; volume and bicarbonate output were reduced by 35% each and amylase output by 73%. The combined administration of antisecretin serum and MK-329 completely abolished the pancreatic exocrine secretion stimulated by camostat. These observations indicate that the camostat-stimulated pancreatic exocrine secretion is mediated by the increased release of both secretin and cholecystokinin in rats.

To determine the effects of luminal protease inhibition on duodenal delivery and the intraluminal fate of pancreatic enzymes, six healthy subjects were intubated with an oro-ileal multilumen tube assembly. By using nonabsorbable markers, cumulative trypsin, chymotrypsin, lipase, and amylase activities were measured as delivered to duodenum, midjejunum, and distal ileum, with or without simultaneous duodenal perfusion of the protease inhibitor camostat at graded doses. Compared with saline, camostat (a) inhibited trypsin activity in the entire small intestinal lumen by up to 99%, and significantly reduced chymotrypsin activity by up to 89%; (b) significantly increased duodenal deliveries of lipase activity, amylase activity and volume; (c) did not influence plasma cholecystokinin concentrations; and (d) significantly increased jejunal and ileal deliveries of lipase but not amylase activity. Small intestinal transit and motility were not affected by camostat. In additional in vitro studies, camostat significantly reduced the spontaneous decline in lipase activity in fresh human duodenal juice incubated at 37 degrees C. These findings demonstrate that duodenal deliveries of lipase and amylase activities increase when intraluminal protease activity is decreased; they suggest that this increase is not caused by slower proteolytic destruction of enzyme protein but by stimulation of pancreatic secretion. Thus, luminal protease-mediated feedback regulation of pancreatic secretion may be operative in humans. Because plasma cholecystokinin concentrations were not affected, these effects may in part be independent of cholecystokinin. The data further suggest that proteolytic digestion plays a major role in the rapid loss of luminal lipase activity on small intestinal transit.

Two models of severe acute pancreatitis were chosen and pancreatitis induced by sodium taurocholate and by a choline-deficient ethionine-supplemented diet, to evaluate the effectiveness of FOY-305 (camostate), a new synthetic trypsin inhibitor. Prophylactic administration of FOY-305 had a significantly favorable effect on the course of the sodium taurocholate-induced disease and on the survival rate of the treated group. A beneficial effect on the amylase and lipase content in serum and ascites was found, but no effect was observed on enzyme concentration in pancreatic tissue or on the degree of histologically detectable organ destruction. Therapeutic administration of FOY-305 had a significantly positive influence when infused directly, 5 and 30 min after the operation, whereas enzyme increase and organ destruction remained unaffected. FOY-305 showed a beneficial effect when given prophylactically or therapeutically at the beginning of the pancreatitis induced by a CDE diet, with no significant change in enzyme increase and degree of organ destruction. The favorable effect on survival time and rate in the early phase of these two severe experimental forms of pancreatitis may justify an evaluation of FOY-305 in a clinically controlled study.

The present study evaluates the effect of atropine and of the cholecystokinin receptor antagonist loxiglumide on feedback regulation of basal pancreatic secretion in 6 healthy volunteers. The intraduodenal instillation of the protease inhibitor camostate reduced enzymatic activities of trypsin and chymotrypsin by 80%. This was accompanied by a strong increase in amylase and lipase output. The intravenous infusion of atropine (5 micrograms/kg.h) completely abolished the stimulatory effect of camostate on enzyme output. The infusion of loxiglumide (10 mg/kg.h) caused no changes in camostate-induced stimulation of enzyme output. Plasma levels of cholecystokinin were not altered after intraduodenal instillation of camostate whether atropine, loxiglumide, or saline were infused. We suggest that the protease inhibitor camostate, by inhibition of the enzymatic activity of trypsin and chymotrypsin, interferes with feedback regulation of basal pancreatic secretion in humans, and this mechanism is predominantly mediated by the cholinergic system.

Chronic feeding of rats with camostate results in pancreatic hypertrophy or hyperplasia, or both. Previous studies suggest that this effect of camostate occurs via an increase in endogenous cholecystokinin due to an enteral feedback mechanism involving the inhibition of trypsin in the duodenum. Studies employing proglumide, a weak and relatively nonspecific cholecystokinin antagonist, have failed to fully abolish camostate-induced pancreas growth. We examined the effects of L-364,718, a new and highly potent cholecystokinin receptor antagonist, on camostate-induced pancreas growth in rats. The pancreas weights and the concentrations of ribonucleic acid, protein, and chymotrypsinogen in the pancreas of rats treated with camostate alone were significantly elevated over those of controls. These effects of camostate were completely abolished in rats treated with camostate + L-364,718. The pancreas weights and the concentrations of deoxyribonucleic acid and ribonucleic acid in the pancreas of rats treated with L-364,718 alone were significantly lower than values in control rats. These data indicate that camostate-induced pancreas growth in rats appears to be dependent on the actions of endogenous cholecystokinin and that cholecystokinin may play a role in the maintenance of pancreatic growth in normal rats.