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Background & Aims: The mechanisms by which reflux of bile acids into the pancreas induces pancreatitis are unknown. We reasoned that key events responsible for this phenomenon might be mediated by Gpbar1, a recently identified and widely expressed G-protein-coupled, cell surface bile acid receptor., Methods: Acute pancreatitis was induced in wild-type and Gpbar1(-/-) mice by either retrograde ductal infusion of taurolithocholic acid-3-sulfate (TLCS) or supramaximal secretagogue stimulation with caerulein. In vitro experiments were performed in which acini obtained from wild-type and Gpbar1(-/-) mice were exposed to either submicellar concentrations of TLCS (200-500 microM) or a supramaximally stimulating concentration of caerulein (10 nM)., Results: Gpbar1 is expressed at the apical pole of acinar cells and its genetic deletion is associated with reduced hyperamylasemia, edema, inflammation, and acinar cell injury in TLCS-induced, but not caerulein-induced, pancreatitis. In vitro, genetic deletion of Gpbar1 is associated with markedly reduced generation of pathological calcium transients, intracellular activation of digestive zymogens, and cell injury when these responses are induced by exposure to TLCS, but not when they are induced by exposure to caerulein., Conclusions: Gpbar1 may play a critical role in the evolution of bile-acid-induced pancreatitis by coupling exposure to bile acids with generation of pathological intracellular calcium transients, intra-acinar cell zymogen activation, and acinar cell injury. Acute biliary pancreatitis may be a "receptor-mediated" disease and interventions that interfere with Gpbar1 function might prove beneficial in the treatment and/or prevention of biliary acute pancreatitis.

Background & Aims: Prior thermal stress induces heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue-induced pancreatitis, but it is not clear that this thermal stress-induced protection is actually mediated by HSP70 since thermal stress may have other, non-HSP related, effects., Methods: In the present study, we have administered antisense (AS) oligonucleotides, which prevent pancreatic expression of HSP70 to rats, in vivo, to evaluate this issue. In a separate series of experiments, designed to examine the role of pancreatitis-induced HSP70 expression in modulating the severity of pancreatitis, rats not subjected to prior thermal stress were given AS-HSP70 before cerulein administration, and trypsinogen activation as well as the severity of pancreatitis were evaluated., Results: Hyperthermia induced HSP70 expression, prevented intrapancreatic trypsinogen activation, and protected against cerulein-induced pancreatitis. Administration of AS-HSP70 but not sense-HSP70 reduced the thermal stress-induced HSP70 expression, restored the ability of supramaximal cerulein stimulation to cause intrapancreatic trypsinogen activation, and abolished the protective effect of prior thermal stress against pancreatitis. In non-thermally stressed animals, pretreatment with AS-HSP70 before the induction of pancreatitis exacerbated all the parameters associated with pancreatitis., Conclusions: These findings lead us to conclude that HSP70 induction, rather than some other thermal stress-related phenomenon, mediates the thermal stress-induced protection against pancreatitis and that it protects against pancreatitis by preventing intrapancreatic activation of trypsinogen. The worsening of pancreatitis, which occurs when non-thermally stressed animals are given AS-HSP70 before cerulein, suggests that cerulein-induced HSP70 expression in nontreated animals acts to limit the severity of pancreatitis.

Background & Aims: The present study was undertaken to evaluate the role of serine proteases in regulating digestive enzyme secretion in pancreatic acinar cells., Methods: Isolated acini were stimulated by various secretagogues in the presence or absence of cell-permeant serine protease inhibitors 4-(2-aminoethyl)-benzenesulfonyl fluoride and N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone. F-actin distribution was studied after staining with rhodamine phalloidin., Results: Both cell-permeant serine protease inhibitors blocked amylase secretion in response to secretagogues that use calcium as a second messenger (e.g., cerulein, carbamylcholine, and bombesin) but not to those that use adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger (e.g., secretin and vasoactive intestinal polypeptide). Incubation of the acini with these inhibitors also resulted in a dramatic redistribution of the F-actin cytoskeleton. This redistribution was energy dependent. Similar redistribution of F-actin from the apical to the basolateral region was also observed when acini were incubated with a supramaximally stimulating concentration of cerulein, which is known to inhibit secretion., Conclusions: These results suggest that a serine protease activity is essential for maintaining the normal apical F-actin distribution; its inhibition redistributes F-actin from the apical to the basolateral region and blocks secretion induced by secretagogues that act via calcium. cAMP reverses the F-actin redistribution and hence cAMP-mediated secretion is not affected.

Background & Aims: Heat shock proteins (Hsps), induced by cell stress, are known to protect against cellular injury. Recent studies have indicated that Hsp60 expression, induced by exposure to water immersion stress, protects against pancreatitis induced by administration of supramaximal doses of cerulein in rats. However, the mechanisms responsible for this protection are not known., Methods: Rats were water-immersed for 3-12 hours. Pancreatitis was induced by cerulein administration., Results: The results confirm that prior induction of Hsp60 expression by water-immersion stress significantly ameliorates the severity of cerulein-induced pancreatitis as judged by the markedly reduced degree of hyperamylasemia, pancreatic edema, and acinar cell necrosis. Water immersion also prevents the subcellular redistribution of cathepsin B from a lysosome-enriched fraction to a heavier, zymogen granule-enriched fraction that is known to occur in this model of pancreatitis. Intra-acinar cell activation of trypsinogen that occurs shortly after exposure to a supramaximally stimulating dose of cerulein both in vivo and in vitro is prevented by prior water-immersion stress and Hsp60 expression. The protection against pancreatitis that follows water-immersion stress is not caused by alterations of cholecystokinin receptors, because water immersion does not alter the typical biphasic amylase secretory response to stimulation with cerulein., Conclusions: Water-immersion stress induces Hsp60 expression, ameliorates cerulein-induced pancreatitis, and prevents intra-acinar cell activation of trypsinogen. We suggest that Hsp60 protects against cerulein-induced pancreatitis by preventing trypsinogen activation within acinar cells.

Background & Aims: Intercellular adhesion molecule 1 (ICAM-1) and neutrophils play important roles in many inflammatory processes, but their importance in both acute pancreatitis and pancreatitis-associated lung injury has not been defined., Methods: To address this issue, mice that do not express ICAM-1 were used and depleted of neutrophils by administration of antineutrophil serum. Pancreatitis was induced by administering either supramaximal doses of the secretagogue cerulein or feeding a choline-deficient, ethionine-supplemented diet. The severity of pancreatitis was evaluated by quantitating serum amylase, pancreatic edema, acinar cell necrosis, and pancreas myeloperoxidase activity (i.e., neutrophil content). Lung injury was evaluated by quantitating lung myeloperoxidase activity and pulmonary microvascular permeability. ICAM-1 was quantitated by enzyme-linked immunosorbent assay and was localized by light-microscopic immunohistochemistry., Results: It was found that serum, pancreas, and lung ICAM-1 levels increase during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in ICAM-1. Neutrophil depletion also reduces the severity of both pancreatitis and lung injury. However, the combination of neutrophil depletion with ICAM-1 deficiency does not reduce the severity of pancreatitis or lung injury to a greater extent than either neutrophil depletion or ICAM-1 deficiency alone. Neither pancreatitis nor pancreatitis-associated lung injury are completely prevented by ICAM-1 deficiency, neutrophil depletion, or combined ICAM-1 deficiency plus neutrophil depletion., Conclusions: The observations indicate that ICAM-1 plays an important, neutrophil-mediated, proinflammatory role in pancreatitis and pancreatitis-associated lung injury. The studies also indicate that ICAM-1 and neutrophil-independent events also contribute to the evolution of pancreatitis and lung injury in these models.

Background & Aims: Recent reports suggest that platelet-activating factor (PAF) plays a role in pancreatitis and pancreatitis-associated lung injury. In this study, the effects on these processes of termination of PAF action by recombinant PAF-acetylhydrolase (rPAF-AH) were investigated., Methods: Rats were given rPAF-AH and then infused with a supramaximally stimulating dose of cerulein to induce mild pancreatitis. Opossums underwent biliopancreatic duct ligation to induce severe pancreatitis, and rPAF-AH administration was begun 2 days later., Results: In mild, secretagogue-induced pancreatitis, rPAF-AH given before the cerulein reduced hyperamylasemia, acinar cell vacuolization, and pancreatic inflammation but did not alter pancreatic edema or pulmonary microvascular permeability. In severe, biliopancreatic duct ligation-induced pancreatitis, rPAF-AH delayed and reduced the extent of inflammation and acinar cell injury/necrosis and completely prevented lung injury even though the rPAF-AH administration was begun after the onset of pancreatitis., Conclusions: PAF plays an important role in the regulation of pancreatic injury but not pancreatic edema or increased pulmonary microvascular permeability in mild, secretagogue-induced pancreatitis. PAF plays a critical role in the regulation of progression of pancreatic injury and mediation of pancreatitis-associated lung injury in severe biliary pancreatitis. Amelioration of pancreatitis and prevention of pancreatitis-associated lung injury can be achieved with rPAF-AH even if treatment is begun after pancreatitis is established.

Background/aims: Cerulein-induced pancreatitis causes a rapid increase in pancreatic enzyme levels in serum and decreases in pancreatic duct secretion and interstitial edema. One mechanism to explain these early events is disruption of the actin tight junction paracellular seal of acinar and intralobular pancreatic duct cells., Methods: To examine the paracellular barrier of the proximal exocrine pancreas, rats were hyperstimulated with 5.0 micrograms.kg-1.h-1 of cerulein. Actin was visualized with rhodamine phalloidin and by electron microscopy and tight junctions were visualized with antibodies to the tight-junction protein ZO-1. Paracellular permeability was measured by movement of horseradish peroxidase from interstitium into duct or acinar lumens., Results: In controls, linear actin and ZO-1 staining occurred along the apical membrane of intralobular duct cells and extended to the apical pole of acinar cells. Hyperstimulation caused progressive disruption of the linear staining of f-actin and ZO-1. Actin disruption in duct cells was confirmed by electron microscopy. Horseradish peroxidase entered intralobular ducts and acinar lumens of hyperstimulated animals more frequently than those of controls., Conclusions: The structure and function of the paracellular barrier of acinar and intralobular pancreatic duct cells are disrupted early during cerulein pancreatitis and may contribute to early clinical features.

Background: The value of early endoscopic or surgical interventions to remove bile duct stones and decompress the biliopancreatic ductal system in gallstone pancreatitis is controversial., Methods: To evaluate this issue, acute hemorrhagic necrotizing pancreatitis was induced in opossums by obstructing the biliopancreatic ductal system with a balloon catheter for 1, 3, or 5 days., Results: A progressive increase in the severity of pancreatitis, as manifested by inflammation, fat necrosis, hemorrhage, acinar cell vacuolization, in vitro lactate dehydrogenase release, and acinar cell necrosis, was noted in these obstructed animals. In contrast, decompression of the obstructed ductal system by removal of the balloon catheter after 1 or 3 days prevented the increase in severity of these parameters of pancreatic injury., Conclusions: We concluded that the severity of biliary pancreatitis in this model is dependent upon the duration of ductal obstruction and that decompression of the ductal system can prevent progression of the disease. These observations support the practice of early attempts to remove obstructing stones in clinical gallstone pancreatitis.

Background: Recent experimental findings have suggested that activation of trypsinogen by cathepsin B within acidic pancreatic acinar cell cytoplasmic vacuoles may be a critical early event in both secretagogue and diet-induced pancreatitis. The weak base chloroquine accumulates within acidic intracellular compartments, raises their pH, and can inhibit proteolysis as well as cathepsin B., Methods: We have investigated the effect of in vivo chloroquine administration on both secretagogue and diet-induced experimental pancreatitis to determine if raising the pH of cytoplasmic vacuoles in these models of pancreatitis would have a protective effect., Results: Infusion of chloroquine (5 mg.kg-1.h-1) resulted in the uptake and concentration of chloroquine in the pancreas, an increase in the pH of acinar cell acidic compartments, and interference with the pH-dependent sorting of lysosomal hydrolases from digestive enzyme zymogens. However, chloroquine administration did not have a protective effect against the hyperamylasemia, the pancreatic edema, the morphological changes or the mortality that is associated with these models of pancreatitis., Conclusions: These observations lead us to conclude that raising the pH of acinar cell acidic compartments by in vivo administration of chloroquine does not prevent either secretagogue or diet-induced pancreatitis.

Background: The common channel theory suggests that bile reflux, through a common biliopancreatic channel, triggers acute pancreatitis. In the present study, this controversial issue was evaluated using an experimental model of hemorrhagic necrotizing pancreatitis., Methods: American opossums underwent ligation of the pancreatic duct alone, bile and pancreatic duct separately, or common biliopancreatic duct; the severity of pancreatitis was evaluated at selected times after ligation., Results: Animals in all three experimental groups developed hemorrhagic necrotizing pancreatitis; the severity of pancreatitis was similar in each group, although only those subjected to common biliopancreatic duct ligation experienced bile reflux., Conclusions: Bile reflux into the pancreatic duct, via a common biliopancreatic channel, is not necessary for the development of pancreatitis and does not worsen the severity of pancreatitis associated with pancreatic duct obstruction in this model.

Acute pancreatitis was induced by ligating the opossum common biliopancreatic duct immediately proximal to its entry into the duodenum, and macroscopic as well as microscopic changes were evaluated during the subsequent 24 hours. Transient pancreatic edema and progressive hyperamylasemia were noted within 6 hours of pancreatic and bile duct ligation. Light microscopic evidence of pancreatic injury including acinar cell necrosis, hemorrhage, fat necrosis, and inflammatory cell infiltration was noted within 12 hours of duct obstruction. Electron microscopic changes included massive dilatation of the rough endoplasmic reticulum and disruption of the apical plasmalemma of acinar cells during the initial 3 hours. These observations indicate that pancreatic and bile duct ligation in the opossum results in the rapid (less than 24 hours) appearance of changes consistent with acute hemorrhagic and necrotizing pancreatitis and that the initial lesion in this model of experimental pancreatitis involves acinar cells.

The short-term effects of rat pancreatic duct obstruction were evaluated and compared with those recently reported to follow obstruction of the rabbit pancreatic duct. In both species pancreatic edema and hyperamylasemia are noted, and the lysosomal hydrolase cathepsin B is redistributed from the lysosome-enriched to the zymogen granule-enriched subcellular fraction. Theoretically, this redistribution phenomenon might lead to digestive enzyme activation because cathepsin B is known to be capable of activating trypsinogen. The hyperamylasemia and pancreatic edema (but not the cathepsin B redistribution) that follow rat pancreatic duct obstruction were increased by infusion of a submaximally stimulating dose of the cholecystokinin analogue cerulein. Administration of the cholecystokinin-receptor antagonist L-364,718 reduced the hyperamylasemia but did not alter the pancreatic edema or cathepsin B redistribution. These observations indicate that cholecystokinin may modulate some but not all of the effects of duct obstruction. Secretin administration increased the degree of pancreatic edema and had no demonstrable protective effect. The rat duct-obstruction model described in this report may prove particularly useful in future studies designed to clarify the early events underlying the development of acute pancreatitis.

Prostaglandins have been noted to have a "protective" effect against gastrointestinal mucosal injury induced by a wide variety of agents although possible protective effects of prostaglandins on injury to other tissues have not been reported. We have tested the effect of prostaglandin E2 (PGE2) on acute experimental pancreatitis induced by feeding young female mice a choline-deficient ethionine-supplemented (CDE) diet for 24 hr. Administration of 0.05--0.20 microgram PGE2/g body wt 1 hr before and 4 hr after institution of the CDE diet lowered the mortality rate of diet-induced pancreatitis from 56% to 31%. Larger and smaller doses of PGE2 were without effect. Administration of PGE2 (0.10 microgram/g body weight) diminished the rise in in-vitro LDH discharge and the increase in "free" Cathepsin D activity which occur during diet-induced pancreatitis. Similarly, PGE2 (0.10 microgram/g body wt) diminished the magnitude of the increase in in-vitro protein discharge and the elevated concentrations of trypsinogen and chymotrypsinogen in pancreas fragments taken from mice given the CDE diet. These findings indicate the PGE2 has a protective effect against CDE diet-induced acute experimental pancreatitis. The Cathespin D and LDH changes noted during CDE diet-induced pancreatitis suggest that this diet may decrease membrane integrity and thus allow these enzymes to leak out of the lysosomes and acinar cell, respectively, during pancreatitis. Although the basis for the protective effect of PGE2 remains unclear, our observations suggest that the prostaglandin may act to reduce the alteration in membrane integrity which occurs during CDE-diet induced pancreatitis.

The role of oxygen-derived free radicals in the pathogenesis of acute pancreatitis was studied by evaluating the effects of catalase, allopurinol, and dimethylsulfoxide on diet-induced acute hemorrhagic pancreatic necrosis in mice. The mortality rate and degree of hyperamylasemia associated with this model of pancreatitis were reduced by catalase but a similar result followed the administration of heat-denatured catalase, suggesting that the apparent protective effect of catalase was not the result of reductions in free radical levels. Neither allopurinol nor dimethylsulfoxide reduced mortality or degree of hyperamylasemia. The increased pancreatic content of amylase and the necrosis that characterize this model of pancreatitis were not altered by any of the agents tested. In contrast, both allopurinol and dimethylsulfoxide reduced peripancreatic edema formation, suggesting that edema, but not the other features that characterize this model of pancreatitis, may result from generation of oxygen-derived free radicals.

Earlier studies have indicated that lysosomal enzymes such as cathepsin B become redistributed within pancreatic acinar cells during the early stages of both diet- and secretagogue-induced acute pancreatitis. As a result, cathepsin B and digestive zymogens became colocalized within large cytoplasmic vacuoles. As cathepsin B can activate trypsinogen, this colocalization could result in intracellular digestive enzyme activation. The present study investigates the protective effects of gabexate mesilate (FOY) and camostate (FOY 305) on both of these noninvasive models of experimental pancreatitis. These esterase inhibitors prevented the hyperamylasemia, pancreatic edema, and acinar cell vacuolization that characterize secretagogue-induced pancreatitis and the hyperamylasemia and mortality that characterize diet-induced pancreatitis. In addition, FOY and FOY 305 were found to significantly decrease the subcellular redistribution of cathepsin B that occurs in both models. These findings indicate that enzyme activity sensitive to inhibition by FOY and FOY 305 may be critical to the redistribution phenomenon that characterizes both of these models of pancreatitis.