Cholecystokinin Analogue Research Articles

Protease-activated Receptor-2 Exerts Contrasting Model-specific Effects on Acute Experimental Pancreatitis

Protease-activated receptor-2 (PAR2) is a 7-transmembrane G-protein-coupled tethered ligand receptor that is expressed by pancreatic acinar and ductal cells. It can be physiologically activated by trypsin. Previously reported studies (Namkung, W., Han, W., Luo, X., Muallem, S., Cho, K. H., Kim, K. H., and Lee, M. G. (2004) Gastroenterology 126, 1844-1859; Sharma, A., Tao, X., Gopal, A., Ligon, B., Andrade-Gordon, P., Steer, M. L., and Perides, G. (2005) Am. J. Physiol. 288, G388-G395) have shown that PAR2 activation exerts a protective effect on the experimental model of pancreatitis induced by supramaximal secretagogue (caerulein) stimulation. We now show that PAR2 exerts a worsening effect on a different model of experimental pancreatitis, i.e. one induced by retrograde pancreatic ductal infusion of bile salts. In vitro studies using freshly prepared pancreatic acini show that genetic deletion of PAR2 reduces bile salt-induced pathological calcium transients, acinar cell injury, and activation of c-Jun N-terminal kinase, whereas genetic deletion of PAR2 has the opposite or no effect on these pancreatitis-related events when they are elicited, in vitro, by caerulein stimulation. Studies employing a combination of trypsin inhibition and activation of PAR2 with the activating peptide SLIGRL show that all these differences indeed depend on the activation of PAR2. These studies are the first to report that a single perturbation can have model-specific and opposite effects on pancreatitis, and they underscore the importance of performing mechanistic pancreatitis studies using two dissimilar models of the disease to detect idiosyncratic, model-specific events. We suggest PAR2 activation exerts a worsening effect on the severity of clinical pancreatitis and that interventions interfering with PAR2 activation may be of benefit in the treatment of patients with severe pancreatitis.

Reply to Lechin and van der Dijs

LETTERS TO THE EDITORReply to Lechin and van der DijsRajinder Dawra, and Ashok SalujaRajinder Dawra, and Ashok SalujaPublished Online:01 Jun 2008https://doi.org/10.1152/ajpgi.90251.2008MoreSectionsPDF (23 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations we appreciate the interest in and thought-provoking discussion about the possible mechanism of l-arginine-induced pancreatitis as it relates to our recently published paper (1). In this study, we hypothesize that excessive peroxynitrite formation as result of high doses of l-arginine could cause acinar cell injury and pancreatitis. This was based on our observations that administering either d-arginine or other amino acids at comparable doses did not induce pancreatitis. Furthermore, l-arginine at lower doses did not induce pancreatitis.Lechin and van der Dijs (2) suggest that l-arginine might induce pancreatitis by the autonomous nervous system. This is based on their observation that a small oral dose of l-arginine (50 mg) is able to provoke significant changes in plasma neurotransmitters. This could cause hypersecretion, which cannot be drained through the pancreatic duct because of the norepinephrine contractile effect by sympathetic nerves. We have not measured plasma neurotransmitters in our experiments, but had this been the mechanism we should have observed some changes in pancreatitis markers with lower doses of l-arginine. Moreover, in our preliminary studies on acinar cells prepared 24 h after l-arginine administration, the secretory response to the cholecystokinin analog caerulein decreased, which again indicates a secretory block at the acinar cell level rather than at the ductal level. In a rat model of l-arginine-induced pancreatitis, antioxidants decrease pancreatic injury. Thus peroxynitrite appears to be a strong contender for mechanism of l-arginine-induced pancreatitis.REFERENCES1 Dawra R, Sharif R, Phillips P, Dudeja V, Dhaulakhandi D, Saluja AK. Development of a new mouse model of acute pancreatitis induced by administration of l-arginine. Am J Physiol Gastrointest Liver Physiol 292: G1009–G1018, 2007.Link | ISI | Google Scholar2 Lechin F, van der Dijs B. Arginine-induced pancreatitis: involvement of the autonomic nervous system? Am J Physiol Gastrointest Liver Physiol. doi: 10.1152/ajpgi.00327.2007.Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: A. K. Saluja, Dept. of Surgery, Univ. of Minnesota, MMC 195, 420 Delaware St. SE, Minneapolis, MN 55455 (e-mail: [email protected]) Download PDF Previous Back to Top FiguresReferencesRelatedInformation More from this issue > Video Abstract Volume 294Issue 6June 2008Pages G1452-G1452 Copyright & PermissionsCopyright © 2008 the American Physiological Societyhttps://doi.org/10.1152/ajpgi.90251.2008History Received 24 March 2008 Accepted 31 March 2008 Published online 1 June 2008 Published in print 1 June 2008 Metrics

Gardenia jasminoides protects against cerulein-induced acute pancreatitis

To investigate the effect of Gardenia jasminoides (GJ) on cerulein-induced acute pancreatitis (AP) in mice. C57BL/6 mice weighing 18-20 g were divided into three groups. (1) Normal saline-treated group, (2) treatment with GJ at a dose of 0.1 g/kg, (3) treatment with GJ at a dose of 1 g/kg. GJ was administered orally (n = 6 per group) for 1 wk. Three hours later, the mice were given an intraperitoneal injection of cerulein (50 microg/kg), a stable cholecystokinin (CCK) analogue, every hour for a total of 6 h as described previously. The mice were sacrificed at 6 h after completion of cerulein injections. Blood samples were obtained to determine serum amylase, lipase and cytokine levels. The pancreas was rapidly removed for morphologic examination and scoring. A portion of pancreas was stored at -70 degree and prepared for the measurement of tissue myeloperoxidase (MPO) activity, an indicator of neutrophil sequestration, and for reverse-transcriptase PCR (RT-PCR) and real-time PCR measurements. Treatment with GJ decreased significantly the severity of pancreatitis and pancreatitis-associated lung injury. Treatment with GJ attenuated the severity of AP compared with saline-treated mice, as shown by reduction in pancreatic edema, neutrophil infiltration, serum amylase and lipase levels, serum cytokine levels, and mRNA expression of multiple inflammatory mediators. These results suggest that GJ attenuated the severity of AP as well as pancreatitis-associated lung injury.

C/EBP homologous protein is crucial for the acceleration of experimental pancreatitis

C/EBP homologous protein (CHOP) is one of the main mediating factors in the ER stress pathway. To elucidate the role of the ER stress-CHOP pathway in experimental pancreatitis, wild-type ( Chop +/+ ) and Chop deficient ( Chop −/− ) mice were administered cerulein, a cholecystokinin analogue, or both cerulein and lipopolysaccharide (LPS). In cerulein-induced acute pancreatitis, ER stress, serum amylase elevation and histological interstitial edema were induced. However, there was no remarkable activation downstream of the CHOP pathway regardless of the presence or absence of CHOP. Whereas, in the cerulein and LPS model, inflammation-associated caspases (caspase-11, caspase-1) and IL-1β, but not apoptosis-associated caspases, were activated. In Chop −/− mice, the expression levels of these mediators returned to basal levels resulting in a milder pancreatitis and decreased serum amylase level. These results indicated that the ER stress-CHOP pathway has a pivotal role in the acceleration of pancreatitis through the induction of inflammation-associated caspases and IL-1β.

Differential anti-inflammatory properties of peroxisome proliferator-activated receptors (PPAR) α and γ in experimental pancreatitis

Perfluorooctanoic acid (PFOA) has anti-inflammatory effects in models of cutaneous inflammation, possibly via activation of PPAR-α and PPAR-γ. We have therefore investigated whether PFOA has similar effects in a model of acute oedematous pancreatitis and whether such effects could be explained by agonism at PPAR-α or PPAR-γ. Acute pancreatitis was induced in anesthetized rats by i.v. infusion of the cholecystokinin analogue, caerulein. The PPAR-α agonist clofibrate or the PPAR-γ agonist rosiglitazone were injected s.c. before the experiments. Pancreatic oedema, neutrophil activation and production of prostaglandin (PG) E2 and prostacyclin (via 6-keto-PGF1α) were assessed in the pancreas. Acute pancreatitis caused significant oedema formation, neutrophil activation as assessed by myeloperoxidase activity in the tissue, and increased synthesis of pro-inflammatory prostanoids. Neutrophil activation was unaffected by clofibrate but was abolished by rosiglitazone. In contrast, prostanoid synthesis was unaffected by rosiglitazone but was inhibited by clofibrate. In conclusion, our data demonstrate that activation of PPAR-α and PPAR-γ has differential anti-inflammatory effects in acute interstitial-oedematous pancreatitis. Neutrophil activation is sensitive to inhibition by PPAR-γ activation while the production of pro-inflammatory prostanoids can be attenuated by activation of PPAR-α. Thus, the anti-inflammatory potential of PPAR-α and PPAR-γ ligands should be further investigated. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Renal uptake and retention of radiolabeled somatostatin, bombesin, neurotensin, minigastrin and CCK analogues: species and gender differences

During therapy with radiolabeled peptides, the kidney is most often the critical organ. Newly developed peptides are evaluated preclinically in different animal models before their application in humans. In this study, the renal retention of several radiolabeled peptides was compared in male and female rats and mice. After intravenous injection of radiolabeled peptides [somatostatin, cholecystokinin (CCK), minigastrin, bombesin and neurotensin analogues], renal uptake was determined in both male and female Lewis rats and C57Bl mice. In addition, ex vivo autoradiography of renal sections was performed to localize accumulated radioactivity. An equal distribution pattern of renal radioactivity was found for all peptides: high accumulation in the cortex, lower accumulation in the outer medulla and no radioactivity in the inner medulla of the kidneys. In both male rats and mice, an increasing renal uptake was found: [(111)In-DTPA]CCK8<[(111)In-DTPA-Pro(1),Tyr(4)]bombesin approximately [(111)In-DTPA]neurotensin<[(111)In-DTPA]octreotide<<[(111)In-DTPA]MG0. Renal uptake of [(111)In-DTPA]octreotide in rats showed no gender difference, and renal radioactivity was about constant over time. In mice, however, renal uptake in females was significantly higher than that in males and decreased rapidly over time in both genders. Moreover, renal radioactivity in female mice injected with [(111)In-DTPA]octreotide showed a different localization pattern. Regarding the renal uptake of different radiolabeled peptides, both species showed the same ranking order. Similar to findings in patients, rats showed comparable and constant renal retention of radioactivity in both genders, in contrast to mice. Therefore, rats appear to be the more favorable species for the study of the renal retention of radioactivity.

Effect of compound GB-115 on morphine-induced analgesia

Experiments on outbred mice showed that compound GB-115, a retropeptide analogue of the tetrapeptide cholecystokinin, produced a naloxone-dependent potentiating effect on morphine-induced analgesia in the hot-plate test, but did not modulate animal behavior in the tail-flick test in outbred mice. This potentiation of antinociceptive activity of morphine was probably related to the interaction of GB-115 with supraspinal opioidergic mechanisms.

Ethanol induces secretion of oxidized proteins by pancreatic acinar cells

The pancreas is vulnerable to ethanol toxicity, but the pathogenesis of alcoholic pancreatitis is not fully defined. The intracellular oxidative balance and the characteristics of the secretion of isolated rat pancreatic acinar cells stimulated with the cholecystokinin analogue cerulein were assayed after acute oral ethanol (4 g/kg) load. Pancreatic acinar cells from ethanol-treated rats showed a significant (p < 0.02) lower content of total glutathione and protein sulfhydryls, and higher levels of oxidized glutathione (p < 0.03), malondialdehyde, and protein carbonyls (p < 0.05). Ethanol-intoxicated acinar cells showed a lower baseline amylase output compared to controls, with the difference being significantly exacerbated by cerulein stimulation. After cerulein, the release of protein carbonyls by ethanol-treated cells was significantly increased, whereas that of protein sulfhydryls was significantly decreased. In conclusion, ethanol oxidatively damages pancreatic acinar cells; cerulein stimulation is followed by a lower output of amylase and by a higher release of oxidized proteins by pancreatic acinar cells from ethanol-treated rats. These findings may account for the decreased exocrine function, intraductular plug formation, and protein precipitation in alcoholic pancreatitis.

Diphenyleneiodonium suppresses apoptosis in cerulein-stimulated pancreatic acinar cells

NADPH oxidase has been considered a major source of reactive oxygen species in phagocytic and non-phagocytic cells. Apoptosis linked to oxidative stress has been implicated in pancreatitis. Recently, we demonstrated that NADPH oxidase subunits Nox1, p27phox, p47phox, and p67phox are constitutively expressed in pancreatic acinar cells, which are activated by cerulein, a cholecystokinin analogue. Cerulein induces an acute and edematous form of pancreatitis. We investigated whether inhibition of NADPH oxidase by diphenyleneiodonium suppresses the production of reactive oxygen species and apoptosis by determining viable cell numbers, DNA fragmentation, TUNEL staining, caspase-3 activity, and the expression of apoptosis-inducing factor in pancreatic acinar AR42J cells stimulated with cerulein. Inhibition on NADPH oxidase by diphenyleneiodonium was assessed by the alterations in NADPH oxidase activity and translocation of the cytosolic subunits p67phox and p47phox to the membrane. Intracellular Ca2+ level was monitored to investigate the relationship between NADPH oxidase and Ca2+ in cells stimulated with cerulein. As a result, cerulein induced the activation of NADPH, increased production of reactive oxygen species, and apoptotic indices determined by the expression of apoptosis-inducing factor, caspase-3 activation, TUNEL staining, DNA fragmentation, and cell viability. Treatment with DPI inhibited cerulein-induced activation of NADPH oxidase, the production of reactive oxygen species, and apoptosis, but not the increase of intracellular Ca2+ levels in pancreatic acinar cells. These results demonstrate that the cerulein-induced increase in intracellular Ca2+ level may be an upstream event of NADPH oxidase activation. Diphenyleneiodonium, an NADPH oxidase inhibitor, inhibits the expression of apoptosis-inducing factor and caspase-3 activation, and thus apoptosis in pancreatic acinar cells.

In Vitro Evidence for Role of ERK, p38, and JNK in Exocrine Pancreatic Cytokine Production

Elucidation of mechanisms of acinar cell cytokine production is essential for a better understanding of acute pancreatitis pathogenesis. We hypothesize that the stress kinases ERK, p38, and JNK play an important role in acinar cell cytokine production. Rat pancreatic fragments were incubated with 100 nM concentration of the cholecystokinin analog caerulein or 100 nM caerulein and specific ERK inhibitor (100 microM PD98059), specific p38 inhibitor (10 microM SB203580), or specific JNK inhibitor (20 microM SP600125). After 3 hours of caerulein treatment, pancreatic fragments were homogenized and assayed for total and phosphorylated ERK, p38, and JNK, and for tumor necrosis factor-alpha or interleukin-1beta concentrations (ELISA). Pancreatic fragments stimulated with caerulein showed activation of ERK, p38, and JNK and increased cytokine concentrations (ANOVA, P<0.05). Specific stress kinase inhibitors significantly attenuated caerulein-induced activation of the corresponding stress kinase and cytokine production; however, the effect of the JNK inhibitor was comparatively less convincing. Increased activation of ERK, p38, and JNK in pancreatic fragments was not associated with significant increases in total ERK, total p38, or total JNK concentrations. The stress kinases ERK and p38 play an important role in caerulein-stimulated exocrine pancreatic overproduction of cytokines. The role of JNK needs further evaluation in this experimental model.

NEUTRALIZING IL-10 REVERSES THE PROTECTION AGAINST ACUTE PANCREATITIS BY CRAMBENE

Acute pancreatitis is a disease of variable severity in which some patients experience mild, self-limited attacks while others manifest a severe, highly morbid, and frequently lethal attack. It has earlier been shown that mild acute pancreatitis is associated with extensive apoptotic acinar cell death while severe acute pancreatitis involves extensive acinar cell necrosis but very little acinar cell apoptosis. We have earlier shown that induction of the pancreatic acinar cell apoptosis in vivo by crambene (1-cyano-2-hydroxy-3-butene - CHB), a plant nitrile, protects mice against acute necrotizing pancreatitis. In the current study, we report a role for IL-10, an anti-inflammatory mediator, as part of the mechanism by which acinar cell apoptosis induced by CHB protects against acute pancreatitis. Acute pancreatitis was induced in the mouse by administering hourly intraperitoneal injections of a supra maximally stimulating dose (50 μg/kg) of the cholecystokinin analog caerulein for 10 hours. Neutralizing monoclonal anti-IL-10 antibody (2.5 mg/kg) was administered either with or without crambene (70 mg/kg) 12 hours before the first caerulein injection. Severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (MPO), water content, MCP-1 level, and morphological examination. Apoptosis in pancreatic sections was visualized by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling method. CHB administration to normal mice resulted in persistently increased IL-10 levels in pancreas from 18 to 48 hours. While the prophylactic treatment with crambene in the acute pancreatitis model significantly reduced the severity, as evidenced by a significant attenuation of hyperamylasemia, pancreatic edema and, pancreatic MCP-1 levels. Also there was histological evidence of diminished pancreatic injury. The biochemical effects were reversed by the administration of anti- IL-10 together with CHB. There was also was greatly reduced apoptosis and enhanced necrosis with inflammation in mice treated with anit-IL-10 and CHB together. Our study shows that IL-10 plays an important role in the protection against acute pancreatitis by CHB and suggests that anti-inflammatory pathways may be involved in the protective effect of acinar cell apoptosis against acute pancreatitis.

Anti-inflammatory Effects of PPAR-γ Agonists Directly Correlate With PPAR-γ Expression During Acute Pancreatitis

Peroxisome proliferator-activated receptors (PPARs) are ligand-inducible transcription factors that regulate cellular energy and lipid metabolism. PPAR-gamma agonists also have potent anti-inflammatory properties through down-regulation of early inflammatory response genes. The role of PPAR-gamma in acute pancreatitis has not been adequately examined. In this study, we determined the effect of PPAR-gamma agonists on the severity of pancreatitis and sought to correlate PPAR-gamma expression in pancreatic acinar cells and the severity of acute pancreatitis in vivo. Acute pancreatitis was induced in mice by hyperstimulation with the cholecystokinin analog, cerulein. PPAR-gamma agonists were administered by intraperitoneal injection 15-30 minutes before induction of pancreatitis (pretreatment) or at various times after induction of pancreatitis (treatment). Pancreata and serum were harvested over the course of 24 hours. Serum amylase activity and glucose levels were measured. Pancreata were used for histological evaluation as well as protein and mRNA analysis. Pretreatment of mice with the PPAR-gamma agonists 15-deoxy-Delta12, 14-prostaglandin J(2), or troglitazone significantly reduced the severity of pancreatitis in a dose-dependent manner. This reduction was indicated by reduced serum amylase activity and histological damage (leukocyte infiltration, vacuolization, and necrosis). Although cerulein decreased PPAR-gamma expression in the pancreas, pretreatment with agonists maintained PPAR-gamma expression early in acute pancreatitis. The expression of PPAR-gamma inversely correlated with pancreatitis severity and expression of the proinflammatory cytokines, interleukin-6, and tumor necrosis factor-alpha. Treatment with troglitazone after the induction of pancreatitis reduced serum amylase activity. The results suggest that PPAR-gamma plays a direct role in the inflammatory cascade during the early events of acute pancreatitis. Our data are the first to demonstrate that PPAR-gamma agonists represent a promising therapeutic strategy for acute pancreatitis.

Liver Disease in Dogs with Tracheal Collapse

Background: Hepatopathy in dogs with chronic respiratory diseases is poorly recognized. The aim of this study was to evaluate liver parameters alanine transferase, alkaline phosphatase, and glutamate dehydrogenase, as well as basal and stimulated bile acid concentration, in dogs with tracheal collapse. Hypothesis: Dogs with tracheal collapse have hepatopathy. Animals: 26 dogs with tracheal collapse. Materials and Methods: Gall bladder contraction was stimulated by intramuscular injection of a synthetic cholecystokinin analogue (ceruletide). Twelve healthy Beagle dogs and 30 dogs of various breeds investigated previously without evidence of hepatic, gastrointestinal, or respiratory diseases served as control. Amelioration of liver variables was assessed after stent implantation. Results: Twelve of 26 (46%) dogs had increased serum activity of 2 or more liver enzymes. Serum basal bile acid concentrations were high in 24 of 26 dogs. Twenty‐and 40‐minute stimulated bile acids were significantly higher in dogs with tracheal collapse (64.2 + 130.0/‐43.0 μmol/L and 82.6 + 164.0/‐57.1 μmol/L) compared to the control dogs (7.0 ± 3.6 μmol/L and 6.4 ± 3.5 μmol/L). All twelve dogs reevaluated after a median of 58 days (48–219 days) had a normal breathing pattern and significantly decreased 20 and 40 minutes stimulated bile acids (50.0 + 92.7/‐32.8 μmol/L, 52.8 + 97.67–34.3 μmol/L; P= .0043), whereas plasma liver enzyme activities were not significantly influenced. Conclusion and Clinical Importance: There was a significant hepatic dysfunction in the majority of dogs with a tracheal collapse. Liver function should be routinely assessed in dogs with severe respiratory disease.

Liver Disease in Dogs with Tracheal Collapse

Hepatopathy in dogs with chronic respiratory diseases is poorly recognized. The aim of this study was to evaluate liver parameters alanine transferase, alkaline phosphatase, and glutamate dehydrogenase, as well as basal and stimulated bile acid concentration, in dogs with tracheal collapse. Dogs with tracheal collapse have hepatopathy. 26 dogs with tracheal collapse. Gall bladder contraction was stimulated by intramuscular injection of a synthetic cholecystokinin analogue (ceruletide). Twelve healthy Beagle dogs and 30 dogs of various breeds investigated previously without evidence of hepatic, gastrointestinal, or respiratory diseases served as control. Amelioration of liver variables was assessed after stent implantation. Twelve of 26 (46%) dogs had increased serum activity of 2 or more liver enzymes. Serum basal bile acid concentrations were high in 24 of 26 dogs. Twenty- and 40-minute stimulated bile acids were significantly higher in dogs with tracheal collapse (64.2 +130.0/-43.0 micromol/L and 82.6 +164.0/-57.1 micromol/L) compared to the control dogs (7.0 +/- 3.6 micromol/L and 6.4 +/- 3.5 micromol/L). All twelve dogs reevaluated after a median of 58 days (48-219 days) had a normal breathing pattern and significantly decreased 20 and 40 minutes stimulated bile acids (50.0 +92.7/-32.8 micromol/L, 52.8 +97.6/-34.3 micromol/L; P = .0043), whereas plasma liver enzyme activities were not significantly influenced. There was a significant hepatic dysfunction in the majority of dogs with a tracheal collapse. Liver function should be routinely assessed in dogs with severe respiratory disease.

A Proinflammatory, Antiapoptotic Phenotype Underlies the Susceptibility to Acute Pancreatitis in Cystic Fibrosis Transmembrane Regulator (−/−) Mice

A Proinflammatory, Antiapoptotic Phenotype Underlies the Susceptibility to Acute Pancreatitis in Cystic Fibrosis Transmembrane Regulator (−/−) Mice

Role of NAADP and cADPR in the Induction and Maintenance of Agonist-Evoked Ca2+ Spiking in Mouse Pancreatic Acinar Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate ribose (cADPR) were first demonstrated to mobilize Ca2+ in sea urchin eggs. In the absence of direct measurements of these messengers, pharmacological studies alone have implicated these molecules as intracellular second messengers for specific cell surface receptor agonists. We now report that in mouse pancreatic acinar cells, cholecystokinin, but not acetylcholine, evokes rapid and transient increases in NAADP levels in a concentration-dependent manner. In contrast, both cholecystokinin and acetylcholine-mediated production of cADPR followed a very different time course. The rapid and transient production of NAADP evoked by cholecystokinin precedes the onset of the Ca2+ signal and is consistent with a role for NAADP in the initiation of the Ca2+ response. Continued agonist-evoked Ca2+ spiking is maintained by prolonged elevations of cADPR levels through sensitization of Ca2+ -induced Ca2+ -release channels. This study represents the first direct comparison of NAADP and cADPR measurements, and the profound differences observed in their time courses provide evidence in support of distinct roles of these Ca2+ -mobilizing messengers in shaping specific Ca2+ signals during agonist stimulation.

THE ABSENCE OF AN EXOCRINE SPECIFIC TRANSCRIPTION FACTOR, MIST1, LEADS TO INCREASED SEVERITY IN SECRETAGOGUE INDUCED PANCREATITIS.

Gallstone migration into the common duct and alcohol abuse are the most common causes of pancreatitis. There is also evidence for genetic basis for the susceptibility to the disease. Unfortunately, with the exception of the cationic trypsinogen gene and SPINK, there is a lack of knowledge of the factors leading to increased disease susceptibility and severity. In an effort to link factors that regulate pancreatic development and function to disease susceptibility, we have examined of the role of Mist1 in pancreatitis. Mist1 is a basic helix-loop-helix (bHLH) transcription factor, highly expressed in acinar cells and the absence of Mist1 in mice (Mist1KO) results in incomplete maturation of acinar cells. In addition, Mist1KO mice exhibit premature enzyme activation and an increased presence of stellate cells, which are hallmarks of pancreatitis yet these mice do not develop the disease. We hypothesize that Mist1KO mice will develop more severe pancreatitis than wild type litter mates. To address this hypothesis, we have injected supramaximal amounts of caerulein, a cholecystokinin analogue. Analysis of serum following initiation of pancreatitis reveal higher levels of amylase in Mist1KO mice suggesting a more severe form of pancreatitis. Histological analysis confirms this suggestion as Mist1KO pancreatic tissue exhibits a progressive degeneration characterized by distended duct formation, vacuolization, pronounced fibrosis and a decrease in cells undergoing apoptosis. To identify additional factors that may play a role in disease susceptibility, widespread analysis by gene microarray was used to compare the molecular profile of wild type and Mist1KO mice. We have identified a number of genes that are differentially expressed in wild type and Mist1KO mice that may be linked to either protection or severity to pancreatitis. Current studies are underway to determine the importance of these factors in the disease process and their potential regulation by Mist1. Our results indicate that the transcription factor, Mist1 plays a role in dictating the severity of pancreatitis making the Mist1KO mouse a good model for studying the factors involved in the modulation of the disease process.

Disruption of paracellular sealing is an early event in acute caerulein-pancreatitis.

Caerulein-induced pancreatitis is a widely used experimental model for studies on acute pancreatitis, however, the molecular mechanisms underlying pancreatitis in response to caerulein hyperstimulation are incompletely understood. We therefore studied early effects of caerulein on tight junctional integrity. Mice were injected with the cholecystokinin analogue caerulein (50microg/kg BW/h) to induce pancreatitis. In pancreatic tissue occludin, claudin 1, zonula occludens protein 1 (ZO-1) were stained immunohistochemically and F-actin was visualized with phalloidin-TRITC. Stained sections and isolated acini were studied by confocal laser scanning microscopy. Under control conditions occludin, claudin1, ZO-1, and F-actin showed a linear staining pattern delineating the apical membranes of intralobular duct cells and of acinar cells. While in vitro caerulein hyperstimulation induced within 10 minutes disassembly of both occludin and ZO-1, in vivo caerulein hyperstimulation induced disassembly of occludin and claudin1 but not of ZO-1 from the tight junctions. Subsequent progressive disruption of ZO-1 was detected in a time dependent manner. Disruption of the transmembrane tight junction proteins occludin and claudin1 is an early event of caerulein hyperstimulation and may allow evasion of noxious luminal content into the interstitium, which may augment edema formation in acute pancreatitis.

Expression of galectin-3 in the rat pancreas during regeneration following hormone-induced pancreatitis.

Supramaximal dosage of the cholecystokinin analog caerulein leads to edematous pancreatitis with subsequent acinar cell destruction predominantly by apoptosis. We have used immunohistochemistry to reveal the expression of the anti-apoptotic protein galectin-3 in pancreatic acinar cells. Galectin-3, which occurs only in duct cells under physiological conditions, is expressed in a subset of acinar cells after the end of a 12-h caerulein infusion, giving rise to a "patchy" staining pattern. During the subsequent period of inflammation and regeneration, galectin-3 expression increases in those acinar cells that undergo apoptosis. By 48 h after the end of caerulein infusion, morphologically normal cells do not contain galectin-3 and participate in regeneration by proliferation. Tubular complexes, being transient structures from degenerative acini, accumulate galectin-3 in the remnants of the epithelium cells. Stimulation with supramaximal dosages of caerulein of the cell line AR4-2J, which is derived from rat pancreatic acinar cells, also results in a marked increase of galectin-3, confirming the in vivo results. We postulate that the high expression of the anti-apoptotic protein galectin-3 regulates the time course of the apoptotic process in pancreatic acinar cells.

Peptide YY exhibits a mitogenic effect on pancreatic cells while improving acute pancreatitis in vitro

Background Peptide YY (PYY), a gastrointestinal regulatory peptide, improves survival and histologic parameters in animal models of acute pancreatitis. Its effects on pancreatic cell growth and acute pancreatitis in pancreatic acinar and ductal cells are unknown. We hypothesized that PYY would affect cell growth and attenuate acute pancreatitis in pancreatic acinar and ductal cells in vitro. Methods Rat pancreatic acinar and ductal cells were cultured in the presence of 1) cerulein, a synthetic cholecystokinin analog that induces pancreatitis, 2) PYY, or 3) a combination group pretreated with PYY prior to addition of cerulein. Cell survival was measured at 48 h using MTT assay. Amylase secretion, as marker for pancreatitis, was measured at 48 h using an amylase activity assay. Statistical significance was calculated using analysis of variance and the Student’s t test. Results Peptide yy significantly increased cell growth and decreased amylase secretion compared with control and cerulein groups. Pretreatment with PYY significantly protected against the pancreatitis effects of cerulein. Conclusions We have shown for the first time that PYY has a mitogenic effect on pancreatic acinar and ductal cells in vitro. In addition, it directly protects against cerulein-induced pancreatitis. Its potential therapeutic benefit in acute pancreatitis would therefore be twofold: amelioration of the inflammatory process, and augmenting growth of normal pancreas to replace necrotic or apoptotic cell loss.