Alcohols enhance caerulein-induced zymogen activation in pancreatic acinar cells

Activation of zymogens within the pancreatic acinar cell is an early feature of acute pancreatitis. Supraphysiological concentrations of cholecystokinin (CCK) cause zymogen activation and pancreatitis. The effects of the CCK analog, caerulein, and alcohol on trypsin and chymotrypsin activation in isolated pancreatic acini were examined. Caerulein increased markers of zymogen activation in a time- and concentration-dependent manner. Notably, trypsin activity reached a peak value within 30 min, then diminished with time, whereas chymotrypsin activity increased with time. Ethanol (35 mM) sensitized the acinar cells to the effects of caerulein (10(-10) to 10(-7) M) on zymogen activation but had no effect alone. The effects of ethanol were concentration dependent. Alcohols with a chain length of >or=2 also sensitized the acinar cell to caerulein; the most potent was butanol. Branched alcohols (2-propanol and 2-butanol) were less potent than aliphatic alcohols (1-propanol and 1-butanol). The structure of an alcohol is related to its ability to sensitize acinar cells to the effects of caerulein on zymogen activation.     

PAR2 exerts local protection against acute pancreatitis via modulation of MAP kinase and MAP kinase phosphatase signaling

During acute pancreatitis, protease-activated receptor 2 (PAR2) can be activated by interstitially released trypsin. In the mild form of pancreatitis, PAR2 activation exerts local protection against intrapancreatic damage, whereas, in the severe form of pancreatitis, PAR2 activation mediates some systemic complications. This study aimed to identify the molecular mechanisms of PAR2-mediated protective effects against intrapancreatic damage. A mild form of acute pancreatitis was induced by an intraperitoneal injection of caerulein (40 microg/kg) in rats. Effects of PAR2 activation on intrapancreatic damage and on mitogen-activated protein (MAP) kinase signaling were assessed. Caerulein treatment activated extracellular signal-regulated kinase (ERK) and c-Jun NH(2)-terminal kinase (JNK) within 15 min and maintained phosphorylation of ERK and JNK for 2 h in the rat pancreas. Although PAR2 activation by the pretreatment with PAR2-activating peptide (AP) itself increased ERK phosphorylation in rat pancreas, the same treatment remarkably decreased caerulein-induced activation of ERK and JNK principally by accelerating their dephosphorylation. Inhibition of ERK and JNK phosphorylation by the pretreatment with MAP/ERK kinase (MEK) or JNK inhibitors decreased caerulein-induced pancreatic damage that was similar to the effect induced by PAR2-AP. Notably, in caerulein-treated rats, PAR2-AP cotreatment highly increased the expression of a group of MAP kinase phosphatases (MKPs) that deactivate ERK and JNK. The above results imply that downregulation of MAP kinase signaling by MKP induction is a key mechanism involved in the protective effects of PAR2 activation on caerulein-induced intrapancreatic damage.     

The proteinase inhibitor camostat mesilate suppresses pancreatic pain in rodents

Camostat mesilate, an orally available proteinase inhibitor, is clinically used for treatment of pancreatitis. Given recent evidence that pancreatic proteinases including trypsin and/or proteinase-activated receptor-2 (PAR2) might be involved in pancreatic pain, we examined if camostat mesilate could suppress spinal Fos expression, a marker for neuronal activation, following specific application of trypsin to the pancreas, and pancreatitis-related referred allodynia. Trypsin, administered into the pancreatic duct, caused delayed expression of Fos proteins in the superficial layer of the bilateral T8 and T9 spinal dorsal horns in rats. The trypsin-induced spinal Fos expression was completely abolished by oral pre-administration of camostat mesilate at 300 mg/kg. After hourly repeated (6 times in total) administration of caerulein, mice showed typical symptoms of pancreatitis, accompanied by mechanical allodynia in the upper abdomen (i.e., referred hyperalgesia/allodynia), as assessed by use of von Frey filaments. Camostat mesilate at 100-300 mg/kg, given orally twice before the 1st and 4th doses of caerulein, abolished the pancreatitis-related abdominal allodynia, while it partially prevented the inflammatory signs. The same doses of camostat mesilate, when administered once after the final dose of caerulein, also revealed significant anti-allodynic effect. These data suggest that camostat mesilate prevents and/or depresses pancreatitis-induced pain and/or referred hyperalgesia/allodynia, in which proteinases including trypsin would play a critical role.     

Vacuolar ATPase regulates zymogen activation in pancreatic acini

Supramaximal concentrations of cholecystokinin or its analogue caerulein have been shown to stimulate the proteolytic activation of zymogens within the pancreatic acinar cell and initiate acute pancreatitis. Previous studies suggest that a low pH compartment might be required for activation. To test this hypothesis, the effects of agents that modulate intracellular pH on caerulein-induced trypsin and chymotrypsin activation were studied. Pretreatment of pancreatic acini with the proto-ionophore monensin (10 microM) and the weak base chloroquine (40 microM) inhibited activation. Pre-incubation with the vacuolar ATPase (V-ATPase) inhibitors bafilomycin A(1) and concanamycin A also decreased activation in a concentration-dependent manner with 50% inhibition at approximately 50 and 25 nM, respectively. Caerulein stimulation caused a time- and concentration-dependent translocation of soluble V-ATPase V(1) subunits to a membrane fraction, a marker of V-ATPase activation. Carbachol also stimulated translocation at supramaximal concentrations. Elevation of cytosolic Ca(2+) by thapsigargin was sufficient to induce translocation. Thus, stimulation of V-ATPase activity appears to be required for agonist-induced zymogen activation in the pancreatic acinar cell.     

Secretin differentially sensitizes rat pancreatic acini to the effects of supramaximal stimulation with caerulein

Supramaximal stimulation of the rat pancreas with CCK, or its analog caerulein, triggers acute pancreatitis and a number of pancreatitis-associated acinar cell changes including intracellular activation of digestive enzyme zymogens and acinar cell injury. It is generally believed that some of these various acinar cell responses to supramaximal secretagogue stimulation are interrelated and interdependent. In a recent report, Lu et al. showed that secretin, by causing generation of cAMP and activation of PKA, sensitizes acinar cells to secretagogue-induced zymogen activation, and, as a result, submaximally stimulating concentrations of caerulein can, in the presence of secretin, trigger intracellular zymogen activation. We found that secretin also sensitizes acinar cells to secretagogue-induced cell injury and to subapical F-actin redistribution but that it did not alter the caerulein concentration dependence of other pancreatitis-associated changes such as the induction of a peak plateau intracellular [Ca(2+)] rise, inhibition of secretion, activation of ERK1/2, and activation of NF-kappaB. The finding that secretin sensitizes acinar cells to both intracellular zymogen activation and cell injury is consistent with the concept that these two early events in pancreatitis are closely interrelated and, possibly, interdependent. On the other hand, the finding that, in the presence of secretin, caerulein can trigger subapical F-actin redistribution without inhibiting secretion challenges the concept that disruption of the subapical F-actin web is causally related to high-dose secretagogue-induced inhibition of secretion in pancreatic acinar cells.     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

The novel protein kinase C isoforms -delta and -epsilon modulate caerulein-induced zymogen activation in pancreatic acinar cells

Isoforms of protein kinase C (PKC) have been shown to modulate some cellular responses such as pathological secretion and generation of inflammatory mediators during acute pancreatitis (AP). We propose that PKC also participates in premature zymogen activation within the pancreatic acinar cell, a key event in the initiation of AP. This hypothesis was examined in in vivo and cellular models of caerulein-induced AP using PKC activators and inhibitors. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA, 200 nM), a known activator of PKC, enhanced zymogen activation at both 0.1 nM and 100 nM caerulein, concentrations which mimic physiological and supraphysiological effects of the hormone cholecystokinin, respectively, in preparations of pancreatic acinar cells. Isoform-specific PKC inhibitors for PKC-delta and PKC-epsilon reduced supraphysiological caerulein-induced zymogen activation. Using a cell-free reconstitution system, we showed that inhibition of PKC-delta and -epsilon, reduced zymogen activation in both zymogen granule-enriched and microsomal fractions. In dispersed acinar cells, 100 nM caerulein stimulation caused PKC-delta and -epsilon isoform translocation to microsomal membranes using cell fractionation and immunoblot analysis. PKC translocation was confirmed with in vivo studies and immunofluorescence microscopy in pancreatic tissues from rats treated with or without 100 nM caerulein. PKC-epsilon redistributed from an apical to a supranuclear region following caerulein administration. The signal for PKC-epsilon overlapped with granule membrane protein, GRAMP-92, an endosomal/lysosomal marker, in a supranuclear region where zymogen activation takes place. These results indicate that PKC-delta and -epsilon isoforms translocate to specific acinar cell compartments and modulate zymogen activation.     

Effect of ligands that increase cAMP on caerulein-induced zymogen activation in pancreatic acini

The pathological activation of proteases within the pancreatic acinar cell is critical to initiating pancreatitis. Stimulation of acinar cells with supraphysiological concentrations of the CCK analog caerulein (CER) leads to protease activation and pancreatitis. Agents that sensitize the acinar cell to the effects of CCK might contribute to disease. The effects of physiological ligands that increase acinar cell cAMP [secretin, VIP, and pituitary adenylate cyclase activating peptide (PACAP)] on CER-induced responses were examined in isolated rat pancreatic acini. Each ligand sensitized the acinar cell to zymogen activation by physiological concentrations of CER (0.1 nM). VIP and PACAP but not secretin also enhanced activation by supraphysiological concentrations of CER (0.1 muM). A cell-permeable cAMP analog also sensitized the acinar cell to CER-induced activation. The cAMP antagonist Rp-8-Br-cAMP inhibited these sensitizing effects. These findings suggest that ligands that increase acinar cell cAMP levels can sensitize the acinar cell to the effects of CCK-induced zymogen activation.     

Dynamics of pancreatic tissue cells in the rat exposed to long-term caerulein treatment. 2. Comparative analysis of the various cell types and their growth

This study was undertaken to evaluate the effects of caerulein, a CCK analog, on the different cell populations of the pancreatic tissue and their respective turnover. Rats received saline or caerulein subcutaneously and 3H-thymidine intraperitoneally three times a day for 4 days. They were sacrificed immediately after termination of treatment and 2, 15 and 50 days later. With age, the proportion of acinar cells decreased significantly whereas those of the ductal and interstitial cells increased. Although caerulein induced preferential acinar cell growth, it did not modify the proportion of this cell population with regard to the other cells. However, at specific times after termination of treatment, caerulein induced modifications in the ductal, endothelial and interstitial cell populations. The growth promoting effect of caerulein was evident from the specific increases in total DNA content and DNA synthesis. The labeling indices indicate that all cell populations except the endocrine system were stimulated to grow in response to caerulein. Furthermore, all new cells remained for at least 50 days after termination of treatment. These data indicate that caerulein induced uniform growth of the pancreatic tissue during intensive treatment. The normal growth rate of these stimulated cells was, however, arrested for the following 50 days while that of the control group cell population proceeded normally.     

Relationship between NF-kappaB and trypsinogen activation in rat pancreas after supramaximal caerulein stimulation

Intra-acinar cell nuclear factor-kappaB (NF-kappaB) and trypsinogen activation are early events in secretagogue-induced acute pancreatitis. We have studied the relationship between NF-kappaB and trypsinogen activation in rat pancreas. CCK analogue caerulein induces early (within 15 min) parallel activation of both NF-kappaB and trypsinogen in pancreas in vivo as well as in pancreatic acini in vitro. However, NF-kappaB activation can be induced without trypsinogen activation by lipopolysaccharide in pancreas in vivo and by phorbol ester in pancreatic acini in vitro. Stimulation of acini with caerulein after 6 h of culture results in NF-kappaB but not trypsinogen activation. Protease inhibitors (AEBSF, TLCK, and E64d) inhibit both intracellular trypsin activity and NF-kappaB activation in caerulein stimulated acini. A chymotrypsin inhibitor (TPCK) inhibits NF-kappaB activation but not trypsin activity. The proteasome inhibitor MG-132 prevents caerulein-induced NF-kappaB activation but does not prevent trypsinogen activation. These findings indicate that although caerulein-induced NF-kappaB and trypsinogen activation are temporally closely related, they are independent events in pancreatic acinar cells. NF-kappaB activation per se is not required for the development of early acinar cell injury by supramaximal secretagogue stimulation.     

Pharmacological and genetic inhibition of calcineurin protects against carbachol-induced pathological zymogen activation and acinar cell injury

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.     

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.     

Bile Acids Induce Pancreatic Acinar Cell Injury and Pancreatitis by Activating Calcineurin

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca²⁺. Thus, it would be clinically relevant to know the targets of this aberrant Ca²⁺ signal. We hypothesized that the Ca²⁺-activated phosphatase calcineurin is such a Ca²⁺ target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca²⁺. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca²⁺ generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.     

Ghrelin attenuates the development of acute pancreatitis in rat.

BACKGROUND: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. METHODS: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. RESULTS: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. CONCLUSIONS: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems Background: Ghrelin, a circulating growth hormone-releasing peptide isolated from human and rat stomach, stimulates growth hormone secretion, food intake and exhibits gastroprotective properties. Ghrelin is predominantly produced by a population of endocrine cells in the gastric mucosa, but its presence in bowel, pancreas, pituitary and hypothalamus has been reported. In human fetal pancreas, ghrelin is expressed in a prominent endocrine cell population. In adult pancreatic islets the population of these cell is reduced. The aim of present study was to investigate the influence of ghrelin administration on the development of acute pancreatitis. Methods: Acute pancreatitis was induced in rat by caerulein injection. Ghrelin was administrated twice (30 min prior to the first caerulein or saline injection and 3 h later) at the doses: 2, 10 or 20 nmol/kg. Immediately after cessation of caerulein or saline injections the following parameters were measured: pancreatic blood flow, plasma lipase activity, plasma interleukin-1beta (IL-1beta) and interleukin 10 (IL-10) concentration, pancreatic DNA synthesis, and morphological signs of pancreatitis. Results: Administration of ghrelin without induction of pancreatitis did not affect significantly any parameter tested. Caerulein led to the development of acute edematous pancreatitis. Treatment with ghrelin at the dose 2 nmol/kg, during induction of pancreatitis, was without effect on pancreatic histology or biochemical and functional parameters. Treatment with ghrelin at the dose 10 and 20 nmol/kg attenuated the development of pancreatitis and the effects of both doses were similar. Administration of ghrelin (10 or 20 nmol/kg) reduced inflammatory infiltration of pancreatic tissue and vacuolization of acinar cells. Also, plasma lipase activity and plasma IL-1beta conc; concentration were reduced, and caerulein-induced fall in pancreatic DNA synthesis was reversed. Administration of ghrelin at the dose 10 and 20 nmol/kg was without effect on caerulein-induced pancreatic edema and pancreatitis-related fall in pancreatic blood flow. Conclusions: (1) Administration of ghrelin attenuates pancreatic damage in caerulein-induced pancreatitis; (2) Protective effect of ghrelin administration seems to be related the inhibition in inflammatory process and the reduction in liberation of pro-inflammatory IL-1beta.     

Cerulein hyperstimulation decreases AMP-activated protein kinase levels at the site of maximal zymogen activation

The premature activation of digestive enzyme zymogens in the pancreatic acinar cell is an important initiating event in acute pancreatitis. We have previously demonstrated that vacuolar ATPase (vATPase) activity is required for zymogen activation. Adenosine monophosphate-activated protein kinase (AMPK) regulates vATPase function in kidney and epididymal clear cells. To determine whether AMPK could affect pancreatitis responses, its effects were first examined in a cellular model of pancreatitis, cerulein-hyperstimulated (100 nM) pancreatic acini. This treatment caused a prominent increase in trypsin and chymotrypsin activities. Pretreatment with AICAR or metformin (AMPK activators) or compound C (an AMPK inhibitor) reduced or increased cerulein-induced zymogen activation, respectively. The association of the vATPase E subunit with membranes, a marker of its activation, tended to be inversely related to AMPK activity (assessed by AICAR and compound C treatments). Cerulein treatment did not change AMPK (α and β) levels but did lead to an increase in its activation (phosphorylation of Thr172) and induced the time-dependent translocation of the enzyme to a Triton-insoluble compartment. Basal in vivo studies showed that AMPK was widely distributed between membrane and soluble fractions generated by differential centrifugation. After cerulein hyperstimulation, AMPK levels selectively decreased in fractions containing the highest levels of active zymogens. These studies suggest that AMPK activity has a protective role in the pancreatic acinar cell that inhibits zymogen activation in the basal state, and this AMPK effect is reduced during pancreatitis. Therapies that prevent the selective reduction of AMPK in compartments that support zymogen activation could reduce injury during pancreatitis.     

Treatment with bindarit, a blocker of MCP-1 synthesis, protects mice against acute pancreatitis

Chemokines are believed to play a key role in the pathogenesis of acute pancreatitis. We have earlier shown that pancreatic acinar cells produce the chemokine monocyte chemotactic protein (MCP)-1 in response to caerulein hyperstimulation, demonstrating that acinar-derived MCP-1 is an early mediator of inflammation in acute pancreatitis. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of inflammatory diseases, such as acute pancreatitis. 2-Methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy]propanoic acid (bindarit) has been shown to preferentially inhibit MCP-1 production in vitro in monocytes and in vivo without affecting the production of the cytokines IL-1, IL-6, or the chemokines IL-8, protein macrophage inflammatory-1alpha, and RANTES. The present study aimed to define the role of MCP-1 in acute pancreatitis with the use of bindarit. In a model of acute pancreatitis induced by caerulein hyperstimulation, prophylactic as well as therapeutic treatment with bindarit significantly reduced MCP-1 levels in the pancreas. Also, this treatment significantly protected mice against acute pancreatitis as evident by attenuated hyperamylasemia neutrophil sequestration in the pancreas (pancreatic MPO activity), and pancreatic acinar cell injury/necrosis on histological examination of pancreas sections.     

CEP-1347 inhibits caerulein-induced rat pancreatic JNK activation and ameliorates caerulein pancreatitis

Pancreatic caerulein-induced activation of c-Jun NH(2)-terminal kinase (JNK) has been reported, and JNK has been proposed as a mediator during induction of hyperstimulated pancreatitis. CEP-1347 has recently been described as a specific JNK inhibitor. We tested whether CEP-1347 inhibits caerulein-induced pancreatic JNK activation in isolated acini and in vivo. CEP-1347 dose dependently inhibited acinar caerulein-induced JNK activation with nearly complete inhibition at 2 microM but had no effect on digestive enzyme release. For in vivo studies, rats were pretreated with CEP-1347 before caerulein hyperstimulation. For assessment of JNK activation and histological alterations, animals were killed 30 min or 2 and 4 h after caerulein hyperstimulation, respectively. Pancreatic wet weight, serum enzyme levels, and pancreatic activity of p38 and extracellular signal-regulated kinase (ERK) were also determined. Caerulein hyperstimulation strongly activated JNK, p38, and ERK. CEP-1347 pretreatment dose dependently reduced caerulein-induced pancreatic JNK activation without p38 or ERK inhibition. JNK inhibition also reduced pancreatic edema formation and reduced histological severity of pancreatitis. Thus we show that CEP-1347 inhibits JNK activation in vivo and ameliorates caerulein-induced pancreatitis.     

Autophagy and acute pancreatitis: a novel autophagy theory for trypsinogen activation

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. Although lysosomal hydrolases, such as cathepsin B, play a key role in intrapancreatic trypsinogen activation, it remains unclear where and how trypsinogen meets these lysosomal enzymes. Autophagy is an intracellular bulk degradation system in which cytoplasmic components are directed to the lysosome/vacuole by a membrane-mediated process. To analyze the role of autophagy in acute pancreatitis, we produced a conditional knockout mouse that lacks the autophagy-related (Atg) gene Atg5 in the pancreatic acinar cells. The severity of acute pancreatitis induced by cerulein is greatly reduced in these mice. In addition, Atg5-deficient acinar cells show a significantly decreased level of trypsinogen activation. These data suggest that autophagy exerts a detrimental effect in pancreatic acinar cells by activation of trypsinogen to trypsin. We propose a theory in which autophagy accelerates trypsinogen activation by lysosomal hydrolases under acidic conditions, thus triggering acute pancreatitis in its early stage.