cholecystokinin and Necrosis

Acute pancreatitis has many causes, all leading to a common pathway of changes within the pancreatic acinar cell. Key amongst these changes is premature intracellular activation of digestive enzymes but this is also accompanied by the appearance of cytosolic vacuoles, co-localization of digestive and lysosomal enzymes, activation of NF-kappaB, and release of pro-inflammatory cytokines. The exact mechanism responsible for enzyme activation remains the subject of much research effort and not a little debate, however it is clear that all of these changes are triggered by an abnormal, sustained rise in cytosolic calcium concentration, which is itself dependent both on release of calcium from endoplasmic reticulum stores and uptake from the extracellular milieu. Activated enzymes are directly damaging to the acinar cell themselves, but recruitment of circulating neutrophils leads to further cellular damage. Cytokines and neutrophil activation are also responsible for the systemic inflammatory response typically seen in severe acute pancreatitis.

Topics: Acute Disease; Apoptosis; Calcium; Cholecystokinin; Cytosol; Humans; Necrosis; Neutrophil Infiltration; Pancreas; Pancreatitis; Phosphatidylinositol 3-Kinases

Topics: Acute Disease; Animals; Apoptosis; Cholecystokinin; Cytoplasmic Granules; Enzyme Activation; Enzyme Precursors; Enzymes; Humans; Necrosis; Pancreatitis

The objective of this study was to test whether pyruvate and glutamine affect the ethanol and cholecystokinin (CCK) effects on the mitochondrial function, viability, and morphology of rat pancreatic acini.. Respiration was measured with Clark oxygen electrode. Mitochondrial membrane potential, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), cell morphology, and viability were studied with fluorescence microscopy.. In vitro, CCK (0.1 nM) caused pyruvate-dependent stimulation of basal and uncoupled respiration, and the effects were abolished by ethanol (20 mM). The combination of ethanol with CCK (2 hours) caused necrosis of approximately 40% acinar cells in medium with glucose, but not with pyruvate and/or glutamine. Cholecystokinin (10 nM) or ethanol with 0.1 nM CCK caused plasma membrane blebbing not related to apoptosis only when both glutamine and pyruvate were present. Glutamine, but not pyruvate, decreased NAD(P)H level and prevented the effects of ethanol with CCK on mitochondrial membrane potential and NAD(P)H, but, in combination with CCK and ethanol, decreased the uncoupled respiration. In vivo, the combination of ethanol (4 g/kg) and CCK (20 pmol/kg) suppressed basal and uncoupled respiration and caused acinar cell blebbing, but not necrosis.. The lack of sufficient substrate supply in vitro makes pancreatic acinar cells susceptible to necrosis caused by ethanol and CCK in clinically relevant concentrations.

Topics: Acinar Cells; Amylases; Animals; Cell Survival; Cells, Cultured; Cholecystokinin; Ethanol; Glutamine; Male; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Mitochondria; Necrosis; Oxidation-Reduction; Oxygen Consumption; Pancreas; Pyruvic Acid; Rats, Wistar

Acute pancreatitis is an inflammatory process of the pancreas and a leading cause of hospitalization amongst gastrointestinal disorders. Previously, cholecystokinin (CCK) has been described to play a role in regeneration of pancreas. The aim of this study was to analyse the function of cholecystokinin octapeptide (CCK-8) during induced pancreatitis in an animal model.. Overall acute pancreatitis was induced in 38 pigs. After the induction of acute pancreatitis, half of the animals were treated with CCK-8. Intraoperative clinical data, postoperative blood parameters, 'Porcine Well-being' (PWB) and fitness score and post-mortal histopathological data were analysed.. At baseline, physiologically parameters of the pigs of both groups were comparable. No differences were observed regarding the overall survival of animals (p = 0.97). Postoperative PWB score were significantly enhanced in animals treated with CCK-8 as compared to the control group (p = 0.029). Moreover, histopathological analysis of the pancreatic tissue revealed that acinar necrosis and edema were significant reduced in the CCK-8 group in comparison to the control group (p = 0.016 and p = 0.019).. In conclusion, we found that CCK-8 treatment reduces acinar necrosis and edema of pancreatic tissue after induction of an acute pancreatitis in pigs.

Topics: Animals; Cholecystokinin; Disease Models, Animal; Necrosis; Pancreas; Pancreatitis; Peptide Fragments; Swine

Pancreatic acinar cell necrosis and inflammatory responses are two key pathologic processes in acute pancreatitis (AP), which determines the severity and outcome of the disease. Recent studies suggest that necroptosis, a programed form of necrosis, is involved in the pathogenesis of AP, but the underlying mechanisms remain unknown. We investigated the expression of necrosome components, including receptor-interacting protein (RIP) 1, RIP3, and mixed lineage kinase domain-like (MLKL), and the molecular mechanisms in pancreatitis-associated necroptosis. We found that RIP3 and phosphorylated MLKL expression was positively related to the degree of necrosis, whereas RIP1 expression was negatively related to the degree of necrosis. Pharmacologic inhibition of RIP1 kinase activity exerted no protection against caerulein/cholecystokinin-8-induced AP, but knockdown of RIP1 with siRNA increased acinar cell necrosis and inhibition of NF-κB activation. RIP1 inhibition led to enhanced RIP3 expression. RIP3 and MLKL inhibition decreased acinar cell necrosis, in which the inhibition of RIP3 reduced the phosphorylation level of MLKL. RIP3 inhibition had no effect on trypsinogen activation but partly inhibited inflammasome activation. Our study strongly suggests that the imbalance between RIP1 and RIP3 shifts the cell death to necrosis, which unravels a new molecular pathogenesis of mechanism of AP and may provide insight into the development of novel therapeutic agent for other necrosis-related diseases.

Topics: Acinar Cells; Acute Disease; Animals; Apoptosis; Ceruletide; Cholecystokinin; Irritants; Male; Mice, Inbred BALB C; Mice, Inbred C57BL; Necrosis; Pancreatitis; Peptide Fragments; Phosphorylation; Protein Kinase Inhibitors; Rats, Sprague-Dawley; Receptor-Interacting Protein Serine-Threonine Kinases

In pancreatic acinar cells, the Src family of kinases (SFK) is involved in the activation of several signaling cascades that are implicated in mediating cellular processes (growth, cytoskeletal changes, apoptosis). However, the role of SFKs in various physiological responses such as enzyme secretion or in pathophysiological processes such as acute pancreatitis is either controversial, unknown, or incompletely understood. To address this, in this study, we investigated the role/mechanisms of SFKs in acute pancreatitis and enzyme release. Enzyme secretion was studied in rat dispersed pancreatic acini, in vitro acute-pancreatitis-like changes induced by supramaximal COOH-terminal octapeptide of cholecystokinin (CCK). SFK involvement assessed using the chemical SFK inhibitor (PP2) with its inactive control, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine (PP3), under experimental conditions, markedly inhibiting SFK activation. In CCK-stimulated pancreatic acinar cells, activation occurred of trypsinogen, various MAP kinases (p42/44, JNK), transcription factors (signal transducer and activator of transcription-3, nuclear factor-κB, activator protein-1), caspases (3, 8, and 9) inducing apoptosis, LDH release reflective of necrosis, and various chemokines secreted (monocyte chemotactic protein-1, macrophage inflammatory protein-1α, regulated on activation, normal T cell expressed and secreted). All were inhibited by PP2, not by PP3, except caspase activation leading to apoptosis, which was increased, and trypsin activation, which was unaffected, as was CCK-induced amylase release. These results demonstrate SFK activation is playing a dual role in acute pancreatitis, inhibiting apoptosis and promoting necrosis as well as chemokine/cytokine release inducing inflammation, leading to more severe disease, as well as not affecting secretion. Thus, our studies indicate that SFK is a key mediator of inflammation and pancreatic acinar cell death in acute pancreatitis, suggesting it could be a potential therapeutic target in acute pancreatitis.

Topics: Acinar Cells; Animals; Apoptosis; Caspases; Cells, Cultured; Chemokines; Cholecystokinin; Male; MAP Kinase Signaling System; Necrosis; Pancreatitis, Acute Necrotizing; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; src-Family Kinases; Transcription Factors; Trypsinogen

Although oxidative stress has been strongly implicated in the development of acute pancreatitis (AP), antioxidant therapy in patients has so far been discouraging. The aim of this study was to assess potential protective effects of a mitochondria-targeted antioxidant, MitoQ, in experimental AP using in vitro and in vivo approaches. MitoQ blocked H2O2-induced intracellular ROS responses in murine pancreatic acinar cells, an action not shared by the control analogue dTPP. MitoQ did not reduce mitochondrial depolarisation induced by either cholecystokinin (CCK) or bile acid TLCS, and at 10 µM caused depolarisation per se. Both MitoQ and dTPP increased basal and CCK-induced cell death in a plate-reader assay. In a TLCS-induced AP model MitoQ treatment was not protective. In AP induced by caerulein hyperstimulation (CER-AP), MitoQ exerted mixed effects. Thus, partial amelioration of histopathology scores was observed, actions shared by dTPP, but without reduction of the biochemical markers pancreatic trypsin or serum amylase. Interestingly, lung myeloperoxidase and interleukin-6 were concurrently increased by MitoQ in CER-AP. MitoQ caused biphasic effects on ROS production in isolated polymorphonuclear leukocytes, inhibiting an acute increase but elevating later levels. Our results suggest that MitoQ would be inappropriate for AP therapy, consistent with prior antioxidant evaluations in this disease.

Topics: Acinar Cells; Acute Disease; Animals; Antioxidants; Apoptosis; Ceruletide; Cholecystokinin; Disease Models, Animal; Inflammation; Male; Membrane Potential, Mitochondrial; Mice; Mitochondria; Necrosis; Organophosphorus Compounds; Oxidative Stress; Pancreas; Pancreatitis; Reactive Oxygen Species; Taurolithocholic Acid; Ubiquinone

To investigate chronic stress as a susceptibility factor for developing pancreatitis, as well as tumor necrosis factor-α (TNF-α) as a putative sensitizer.. Rat pancreatic acini were used to analyze the influence of TNF-α on submaximal (50 pmol/L) cholecystokinin (CCK) stimulation. Chronic restraint (4 h every day for 21 d) was used to evaluate the effects of submaximal (0.2 μg/kg per hour) cerulein stimulation on chronically stressed rats.. In vitro exposure of pancreatic acini to TNF-α disorganized the actin cytoskeleton. This was further increased by TNF-α/CCK treatment, which additionally reduced amylase secretion, and increased trypsin and nuclear factor-κB activities in a protein-kinase-C δ and ε-dependent manner. TNF-α/CCK also enhanced caspases' activity and lactate dehydrogenase release, induced ATP loss, and augmented the ADP/ATP ratio. In vivo, rats under chronic restraint exhibited elevated serum and pancreatic TNF-α levels. Serum, pancreatic, and lung inflammatory parameters, as well as caspases'activity in pancreatic and lung tissue, were substantially enhanced in stressed/cerulein-treated rats, which also experienced tissues' ATP loss and greater ADP/ATP ratios. Histological examination revealed that stressed/cerulein-treated animals developed abundant pancreatic and lung edema, hemorrhage and leukocyte infiltrate, and pancreatic necrosis. Pancreatitis severity was greatly decreased by treating animals with an anti-TNF-α-antibody, which diminished all inflammatory parameters, histopathological scores, and apoptotic/necrotic markers in stressed/cerulein-treated rats.. In rats, chronic stress increases susceptibility for developing pancreatitis, which involves TNF-α sensitization of pancreatic acinar cells to undergo injury by physiological cerulein stimulation.

Topics: Actins; Adenosine Diphosphate; Adenosine Triphosphate; Amylases; Animals; Antibodies; Calcium Signaling; Caspases; Ceruletide; Cholecystokinin; Chronic Disease; Cytoskeleton; Disease Models, Animal; Enzyme Activation; Lung Injury; Male; Necrosis; NF-kappa B; Pancreas, Exocrine; Pancreatitis; Protein Kinase C-delta; Protein Kinase C-epsilon; Protein Transport; Rats; Rats, Wistar; Restraint, Physical; Severity of Illness Index; Stress, Psychological; Tissue Culture Techniques; Trypsin; Tumor Necrosis Factor-alpha

The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([Ca(2+)](c)), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [Ca(2+)](c), which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [Ca(2+)](c) that declined to a plateau, which remained above the basal level 20 min after application and was dependent on external Ca(2+) entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [Ca(2+)](c) at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone.

Topics: Amylases; Animals; Calcium Signaling; Cell Differentiation; Cholecystokinin; Enzyme Precursors; Exocytosis; Humans; Membrane Potentials; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Mitochondria; Necrosis; Pancreas, Exocrine; Patch-Clamp Techniques; Peptide Fragments; Time Factors

Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.

Topics: Amylases; Animals; Apoptosis; Blotting, Western; Calcium; Caspase 3; Caspase 8; Caspase 9; Caspases; Cells, Cultured; Cholecystokinin; Cytochrome c Group; Cytosol; DNA; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Isoenzymes; L-Lactate Dehydrogenase; Mitochondria; Necrosis; Pancreas; Pancreatitis; Protein Binding; Protein Kinase C; Protein Kinase C-delta; Rats; Signal Transduction; Trypsin

In healthy rats, combined bile and pancreatic juice diversion from gut has a synergistic rather than additive effect on stimulation of exocrine pancreatic protein secretion. We hypothesized that exclusion of combined bile and pancreatic juice from gut exacerbates bile and pancreatic-duct ligation-induced acute pancreatitis in rats to a greater extent than exclusion of either bile or pancreatic juice alone.. Bile and pancreatic juice (obtained fresh from donor rats) were replaced, separately or together, via a duodenal fistula beginning immediately before 6 hours of duct ligation. Pancreatic morphologic changes were evaluated with an acute pancreatitis histology score and morphometric quantitation of acinar-cell necrosis. Plasma amylase and cholecystokinin concentrations and pancreatic subcellular distribution of cathepsin B activity were determined. Characteristics of bile and pancreatic juice obtained from donor rats were also studied.. Combined bile and pancreatic juice replacement limited the increase in acute pancreatitis histology score by 77%, acinar cell necrosis by 95%, hyperamylasemia by 77%, and hypercholecystokininemia by 99%, while preventing subcellular redistribution of cathepsin B. Amelioration of pancreatic morphologic changes was significantly greater with combined bile and pancreatic juice replacement than with replacement of either bile or pancreatic juice alone.. In this experimental corollary of early gallstone-induced acute pancreatitis, combined bile and pancreatic juice exclusion from gut contributes to disease pathogenesis to a greater extent than exclusion of either bile or pancreatic juice alone.

Topics: Acute Disease; Amylases; Animals; Bile; Cathepsin B; Cholecystokinin; Common Bile Duct; Duodenostomy; Infusion Pumps; Ligation; Male; Necrosis; Pancreas; Pancreatic Ducts; Pancreatic Juice; Pancreatitis; Rats; Rats, Sprague-Dawley

To determine whether a synthetic somatostatin analogue, octreotide, and a cholecystokinin receptor antagonist, L-364,718, may be beneficial in acute pancreatitis, 33 dogs were assigned to four groups. Each dog underwent laparotomy with injection of autologous bile into the dorsal pancreatic duct. Thirty minutes after the induction of pancreatitis, Group 1 received a subcutaneous injection of octreotide (200 micrograms/kg), Group 2 received an equal volume of the octreotide carrier, Group 3 received an hourly intravenous bolus of L-364,718 (60 micrograms/kg), and Group 4 received an equal volume of the L-364,718 carrier. Hemodynamic profiles, arterial blood gases, plasma glucose, and serum amylase were obtained before laparotomy, at bile injection, and at hourly intervals. The pancreas was removed after 8 hours for gross evaluation, measurement of water content, and histologic examination. A significant decrease in cardiac index and a significant increase in serum amylase and pancreatic edema occurred in all four groups 8 hours after the induction of pancreatitis (P less than 0.05), but there was no statistical difference between any group. Likewise, there was no difference in gross or histologic changes in the pancreas of any group. The somatostatin analogue, octreotide, and the cholecystokinin receptor antagonist, L-364,718, did not ameliorate the effects of severe, bile-induced pancreatitis in dogs.

Topics: Acute Disease; Amylases; Animals; Benzodiazepinones; Bile; Body Water; Cardiac Output; Cholecystokinin; Devazepide; Dogs; Edema; Hemorrhage; Injections, Intravenous; Injections, Subcutaneous; Necrosis; Octreotide; Pancreas; Pancreatitis

The article deals with clinical experience in early diagnosis of pancreatonecrosis in which enzymological methods of examination acquire importance. The most informative test in the diagnosis of fatty pancreatonecrosis is the increase of the pancreatic lipase content in blood serum, particularly in stimulation of pancreatic secretion by pancreozymin. A new Soviet-produced peptide agent--dalargin was included in the complex drug treatment of fatty pancreatonecrosis in 74 patients. With the use of dalargin as an inhibitor of the activity of pancreatic enzymes and kinins in the treatment of fatty pancreatonecrosis a marked positive effect was produced. The mortality in fatty pancreatonecrosis was 5.5%.

Topics: Cholecystokinin; Clinical Enzyme Tests; Enkephalin, Leucine-2-Alanine; Humans; Lipase; Necrosis; Pancreas; Pancreatitis

The management of acute pancreatitis has not changed appreciably throughout several decades. Recent evidence has suggested that cholecystokinin (CCK) may play an important role in pancreatic disease. Investigations into the precise role of CCK in acute pancreatitis have been hampered by the lack of a specific CCK receptor antagonist. Using a newly described, highly potent and specific CCK receptor antagonist, L-364,718, the effect of CCK in two models of acute "surgical" pancreatitis was examined: (1) the bile salt ductal perfusion model in the rat and (2) a traumatic model in the guinea pig. At a suboptimal dose for pancreatic enzyme secretion (25 pmol/kg/h), CCK was found to potentiate the severity of the ensuing pancreatitis in both models. Continuous CCK receptor blockade with L-364,718 (25 nmol/kg/h) improved biochemical, morphologic, and survival indexes. This study suggests that physiologic levels of CCK play an important permissive role in the evolution of acute pancreatitis. The use of L-364,718 as an investigative probe or therapeutic tool for acute pancreatitis is worthy of further consideration.

Topics: Acute Disease; Amylases; Animals; Benzodiazepinones; Cholecystokinin; Devazepide; Female; Guinea Pigs; Lipase; Male; Necrosis; Organ Size; Pancreas; Pancreatitis; Rats; Rats, Inbred Strains

The effects of long-term alcohol intake and pancreatic secretory stimulation on pancreatic necrosis were studied in rats with acute pancreatitis induced by an intraductal injection of sodium taurocholate. Neither alcohol nor pancreozymin alone influenced the extent of pancreatic tissue damage in this experimental model. However, when animals were exposed to both alcohol and pancreozymin, the tissue lesions induced by the bile salt were wider than those in the presence of either alcohol or secretory stimulation alone. The results indicate that secretory stimulation with pancreozymin superimposed on chronic alcohol intake sensitizes the pancreas to the injury caused by intraductal bile salt administration.

Topics: Acute Disease; Alcoholism; Animals; Cholecystokinin; Male; Necrosis; Pancreas; Pancreatitis; Rats; Rats, Inbred Strains; Reference Values; Taurocholic Acid

A follow-up investigation of 20 patients, surgically treated for acute haemorrhagic necrotising pancreatitis, was performed in an average of 2 3/4 years after the operation. Twelve patients showed manifest diabetes mellitus, four further cases had a suspicious oral glucose tolerance test. Only one patient was insulin dependent. A secretin-pancreozymin test performed in 15 patients showed a dissociated or global pancreatic insufficiency in 13 cases. The extent of the endocrine and exocrine functional disturbance did not correlate with the extent of surgery. Postoperative functional defects were readily improved therapeutically in most cases. Only in patients who continued to consume alcohol were there digestive disturbances. The results indicate that the functional state of the remaining pancreas does not only depend on the extent of surgery but also on the extent of already existing or persisting toxic inflammatory damage and on the regenerative capacity of the remaining parenchyma.

Topics: Acute Disease; Cholecystokinin; Diabetes Mellitus; Female; Humans; Male; Middle Aged; Necrosis; Pancreatic Diseases; Pancreatitis; Postoperative Complications; Secretin

Topics: Amylases; Calcinosis; Cholecystokinin; Clinical Enzyme Tests; Humans; Lipase; Lipomatosis; Necrosis; Pancreas; Pancreatic Diseases; Pancreatitis; Sclerosis; Secretin