trypsinogen and Necrosis

Topics: Acute Disease; Alcoholism; Apoptosis; Cathepsin B; Chemokines; Cholelithiasis; Cytokines; Humans; Necrosis; Oxidative Stress; Pancreatitis; Regeneration; Risk Factors; Trypsin; Trypsinogen

Topics: Amylases; Aprotinin; Cathepsins; Kallikreins; Kinins; Lipase; Models, Theoretical; Necrosis; Pancreas; Pancreatic Elastase; Pancreatic Juice; Pancreatitis; Peptide Hydrolases; Trypsin; Trypsinogen

Alcohol abuse, a main cause of pancreatitis, has been known to augment NF-κB activation and cell necrosis in pancreatitis. However, the underlying mechanisms are unclear. We recently reported that inhibition of protein kinase D (PKD) alleviated NF-κB activation and severity of experimental pancreatitis. Here we investigated whether PKD signaling mediated the modulatory effects of alcohol abuse on pathological responses in alcoholic pancreatitis.. Alcoholic pancreatitis was provoked in two rodent models with pair-feeding control and ethanol-containing Lieber-DeCarli diets for up to 8 weeks followed by up to 7 hourly intraperitoneal injections of cerulein at 1 μg/kg (rats) or 3 μg/kg (mice). Effects of PKD inhibition by PKD inhibitors or genetic deletion of pancreatic PKD isoform (PKD3Δpanc mice) on alcoholic pancreatitis parameters were determined.. Ethanol administration amplified PKD signaling by promoting expression and activation of pancreatic PKD, resulted in augmented/promoted pancreatitis responses. Pharmacological inhibition of PKD or with PKD3Δpanc mice prevented the augmenting/sensitizing effect of ethanol on NF-κB activation and inflammatory responses, cell necrotic death and the severity of disease in alcoholic pancreatitis. PKD inhibition prevented alcohol-enhanced trypsinogen activation, mRNA expression of multiple inflammatory molecules, the receptor-interacting protein kinase activation, ATP depletion, and downregulation of pro-survival Bcl-2 protein in alcoholic pancreatitis. Furthermore, PKD inhibitor CID755673 or CRT0066101, administrated after the induction of pancreatitis in mouse and rat alcoholic pancreatitis models, significantly mitigated the severity of pancreatitis.. PKD mediates effect of alcohol abuse on pathological process of pancreatitis and constitutes a novel therapeutic target to treat this disease.

Topics: Adenosine Triphosphate; Alcoholism; Animals; Ceruletide; Ethanol; Mice; Necrosis; NF-kappa B; Pancreatitis, Alcoholic; Protein Kinase C; Proto-Oncogene Proteins c-bcl-2; Rats; RNA, Messenger; Trypsinogen

Acute pancreatitis is characterized by premature intracellular activation of digestive proteases within pancreatic acini and a consecutive systemic inflammatory response. We investigated how these processes interact during severe pancreatitis in mice.. Pancreatitis was induced in C57Bl/6 wild-type (control), cathepsin B (CTSB)-knockout, and cathepsin L-knockout mice by partial pancreatic duct ligation with supramaximal caerulein injection, or by repetitive supramaximal caerulein injections alone. Immune cells that infiltrated the pancreas were characterized by immunofluorescence detection of Ly6g, CD206, and CD68. Macrophages were isolated from bone marrow and incubated with bovine trypsinogen or isolated acinar cells; the macrophages were then transferred into pancreatitis control or cathepsin-knockout mice. Activities of proteases and nuclear factor (NF)-κB were determined using fluorogenic substrates and trypsin activity was blocked by nafamostat. Cytokine levels were measured using a cytometric bead array. We performed immunohistochemical analyses to detect trypsinogen, CD206, and CD68 in human chronic pancreatitis (n = 13) and acute necrotizing pancreatitis (n = 15) specimens.. Macrophages were the predominant immune cell population that migrated into the pancreas during induction of pancreatitis in control mice. CD68-positive macrophages were found to phagocytose acinar cell components, including zymogen-containing vesicles, in pancreata from mice with pancreatitis, as well as human necrotic pancreatic tissues. Trypsinogen became activated in macrophages cultured with purified trypsinogen or co-cultured with pancreatic acini and in pancreata of mice with pancreatitis; trypsinogen activation required macrophage endocytosis and expression and activity of CTSB, and was sensitive to pH. Activation of trypsinogen in macrophages resulted in translocation of NF-kB and production of inflammatory cytokines; mice without trypsinogen activation (CTSB-knockout mice) in macrophages developed less severe pancreatitis compared with control mice. Transfer of macrophage from control mice to CTSB-knockout mice increased the severity of pancreatitis. Inhibition of trypsin activity in macrophages prevented translocation of NF-κB and production of inflammatory cytokines.. Studying pancreatitis in mice, we found activation of digestive proteases to occur not only in acinar cells but also in macrophages that infiltrate pancreatic tissue. Activation of the proteases in macrophage occurs during endocytosis of zymogen-containing vesicles, and depends on pH and CTSB. This process involves macrophage activation via NF-κB-translocation, and contributes to systemic inflammation and severity of pancreatitis.

Topics: Adoptive Transfer; Animals; Cathepsin B; Cathepsin L; Cells, Cultured; Ceruletide; Coculture Techniques; Cytokines; Disease Models, Animal; Endocytosis; Enzyme Activation; Genetic Predisposition to Disease; Humans; Hydrogen-Ion Concentration; Inflammation Mediators; Macrophages; Mice, Inbred C57BL; Mice, Knockout; Necrosis; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Pancreas; Pancreatectomy; Pancreatitis, Acute Necrotizing; Phagocytosis; Phenotype; Severity of Illness Index; Time Factors; Trypsinogen

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

Severe acute pancreatitis (SAP) is characterized by trypsinogen activation, infiltration of leucocytes and tissue necrosis but the intracellular signalling mechanisms regulating organ injury in the pancreas remain elusive. Rho-kinase is a potent regulator of specific cellular processes effecting several pro-inflammatory activities. Herein, we examined the role of Rho-kinase signalling in acute pancreatitis.. Pancreatitis was induced by infusion of taurocholate into the pancreatic duct in C57BL/6 mice. Animals were treated with a Rho-kinase inhibitor Y-27632 (0.5-5 mg·kg⁻¹) before induction of pancreatitis.. Taurocholate infusion caused a clear-cut increase in blood amylase, pancreatic neutrophil infiltration, acinar cell necrosis and oedema formation in the pancreas. Levels of pancreatic myeloperoxidase (MPO), macrophage inflammatory protein-2 (MIP-2), trypsinogen activation peptide (TAP) and lung MPO were significantly increased, indicating local and systemic disease. Inhibition of Rho-kinase activity dose-dependently protected against pancreatitis. For example, 5 mg·kg⁻¹ Y-27632 reduced acinar cell necrosis, leucocyte infiltration and pancreatic oedema by 90%, 89% and 58%, respectively, as well as tissue levels of MPO by 75% and MIP-2 by 84%. Moreover, Rho-kinase inhibition decreased lung MPO by 75% and blood amylase by 83%. Pancreatitis-induced TAP levels were reduced by 61% in Y-27632-treated mice. Inhibition of Rho-kinase abolished secretagogue-induced activation of trypsinogen in pancreatic acinar cells in vitro.. Our novel data suggest that Rho-kinase signalling plays an important role in acute pancreatitis by regulating trypsinogen activation and subsequent CXC chemokine formation, neutrophil infiltration and tissue injury. Thus, these results indicate that Rho-kinase may constitute a novel target in the management of SAP.

Topics: Amides; Amylases; Animals; Chemokine CXCL2; Cholagogues and Choleretics; Enzyme Inhibitors; Flow Cytometry; Male; Mice; Mice, Inbred C57BL; Necrosis; Neutrophil Infiltration; Pancreas; Pancreatitis; Peroxidase; Pyridines; rho-Associated Kinases; Signal Transduction; Taurocholic Acid; Trypsinogen

Severe impairment of exocrine pancreatic secretion has recently been demonstrated in a clinical study in sepsis and septic shock patients. The purpose of this study was to further evaluate involvement of the pancreas in the acute phase reaction in sepsis. Using a normotensive rat model of Pseudomonas pneumonia-induced sepsis, we assessed the expression of PAP-I, amylase and trypsinogen mRNA, PAPI protein levels, and cytokine expression in the pancreas by Northern and Western blot analysis and RT-M PCR, respectively. Presence of several well-established features of pancreatitis in sepsis-induced animals were examined by biochemical and histopathological methods as well as by a determination of both water and myeloperoxidase content. Sepsis resulted in an up-regulation of PAP-I gene expression and increase in its protein level in pancreas while the mRNA levels of amylase and trypsinogen were down-regulated. Differences in the pancreatic cytokine expression, serum amylase and serum lipase levels, the occurrence of pancreatic edema as well as the severity of inflammatory infiltration and necrosis were not significantly different between sham and pneumonia groups. Acinar cells showed increased vacuolization in pneumonia animals 24 hours after the treatment. These findings demonstrate that the pancreas is actively involved in the acute phase reaction in sepsis of remote origin. This involvement occurs without concomitant biochemical and histopathologic alterations observed in pancreatitis. Taken all together, these features are indicative of a sepsis-specific dysfunction of the pancreas.

Topics: Acute-Phase Reaction; Amylases; Animals; Antigens, Neoplasm; Biomarkers, Tumor; Cytokines; Gene Expression Regulation; Lectins, C-Type; Leukocyte Count; Lipase; Male; Necrosis; Pancreas; Pancreatitis; Pancreatitis-Associated Proteins; Peroxidase; Pneumonia, Bacterial; Pseudomonas Infections; Rats; Rats, Sprague-Dawley; RNA, Messenger; Sepsis; Time Factors; Trypsinogen; Vacuoles

The relative role of endothelin-1 receptors, ET(A) and ET(B) blockade in acute pancreatitis (AP) remains controversial. The aim of the study was to compare the effect of nonselective ET(A/B) antagonist (LU 302872) and selective ET(A)antagonist (LU 302146) in severe taurocholate AP in rats. Male Wistar rats with AP were treated with increasing doses: 1, 5 or 10 mg/kg b.w. of antagonists i.p. at 0, 6, 12, 18 h after induction of AP. In 24 h survivors, free active trypsin (FAT) and total potential trypsin (TPT), chymotrypsin and lipase in 12,000 x g supernatants of the pancreases were assayed. The index of trypsinogen activation (% FAT/TPT) was elevated in untreated AP to 29.2 +/- 5.0 vs 5.4 +/- 0.9 in the control (p < 0.001). ET(A/B) antagonist at increasing doses, diminished this index to 9.8 +/- 2.7, 10.3 +/- 1.6 and 10.1 +/- 2.0 respectively (p < 0.005). ET(A) antagonist reduced % FAT/TPT ratio to 10.6 +/- 1.9 (p < 0.005), 13.4 +/- 0.5 (p < 0.001) and 10.2 +/- 2.4 (p < 0.005) at respective doses. Both antagonists to a similar degree reduced the histological scores of inflammation, hemorrhages and necrosis. The increase in chymotrypsin and lipase activities after 24 h was not significant. In conclusion, both nonselective ET(A/B) and selective ET(A) antagonists attenuated to similar degree the augmented trypsinogen activation and pancreatic injury in taurocholate acute experimental pancreatitis in rats. Endothelin-1 receptor antagonists could be beneficial in the course of acute pancreatitis by the attenuation of trypsinogen activation.

Topics: Animals; Benzhydryl Compounds; Chymotrypsinogen; Disease Models, Animal; Dose-Response Relationship, Drug; Endothelin Receptor Antagonists; Injections, Intraperitoneal; Lipase; Male; Necrosis; Pancreas; Pancreatitis, Acute Necrotizing; Propionates; Pyrimidines; Rats; Rats, Wistar; Taurocholic Acid; Trypsin; Trypsinogen

Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.

Topics: Acute Disease; Androstadienes; Animals; Cells, Cultured; Ceruletide; Chromones; Cytoskeleton; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Lysosomes; Male; Mice; Morpholines; Necrosis; NF-kappa B; Pancreatitis; Phosphatidylinositol 3-Kinases; Phosphatidylinositol Phosphates; Phosphorylation; Rats; Time Factors; Trypsinogen; Wortmannin

Summary. The understanding of the pathogenesis of chronic pancreatitis is limited. Several theories (i. e. obstruction hypothesis) were suggested in the past but could not be confirmed by experimental data. As a formal description of the course of the disease, the necrosis-fibrosis concept seems to be very attractive. According to this theory, there is no significant difference in the pathogenesis of acute and chronic pancreatitis. A major step was the identification of mutations of the cationic trypsinogen, the secretory trypsin inhibitor (SPINK 1) and the cystic-fibrosis protein (CFTR) in some patients. Investigation of these mutations may significantly contribute to a better understanding of the pathogenesis of chronic pancreatitis.

Topics: Adult; Chronic Disease; Cystic Fibrosis Transmembrane Conductance Regulator; Fibrosis; Humans; Mutation; Necrosis; Pancreas; Pancreatic Neoplasms; Pancreatitis; Risk Factors; Trypsin Inhibitors; Trypsinogen

Autodigestion of the pancreas by its own prematurely activated digestive proteases is thought to be an important event in the onset of acute pancreatitis. The mechanism responsible for the intrapancreatic activation of digestive zymogens is unknown, but a recent hypothesis predicts that a redistribution of lysosomal cathepsin B (CTSB) into a zymogen-containing subcellular compartment triggers this event. To test this hypothesis, we used CTSB-deficient mice in which the ctsb gene had been deleted by targeted disruption. After induction of experimental secretagogue-induced pancreatitis, the trypsin activity in the pancreas of ctsb(-/-) animals was more than 80% lower than in ctsb(+/+) animals. Pancreatic damage as indicated by serum activities of amylase and lipase, or by the extent of acinar tissue necrosis, was 50% lower in ctsb(-/-) animals. These experiments provide the first conclusive evidence to our knowledge that cathepsin B plays a role in intrapancreatic trypsinogen activation and the onset of acute pancreatitis.

Topics: Acute Disease; Amylases; Animals; Apoptosis; Cathepsin B; Ceruletide; Disease Models, Animal; Edema; Enzyme Activation; Gene Deletion; Gene Targeting; Humans; Lipase; Mice; Mice, Knockout; Necrosis; Pancreas; Pancreatitis; Phenotype; Trypsinogen

Activation of trypsinogen and phospholipase A(2) is an early event in pancreatic inflammation, but little is known about zymogen activation and the severity of human pancreatitis.. Using a new fluoroimmunoassay we measured trypsinogen activation peptide (TAP) and phospholipase A(2) activation peptide (PROP) in plasma and ascites in 25 patients with acute pancreatitis. TAP, PROP, Pro-PROP and pancreatic PLA(2)-I were measured in plasma for 14 days and in pancreatic necroses, ascitic fluid and pleural effusions.. All 16 patients with severe acute pancreatitis (SAP) had pancreatic necrosis, 10 developed systemic complications like sepsis, pulmonary or renal failure, 6 had infected necrosis, and 4 died. All 9 patients with mild pancreatitis (MAP) survived. Plasma TAP on admission was higher in patients with SAP than in those with MAP and increased in infected necroses. It did not correlate with systemic complications. Systemic PROP was not increased in complicated courses but was significantly higher in patients with MAP than in those with SAP on admission. Pro-PROP was higher in patients with SAP than in those with MAP but was not correlated with systemic complications. Plasma pancreatic PLA(2)-I was increased but not different in patients with SAP and those with MAP. In patients with pancreatic necrosis, TAP and PROP were highest, while in those with post-acute pancreatic abscess, only PROP and Pro-PROP were high. In patients with pleural effusion, TAP was low and PROP/ Pro-PROP were high.. Trypsinogen and PLA(2)-I activation are early events in acute pancreatitis and the activation peptides can be detected in plasma. In the pancreas, trypsinogen activation is accompanied by PLA(2)-I activation in patients with pancreatic necrosis. However, in our study, organ complications in SAP patients was not associated with increased plasma PROP.

Topics: Acute Disease; Adult; Aged; Aged, 80 and over; Ascites; Enzyme Induction; Female; Humans; Male; Middle Aged; Necrosis; Oligopeptides; Pancreatitis; Phospholipases A; Pleural Effusion; Proteins; Trypsinogen

Dextrans improve pancreatic microcirculation in acute experimental pancreatitis. They could therefore facilitate the transport of a protease inhibitor to ischemic areas of tissue injury and be of additional benefit.. To compare the effects of dextrans with and without aprotinin, necrotizing pancreatitis was induced in 33 male dextran-resistant Wistar rats by intraductal infusion of low-dose glycodeoxycholic acid (10 mmol/l) followed by intravenous cerulein (5 micrograms/kg/h) for 6 h. Three and four hours after the start of the cerulein infusion the animals received infusions of either Ringer's lactate (RL) (12 ml/kg), 70,000 Da dextran (10%) (DEX-70) (4 ml/kg) alone, or DEX-70 (4 ml/kg) with aprotinin (5000 IU/kg) (DEX-70/A).. The death rate was 60% within 9 h in the RL group (6 of 10) but only 10% in the DEX-70 group (1 of 10) (p < 0.03; Fisher's exact test) and 23% in the DEX-70/A group (3 of 13). Histomorphometry demonstrated a significant reduction of acinar necrosis in both treatment groups compared with control animals (p < 0.014; t test). Total amounts of trypsinogen activation peptides (TAP) in ascites were also significantly lower in these groups (p < 0.05; t test).. DEX-70 given 3 h and 4 h after induction of pancreatitis significantly reduced the levels of TAP, limited acinar necrosis, and improved survival rate in acute necrotizing rodent pancreatitis. There was no additional benefit from the combination with aprotinin.

Topics: Acute Disease; Animals; Aprotinin; Ceruletide; Dextrans; Glycodeoxycholic Acid; Hemodilution; Male; Necrosis; Oligopeptides; Pancreas; Pancreatitis; Plasma Substitutes; Rats; Rats, Wistar; Serine Proteinase Inhibitors; Time Factors; Trypsinogen

Clinical and experimental observations have associated acute and chronic hypercalcemia with pancreatitis. The aim of this study was to determine whether acute hypercalcemia can induce acute pancreatitis and, if so, whether the pathogenesis involves premature protease activation.. Rats given bolus infusions of CaCl2 (200 mg/kg; n = 76) were compared with saline-treated controls (n = 40). Serum [Ca2+], serum amylase activity, trypsinogen activation peptide (TAP) concentration in serum and pancreatic tissue, pancreatic wet/dry weight ratio, and histology were assessed for 24 hours. For dose-response analysis, CaCl2 was injected at a dose of 50-200 mg/kg, and the aforementioned indices were assayed for 1 hour (n = 5 each).. There were no significant changes in the controls. Calcium infusion increased serum [Ca2+] 3-fold after 5 minutes (P < 0.001). Within 1 hour, serum amylase (2.5-fold) and tissue TAP (3-fold) levels increased along with macroscopic and microscopic edema formation and leukocytic infiltration. The extent of the changes at 1 hour correlated with the calcium dose. Amylase and tissue TAP concentrations remained elevated until 24 hours when serum TAP concentration had increased (P < 0.001) and focal acinar necrosis became evident.. Acute experimental hypercalcemia induces dose-dependent morphological alterations characteristic of acute pancreatitis, acute hyperamylasemia, and early ectopic trypsinogen activation. This supports the pathophysiological relevance of excess calcium and offers a possible pathogenetic mechanism for its association with clinical pancreatitis.

Topics: Acute Disease; Amylases; Animals; Calcium; Calcium Chloride; Chi-Square Distribution; Dose-Response Relationship, Drug; Enzyme Activation; Hypercalcemia; Linear Models; Male; Necrosis; Oligopeptides; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsinogen

Extraintestinal trypsinogen activation peptides (TAP) have been shown to correlate with severity of acute pancreatitis in humans as well as in various animal models. Ischemia superimposed on experimental pancreatitis, however, increases acinar cell injury without increasing TAP in plasma. We speculated that TAP generated in the pancreas might not reach the circulation in necrotizing pancreatitis due to decreased pancreatic perfusion. To test the hypothesis that generation of TAP in plasma is related to pancreatic perfusion and that plasma TAP may therefore underestimate acinar cell injury in necrotizing disease, we correlated TAP in pancreatic tissue and body fluids with capillary pancreatic blood flow in necrotizing and edematous pancreatitis. The ratio between necrosis and TAP in tissue was similar in both models; the ratio between TAP in plasma and tissue, however, was significantly lower in necrotizing pancreatitis, indicating that a certain amount of TAP generated in the pancreas did not reach the circulation. Decreased pancreatic perfusion found in necrotizing pancreatitis was consistent with this finding. Our data suggest that TAP in tissue is most reliable to indicate severity of acute pancreatitis, whereas plasma TAP may underestimate pancreatic injury in necrotizing disease due to decreased pancreatic perfusion.

Topics: Acute Disease; Animals; Ceruletide; Disease Models, Animal; Edema; Glycodeoxycholic Acid; Male; Microcirculation; Necrosis; Oligopeptides; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsinogen

Previous reports demonstrated that radiographic contrast medium, as used in contrast-enhanced computed tomography, increases acinar necrosis and mortality in experimental pancreatitis. The authors studied the possibility that these changes may be related to an additional impairment of pancreatic microcirculation.. Fifty Wistar rats had acute pancreatitis induced by intraductal glycodeoxycholic acid (10 mmol/L for 10 min) and intravenous cerulein (5 micrograms/kg/hr for 6 hrs). After rehydration (16 mL/kg), pancreatic capillary perfusion was quantified by means of intravital microscopy at baseline before intravenous infusion of contrast medium (n = 25) or saline (n = 25), and 30 and 60 minutes thereafter. In addition to total capillary flow, capillaries were categorized as high- or low-flow (> or < 1.6 nL/min).. Pancreatic capillary flow did not change in either high- or low-flow capillaries after saline infusion. However, contrast medium infusion induced a significant decrease of total capillary flow (p < 0.001). Analysis according to the relative flow rate revealed that this was primarily because of a significant additional reduction of perfusion in low-flow capillaries (p < 0.0001). Furthermore, complete capillary stasis was observed in 15.9 +/- 3.4% after contrast medium as compared with 3.2 +/- 1.2% after saline infusion (p < 0.006).. Radiographic contrast medium aggravates the impairment of pancreatic microcirculation in experimental necrotizing pancreatitis.

Topics: Acute Disease; Animals; Capillaries; Contrast Media; Enzyme Activation; Iopamidol; Male; Necrosis; Pancreas; Pancreatitis; Rats; Rats, Wistar; Trypsinogen

Although intracellular protease activation is thought to be an early event in acute pancreatitis, factors determining progression from edematous to necrotizing pancreatitis are largely unknown. With enterokinase as a probe and an immunoassay quantifying free trypsinogen activation peptides (TAP), we sought evidence for the presence of interstitial trypsinogen in edematous pancreatitis and documented the effects of its ectopic activation.. Edematous pancreatitis in the rat was induced by supramaximal stimulation with cerulein (5 micrograms/kg/hr) and coupled with enterokinase infused into the pancreatic duct at 30 mm Hg. Blue dextran infusion at this pressure corroborated interstitial delivery. Rats with no stimulation, maximal physiologic stimulation (0.25 microgram/kg/hr of cerulein), or intraductal saline infusion served as controls. TAP levels measured by enzyme-linked immunosorbent assay, 6-hour survival, and histopathology were used as end points.. Intraductal enterokinase infusion alone or in combination with maximal physiologic stimulation generated only slight increases in TAP level and no or minimal pancreatic injury. In contrast, enterokinase superimposed on edematous pancreatitis (supramaximal cerulein stimulation) produced fulminant pancreatitis and rapid death of all animals within 6 hours. Pancreatic histopathology showed severe intrapancreatic hemorrhage, acinar inflammation, and necrosis. TAP levels were significantly higher in plasma (p = 0.02), urine (p = 0.05), and ascites (p < 0.001) when compared with all other groups.. In edematous pancreatitis a large pool of trypsinogen accumulates in the interstitial space. Activation of these proenzymes leads to catastrophic consequences and may underlie progression from mild to necrotizing pancreatitis.

Topics: Animals; Ceruletide; Edema; Enteropeptidase; Enzyme Activation; Male; Necrosis; Oligopeptides; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsinogen

Trypsinogen activation peptides (TAP) can be found in patients with acute pancreatitis (AP) as well as in different models of experimental AP. First experience has suggested that early elevation of TAP indicates development of necrotizing AP and that the amount of TAP correlates with the extent of acinar cell necrosis and mortality. It is however unknown whether TAP similarly assess severity of AP in the later course of the disease. The present study monitores TAP in plasma, urine and ascites during the initial development of pancreatic injury and correlates the amount of TAP and the extent of pancreatic necrosis over 48 h in a rodent model of AP. While there was no elevation of TAP in control animals or animals with edemantous AP, significant amounts of TAP im plasma were found as early as 30 min following induction of severe necrotizing AP. Serum amylase returned to normal values within 24 h, TAP maintained at high levels until the end of the 48 h observation period. During the first 24 h TAP paralleled the development of acinar cell necrosis, but decreased thereafter despite further progression of pancreatic injury. Our results provide further evidence suggesting that TAP initially precede morphological changes in AP. Early in the course of the disease TAP parallel development of pancreatic injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acute Disease; Animals; Male; Necrosis; Oligopeptides; Pancreas; Pancreatitis; Rats; Rats, Sprague-Dawley; Trypsinogen

Inappropriate extraluminal activation of trypsin is assumed to play a part in the pathogenesis of acute pancreatitis (AP), but proof has been elusive because active trypsin is transient and difficult to measure. We have previously shown increased levels of trypsinogen activation peptides (TAP), a direct measure of trypsin activation, to correlate with severity of AP, tissue necrosis, and survival in a rodent model induced by cerulein hyperstimulation and bile salt infusion. The present study seeks to show that increased trypsinogen activation also characterizes three other models of experimental AP in rodents to give credence to the generality of the phenomenon and to its potential relevance to human AP.. Experimental AP was induced in mice by a choline-deficient diet supplemented with ethionine and in rats by creation of a closed duodenal loop or by ligation of the biliopancreatic duct plus physiologic stimulation. TAP were quantified by an immunoassay in tissue and plasma at various time points after onset of AP.. In the group with choline-deficient diet supplemented with ethionine a significant increase in tissue and plasma TAP was found at 48 and 72 hours, respectively. In the group with closed duodenal loop significant TAP elevations were found in plasma as early as 6 hours and in the group with ligation of the biliopancreatic duct plus physiologic stimulation at 24 hours.. These experiments provide further evidence that extraluminal protease activation is a pathophysiologic event common to the evolution of various models of experimental acute pancreatitis and therefore increase the likelihood that this phenomenon is important in the human disease as well.

Topics: Acute Disease; Animals; Choline Deficiency; Diet; Disease Models, Animal; Duodenum; Enzyme Activation; Ethionine; Female; Ligation; Male; Mice; Necrosis; Oligopeptides; Pancreas; Pancreatic Ducts; Pancreatitis; Rats; Trypsinogen

Contrast-enhanced computed tomography (CECT) is used to show areas of decreased pancreatic perfusion in severe acute pancreatitis (AP). To evaluate possible adverse effects of the contrast medium (CM) on the course of AP, the impact of intravenous CM in AP of graded severity in the rat was studied.. Pancreatitis of three levels of severity was induced in Sprague-Dawley rats with intravenous cerulein hyperstimulation plus time- and pressure-controlled intraductal infusion of saline or glycodeoxycholic acid. At 7 hours, control and pancreatitis animals received intravenous ionic CM, nonionic CM, or saline. The principal outcome measures were 24-hour survival, trypsinogen activation peptides (TAP) in ascites, and histological acinar necrosis score.. There was no measurable effect of CM on the index features in control animals or animals with mild or moderate AP. In severe AP, CM caused a significant increase in mortality, ascites TAP, and necrosis score.. Intravenous CM increases pancreatic injury when administered early in the course of severe experimental AP. Because CM may convert borderline ischemia to irreversible necrosis, CECT performed early in pancreatitis to show poor perfusion and predict areas of necrosis may depict a self-fulfilling prophecy. Early CECT should be reconsidered and perhaps avoided.

Topics: Acute Disease; Animals; Ascites; Ceruletide; Contrast Media; Glycodeoxycholic Acid; Injections; Injections, Intravenous; Male; Necrosis; Pancreatic Ducts; Pancreatitis; Peptides; Rats; Rats, Sprague-Dawley; Sodium Chloride; Survival Analysis; Time Factors; Trypsinogen

Acinar necrosis in patients with acute pancreatitis can be due to enzymatic injury, ischemia, or both. We hypothesized that novel therapy aimed at an improvement of pancreatic microcirculation early in the course of pancreatitis may reduce the lethality and acinar damage. Forty-six dextran-resistant rats received controlled intraductal infusion of glycodeoxycholic acid (10 mmol/L), followed by intravenous cerulein (5 micrograms/kg/h) for 6 hours. Beginning 30 minutes after the induction of pancreatitis, all animals were resuscitated with Ringer's lactate (RL) (8 mL/kg/h intravenously for 9 hours). In addition, they were given intra-aortic bolus infusions (2 mL/kg at 30, 60, 90, and 150 minutes) of either RL, sodium chloride (NaCl) (7.5%) and dextran 60,000 (10%) (HHS-60), NaCl (7.5%) and dextran 500,000 (10%) (HHS-500), or NaCl (0.9%) and dextran 500,000 (10%) (DEX-500). Despite high-volume fluid resuscitation in the groups that received RL and HHS-60, 70% of the animals in each of these groups died within 24 hours. In contrast, the mortality rates in the groups of animals that received HHS-500 and DEX-500 were dramatically reduced to 0% and 10%, respectively (p = 0.005, p = 0.02). Histopathologic scores for acinar necrosis were significantly lower in the group of animals that received DEX-500 (p < 0.009) compared with those that received RL and HHS-60. Finally, total amounts of trypsinogen activation peptides in ascites were significantly lower in the animals that received HHS-500 (p < 0.004) and DEX-500 (p < 0.02) compared with those that received RL and HHS-60. Rapid bolus infusion of hyperoncotic ultrahigh molecular weight dextran solution with or without hypertonic saline but not RL or hypertonic-hyperoncotic saline-dextran significantly reduced pathologic trypsinogen activation, prevented acinar necrosis, and improved survival in acute experimental pancreatitis. We speculate that a sustained improvement of pancreatic microcirculation by ultrahigh molecular weight dextran is the mechanism of action.

Topics: Animals; Dextrans; Fluid Therapy; Isotonic Solutions; Male; Microcirculation; Necrosis; Pancreas; Pancreatitis; Rats; Rats, Wistar; Ringer's Lactate; Saline Solution, Hypertonic; Trypsinogen

The cascade enterokinase-trypsinogen-prophospholipase A2 lecithin, generating trypsin, phospholipase A2 and lysolecithin, respectively, was studied in vitro using a novel phospholipase A2 assay. The rate of enterokinase catalysed activation of trypsinogen was maximal at 4 mmol/1 glycodeoxycholic acid; higher concentrations of bile salt progressively inhibited enterokinase activity. Net phospholipase A2 activity in reaction mixtures was critically dependent on the trypsin/prophospholipase A2 molar ratio. Lecithin hydrolysis by phospholipase A2 was dependent on the bile salt/lecithin molar ratio and was optimal at 1.25 to 1. The addition of enterokinase to lecithin and bile salt mixtures, containing trypsinogen and prophospholipase A2 at presumed pathophysiological concentrations, resulted in the generation of concentrations of lysolecithin lytic for pancreatic acinar cells within 5 min. These findings would support the concept that the entry of bile containing active enterokinase into the pancreatic duct system in vivo may in some cases be involved in the initiation of necrotising acute pancreatitis in man.

Topics: Acute Disease; Endopeptidases; Enteropeptidase; Enzyme Activation; Enzyme Precursors; Glycodeoxycholic Acid; Humans; Hydrolysis; Kinetics; Lysophosphatidylcholines; Necrosis; Pancreatitis; Phospholipases; Phospholipases A; Phospholipases A2; Trypsin; Trypsinogen

Acute hemorrhagic pancreatic necrosis (AHPN) is induced in young female mice fed fo 4 days a choline-deficient diet containing diet 0.5% DL-ethionine (CDE). Contrary to females, male mice do not develop AHPN when fed the same diet. For determination of whether estrogens are involved in the induction of AHPN, estradiol-treated male mice were fed the CDE diet. In such estrogen-treated male mice, the mortality rate, incidence of AHPN, and alterations in biochemical parameters of the pancreas and of serum were similar to those induced by the CDE diet in females.

Topics: Acute Disease; Amylases; Animals; Blood Proteins; Cathepsin B; Cathepsins; Choline Deficiency; Diet; Estradiol; Female; Male; Mice; Necrosis; Pancreas; Pancreatitis; Trypsin; Trypsinogen