NFATc3 regulates trypsinogen activation, neutrophil recruitment, and tissue damage in acute pancreatitis in mice.

BACKGROUND & AIMS: The signaling mechanisms that regulate trypsinogen activation and inflammation in acute pancreatitis (AP) are unclear. We explored the involvement of the calcium- and calcineurin-dependent transcription factor nuclear factor of activated T cells (NFAT) in development of AP in mice. METHODS: We measured levels of myeloperoxidase and macrophage inflammatory protein 2 (CXCL2), trypsinogen activation, and tissue damage in the pancreas 24 hours after induction of AP by retrograde infusion of taurocholate into the pancreatic ducts of wild-type, NFAT luciferase reporter (NFAT-luc), and NFATc3-deficient mice. We isolated acinar cells and measured NFAT nuclear accumulation, trypsin activity, and expression of NFAT-regulated genes. RESULTS: Infusion of taurocholate increased the transcriptional activity of NFAT in the pancreas, aorta, lung, and spleen of NFAT-luc mice. Inhibition of NFAT with A-285222 blocked taurocholate-induced activation of NFAT in all organs. A-285222 also reduced taurocholate-induced increases in levels of amylase, myeloperoxidase, and CXCL2; activation of trypsinogen; necrosis of acinar cells; edema; leukocyte infiltration; and hemorrhage in the pancreas. NFATc3-deficient mice were protected from these effects of taurocholate. Similar results were obtained using an l-arginine-induced model of AP. Reverse-transcription polymerase chain reaction and confocal immunofluorescence analyses showed that NFATc3 is expressed by acinar cells. NFATc3 expression was activated by stimuli that increase intracellular calcium levels, and activation was prevented by the calcineurin blocker cyclosporin A or A-285222. Activation of trypsinogen by secretagogues in acinar cells was prevented by pharmacologic inhibition of NFAT signaling or lack of NFATc3. A-285222 also reduced expression of inflammatory cytokines such as CXCL2 in acinar cells. CONCLUSIONS: NFATc3 regulates trypsinogen activation, inflammation, and pancreatic tissue damage during development of AP in mice and might be a therapeutic target.

The effect of endothelin-1, endothelin-2 and endothelin-3 in early cerulein-induced acute pancreatitis in rats.

PURPOSE: To assess the effect of endothelins: ET-1, ET-2 and ET-3 on trypsinogen activation, lipase activity and histological changes in the pancreas in early (4 hrs) cerulein acute pancreatitis (AP) in rats. MATERIAL AND METHODS: In 45 Wistar rats with cerulein induced AP (2 x 40 microg/kg i.p. at 1 hour interval, the effect of endothelins at the dose 2 x 0.5 or 2 x 1.0 nmol/kg i.p. was assessed vs untreated AP; 6 healthy rats were control (C). Free active trypsin (FAT), total potential trypsin after activation with enterokinase (TPT), lipase in 12000 xg supernatants of pancreatic homogenates and the plasma alpha-amylase were assayed. The %FAT/TPT was an index of trypsinogen activation. RESULTS: %FAT/TPT increased from 3.0 +/- 0.6 in C to 16.2 +/- 3.1 in AP (p < 0.01). ET-1 decreased this index to 4.8 +/- 1.1 after higher dose (p < 0.01); the effect of lower dose was insignificant. Attenuating effect of ET-2 was significant: 7.3 +/- 1.7 after higher dose (p < 0.05) and 6.1 +/- 0.9 after lower dose (p < 0.01). ET-3 diminished this index to 4.5 +/- 1.5 (p < 0.01) and to 6.3 +/- 2.2 (p < 0.05) respectively. Lipase activity in supernatant increased from 4.1 +/- 0.6 in C to 6.3 +/- 0.7 U/mg protein in untreated AP (p < 0.05) and plasma alpha-amylase from 7.0 +/- 0.6 in C to 25.9 +/- 4.3 U/ml in AP (p < 0.001), without essential changes in treated groups vs untreated AP. Higher doses of endothelins decreased inflammatory cell infiltration score in AP. CONCLUSIONS: The exogenous endothelins, especially ET-2 and ET-3 and to lesser extent ET-1 exerted some protective effect in early, edematous acute pancreatitis by the attenuation of trypsinogen activation and inflammatory cell infiltration in the pancreas.

The bifunctional rat pancreatic secretory trypsin inhibitor/monitor peptide provides protection against premature activation of pancreatic juice.

BACKGROUND: In the rat, two forms of the pancreatic secretory trypsin inhibitor, PSTI-I and PSTI-II, are secreted into pancreatic juice. It is assumed that their role is to protect the pancreas from premature activation of the protease-rich pancreatic juice. In the small intestine, PSTI-I, also called 'monitor peptide', is thought to have a different role: PSTI-I competes with protein for activated trypsin. In the presence of a protein-rich meal, free PSTI induces a release of cholecystokinine from the intestine. METHODS: To investigate whether its role as monitor peptide is compatible with the inhibitory, protective function in the pancreas, PSTI-I was chemically synthesized and then renatured. RESULTS: The peptide was almost completely trypsin resistant and exhibited a dose-dependent inhibitory activity to bovine and partially purified rat trypsin. Furthermore, experiments with trypsin- and endopeptidase-activated pancreatic juice demonstrated that its inhibitory capacity was sufficient to prevent premature activation. Binding studies of (125)I-labeled PSTI-I with the putative intestinal receptor using isolated membranes indicated the presence of high-affinity binding sites (k(d) = 5 x 10(-8)M). Binding of PSTI-I could be competed with excess PSTI-I or trypsin. In a biological assay system, injections of PSTI-I displayed monitor peptide activity by inducing a dose-dependent trypsinogen release from the pancreas. CONCLUSION: Our experiments support a dual function of PSTI-I: monitoring protein in the gut due to its 'moderate' affinity for trypsin and a protective role in the pancreas.

Plasma immunoreactive cationic trypsin(ogen) pattern in reserpinized rat model of cystic fibrosis. Resemblance to humans.

Plasma immunoreactive cationic trypsin (ogen) is elevated in cystic fibrosis during early infancy, before exocrine pancreatic insufficiency is fully developed. The recently developed cystic fibrosis mouse model carrying a mutated gene presents only minor pathologic findings in the pancreas. However, the reserpinized rat model shows cystic fibrosis-like defects in various exocrine glands, including the exocrine pancreas. Plasma immunoreactive cationic trypsin (ogen) has not been studied yet in this model. The present study explored the plasma immunoreactive cationic trypsin (ogen) pattern and possible mechanisms in this rat model. Plasma immunoreactive cationic trypsin (ogen) (RIA), pancreatic juice volume, protein, and trypsin, and pancreas weight were determined in rats treated with reserpine (0.5 mg/kg/day subcutaneously) for four or seven days, following cerulein stimulation (5 micrograms/kg/dose intraperitoneally), versus pair-fed controls. The first of four consecutive 30 min periods revealed peak values in all parameters. Four-day reserpine-treated rats demonstrated significantly higher plasma immunoreactive cationic trypsin (ogen) levels (167.3 +/- 12.8 vs 88.9 +/- 6.1 ng/ml; P < 0.0001) with similar values of pancreatic juice trypsin (8.2 +/- 2.4 vs 6.6 +/- 1.8 units/mg protein; P = NS) and volume (5.6 +/- 1.3 vs 4.2 +/- 1.6 mg/min/g pancreas; P = NS), compared to controls. Rats treated with reserpine for seven days revealed significantly lower values of plasma immunoreactive cationic trypsin (ogen) (39.2 +/- 8.4 vs 66.8 +/- 4.9 ng/ml; P < 0.001), pancreatic juice trypsin (1.9 +/- 0.3 vs 3.2 +/- 0.9 units/mg protein; P < 0.001) and volume (1.6 +/- 0.7 vs 3.1 +/- 0.6 mg/min/g pancreas; P < 0.001) compared to controls. We conclude that the reserpinized rat model resembles human cystic fibrosis as to elevated plasma immunoreactive cationic trypsin (ogen) before exocrine pancreatic insufficiency is fully developed. Since exocrine pancreatic volume secretion is intact at this stage, the mechanism of elevated plasma immunoreactive cationic trypsin is probably not due to ductular obstruction. We suggest that this model be studied further in order to investigate other possible mechanisms.

Cholecystokinin-8 induces edematous pancreatitis in dogs associated with short burst of trypsinogen activation.

To study the early pathogenesis of acute edematous pancreatitis in dogs, we examined the relationship of pancreatic hyperstimulation with cholecystokinin-8 (10 micrograms/kg/hr intravenously for 6 hr) to alterations in circulating pancreatic enzymes and pancreatic morphology with special reference to trypsinogen activation. Cholecystokinin-8 infusion was associated with increases in plasma amylase, lipase, trypsin-like immunoreactivity, and plasma and urine trypsinogen activation peptide. Pancreatic parenchymal swelling and interlobular and subcapsular fluid accumulations were detected ultrasonographically within 2 hr of cholecystokinin-8. Circulating trypsin-like immunoreactivity and trypsinogen activation peptide in urine reached a peak at 2 and 4 hr, respectively, then declined despite progressive increases in circulating amylase and lipase and intrapancreatic fluid. No significant changes were observed in dogs receiving a saline infusion. This study illustrates that cholecystokinin-8 induces edematous pancreatitis in dogs that is associated with a short-lived burst of trypsinogen activation.

Denaturation of free and complexed bovine trypsinogen with the calcium ion, dipeptide Ile-Val and basic pancreatic trypsin inhibitor (Kunitz).

Thermal and chemical denaturation has been used to probe changes in the thermodynamic stability of trypsinogen upon complexation with calcium ion and with ligands, which induce the conformational transition of the zymogen to the trypsin-like form. Chemical and thermal unfolding curves of ligand-free trypsinogen at pH 5.8 are cooperative and yielded the following stability parameters: the free energy change of denaturation delta Gden = 44.8 kJ/mol, the denaturation temperature = 65.7 degrees C, the enthalpy change of denaturation delta Hden at the denaturation temperature Tden = 607 kJ/mol and the heat capacity change of denaturation delta Cp,den = 12.4 kJ.mol-1.K-1. Fast phases of both unfolding and refolding of trypsinogen proceed on a time scale of seconds and fit to a single exponential. At pH 5.8, the calcium ion increases the conformational stability delta Gden by 7.1 kJ/mol, Tden by 2.6 K and delta Hden by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by 1H-NMR spectra. The trypsin-like form of trypsinogen, generated by complexation of the zymogen with the dipeptide Ile-Val and/or basic pancreatic trypsin inhibitor (Kunitz), is characterized by increase of delta Hden by 134 kJ/mol and Tden by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.

Effect of intrinsic CCK and CCK antagonist on pancreatic growth and pancreatic enzyme secretion in pancreaticobiliary diversion rats.

When pancreaticobiliary diversion (PBD) surgery was performed in rats, plasma CCK level increased, the pancreas grew mainly by proliferation, and pancreatic trypsinogen showed a remarkable increase, although amylase and lipase synthesis were somewhat decreased. The sensitivity of amylase release against CCK-8 in the pancreatic acini decreased when plasma CCK level was high. These changes in pancreatic growth and pancreatic enzyme secretion caused by PBD were completely inhibited by the CCK-receptor antagonist loxiglumide. From these results, intrinsic CCK was considered to play an important role in both pancreatic enzyme synthesis and proliferation.

[The effects of cholestyramine on pancreatic secretion]. / Efectos de la colestiramina sobre la secreción pancreática.

The objective was to evaluate the effect of biliary salt depletion on morphology and function of the exocrine pancreas. Cholestyramine (15 g/day) was given during fifteen days to male Wistar rats in order to evaluate changes in pancreas weight as well as in enzymatic content of pancreatic tissue and duodenal juice (amylase, lipase and trypsinogen); in duodenal juice, bile salt concentration was also measured; Moreover ultrastructure of the exocrine pancreas was studied. Our results show an increase in pancreas weight in rats treated with cholestyramine, with a significant increase of amylase (p < 0.05) and trypsinogen in pancreatic tissue (p < 0.01), and of lipase in duodenal juice (p < 0.05). Ultrastructural changes were absent. It is concluded that cholestyramine, probably through a bile salt depletion, stimulates pancreatic function and growth, as well as lipase secretion.