Gabryelewicz A

Reactive oxygen species activate mitogen-activated protein kinases in pancreatic acinar cells.

It has been recently reported that kinases that belong to the mitogen-activated protein kinase (MAPK) family are rapidly activated by cholecystokinin (CCK) in rat pancreas both in vitro and in vivo. It is known that reactive oxygen species (ROS) play an important role in the pathogenesis of acute pancreatitis induced by supraphysiologic stimulation with CCK analogue, cerulein. The aim of our study was to evaluate whether MAPKs are activated by ROS in pancreatic acini. The activity of MAPK, c-Jun amino-terminal kinase (JNK), and p38 MAPK was determined in isolated rat pancreatic acinar cells by means of Western blotting, with the use of specific antibody that recognizes active, dually phosphorylated kinases. Incubation of acini with ROS donors, hydrogen peroxide (H2O2) and/or menadione (MND), strongly activated all three kinases. Activation of these kinases by ROS, but not by CCK, was substantially inhibited by pretreatment of acini with antioxidant N-acetylo-l-cysteine (NAC). Whereas CCK-induced activation of MAPK or JNK was totally or partially blocked by protein kinase C (PKC) inhibitor GF-109203X, ROS-induced activation of MAPK, JNK, and p38 MAPK was PKC independent. In conclusion, ROS strongly activate MAPK, JNK, and p38 MAPK in pancreatic acinar cells. It may be of importance in acute pancreatitis, because ROS are involved in the pathogenesis of this disease.

The results of severe acute pancreatitis treatment with continuous regional arterial infusion of protease inhibitor and antibiotic: a randomized controlled study.

Objectives: A randomized controlled trial was conducted to clarify whether continuous regional arterial infusion (CRAI) of protease inhibitor and antibiotic could reduce mortality rate of severe acute pancreatitis (SAP). Methods: Seventy-eight patients with SAP were included in the study. Thirty-nine patients were treated with CRAI, 31 patients completed the study; and another group of 39 patients was treated without CRAI therapy. Groups were well matched in clinical characteristics. The CRAI patients were treated continuously with nafamostat mesylate 240 mg/d and imipenem 1 g/d for 5 days via one of the arteries perfusing the pancreas. Later, imipenem was given intravenously (0.5 g every 8 hours) for 9 days. The non-CRAI patients received imipenem (0.5 g every 8 hours) intravenously for 14 days. Statistical analysis of the intention-to-treat (ITT) group was performed. Results: Lack of septic complications was observed in 23 patients with CRAI therapy and 20 non-CRAI patients (not significant). The additional antibiotics were applied in 8 of CRAI patients and in 18 non-CRAI (ITT, P = 0.02). Mortality rate was 5.1% in CRAI and 23.1% in non-CRAI group (ITT, P = 0.02). Urgent surgical intervention was necessary in 10.3% CRAI patients and in 33.3% non-CRAI (ITT, P = 0.01). Conclusions: The results show that CRAI of protease inhibitor and antibiotic is effective in preventing complications and in reducing mortality rate in SAP.

Serum profiles of E-selectin, interleukin-10, and interleukin-6 and oxidative stress parameters in patients with acute pancreatitis and nonpancreatic acute abdominal pain.

Introduction Excessive inflammatory response is one of the major causes of early mortality in acute pancreatitis (AP). Aim To evaluate the serum profiles of E-selectin, interleukin (IL)–6, and IL-10 along with their correlation to the markers of oxidative stress and neutrophil activation in patients with AP and patients with nonpancreatic acute abdominal pain (NPAAP). Methodology This prospective clinical study included 56 patients with AP (28 with mild AP and 28 with severe AP) as well as 15 patients with NPAAP. Results Serum concentrations of E-selectin, IL-10, and IL-6 and plasma concentrations of polymorphonuclear leukocyte elastase (determined on days 1–3, 5, and 10 after admission) were the highest in severe AP during the first 3 days and then declined. At day 10, the E-selectin level in severe AP was still higher than that in mild AP, and the IL-10 concentration increased again. There was no elevation in the E-selectin concentration in NPAAP patients, and IL-10 levels remained unchanged in mild AP. Oxidative stress, measured by serum malondialdehyde and 4-hydroxyalkenals levels, was the most pronounced in severe AP. Conclusions The serum E-selectin concentration is markedly elevated in severe AP and is less in mild AP but not in NPAAP. It may result from stimulation with different inflammatory mediators or indicate vascular endothelium injury mediated by oxidative stress, especially in the severe form of AP.

The effect of platelet activating factor antagonist (BN 52021) on cerulein-induced acute pancreatitis with reference to oxygen radicals

SummaryAcute edematous pancreatitis was induced in Wistar male rats by iv infusion of cerulein (CR) in the dose of 5.10-6g.kg-1.h-1 during 3 or 6 h. The effect of BN 52021—platelet activating factor (PAF) receptor antagonist, against this model of disease was examined. BN 52021 was applied iv as a bolus injection in the dose of 5.10-3g.kg-1 at 0 time. Treatment with this agent significantly ameliorates cerulein-induced acute pancreatitis in rats. The effect of BN 52021 was expressed by significant reduction of pancreas edema, diminution of hyperamylasemia, lack of superoxide dismutase activity depletion, and inhibition of lipid peroxidation in pancreatic tissue. These changes were accompanied by significant reduction of acinar cells vacuolization and remarkable inhibition of infiltration with inflammatory cells in the interacinar space. We suppose that beneficial effect of BN 52021 against cerulein-induced acute pancreatitis in rats depends on the prevention of inflammatory cells activation and subsequent generation of oxygen radicals within pancreatic tissue.

Feedback regulation of stimulated pancreatic enzyme secretion during intraduodenal perfusion of trypsin in man

Abstract. The pancreatic enzyme secretion in several species is controlled by a negative feedback mechanism induced by the presence of active proteases in the duodenum. The existence of this mechanism in man is controversial. The purpose of the present study was to evaluate the effect of tryptic activity in the duodenum on phenylalanine‐stimulated pancreatic enzyme secretion in healthy volunteers. A double‐balloon, multilumen tube was used for the collection of duodenal juice containing pancreatic enzymes. The continuous infusion of phenylalanine (100 mM) into the duodenum evoked an almost constant secretion of trypsin, amylase and lipase for 160 min. The infusion of trypsin (150 mg h‐1; 1·25 g l‐1) caused a reduction of phenylalanine‐stimulated amylase and lipase output by 25%. The subsequent infusion of aprotinin at a dose of 1·5 × 106 KIU for 30 min led to an almost complete inhibiton of trypsin. Simultaneously, the amylase and lipase output returned to the values seen before trypsin perfusion. Infusion of a higher dose of trypsin (300 mg h‐1; 2·5 g l‐1) caused a more pronounced decrease in phenylalanine‐stimulated amylase and lipase output by 45%. These data indicate that active trypsin in the duodenum is responsible for the inhibition of phenylalanine‐stimulated pancreatic enzyme secretion in man in a dose‐dependent fashion, thus confirming the existence of a feedback control of pancreatic secretion regulated by the amount of proteases in the gut.

The effect of platelet activating factor antagonist (BN 52021) on acute experimental pancreatitis with reference to multiorgan oxidative stress

SummaryAcute hemorrhagic pancreatitis was induced in Wistar rats using a retrograde intraductal injection of 5% Na-taurocholate. Rats were treated with platelet-activating factor receptor (PAF) antagonist—BN 52021 (5 mg/kg) and sacrificed at 1 and 3 h after induction of acute pancreatitis. Malondialdehyde and sulfhydryl groups concentration were measured in pancreatic, lung, and liver tissue as a parameters of oxidant-antioxidant balance. We have shown that BN 52021 exerts only partial protecting effect against Na-TC-induced AP in rats. The positive effects of BN 52021 were expressed by: (1) Significant reduction of hyperamylasemia accompanied by lower malondialdehyde accumulation in pancreatic tissue; (2) Prevention of sulfl hydryl groups depletion in lung tissue; (3) Diminution of necrotic and inflammatory changes in pancreatic tissue; and (4) Improvement of survival rate. We suggest that these effects may depend on the inhibition of PAF-mediated activation and oxidant generation by phagocytes.

Lysosomal activity of pulmonary alveolar macro phages in acute experimental pancreatitis in rats with reference to positive PAF-antagonist (BN 52021) effect

The activation of pulmonary alveolar macrophages (PAMs), might play an important role in severe complications of acute pancreatitis. The aim of our study was to assess the labilization of macrophage lysosomal membranes and release of lysosomal cathepsin B (CB) and N-acetyl-beta-D-hexosaminidase (NAH) into bronchoalveolar lavage fluid (BALF) during taurocholate acute pancreatitis (AP) in rats treated with PAF-antagonist--BN 52021. Total activity of CB increased by 374% after 6 h and by 237% after 12 h of AP in lysosomal enriched fraction of PAMs. Fractional free activity of CB increased to 40% after 6 h and to 38% after 12 h of AP. Free activity of CB was increased 5 fold in the supernatant of macrophage homogenate, and 10 fold in the supernatant of BALF after 6 h of AP. The values of NAH activity roughly paralleled that of CB. Treatment with BN 52021 (5 mg x kg(-1) every 6 h i.v.) partially normalized the measured parameters. Our results indicate that the PAF-antagonist BN 52021 reduced the increase of total and free activity of lysosomal hydrolases of PAMs and partly prevented the labilization of their lysosomal membranes. Therefore, an important mechanism of BN 52021 beneficial effect in pulmonary complications of acute pancreatitis could be dependent on the stabilization of PAMs lysosomes.

Tissue plasminogen activator, plasminogen activator inhibitor, and other parameters of fibrinolysis in the early stages of taurocholate acute pancreatitis in rats

SummaryIt is well known that fibrinolytic activity in the early stages of acute experimental pancreatitis (AEP) as assessed by euglobulin lysis time (ELT) is depressed. The aim of this study was to evaluate changes in the fibrinolytic system in the early stages of taurocholate AEP in rats. Tissue plasminogen activator (t-PA) activity, plasminogen activator inhibitor 1 (PAI-1) activity, plasminogen, α1 proteinase inhibitor (α1PI), α2 antiplasmin (α2AP), antithrombin III (AT III), fibrinogen, and ELT were measured 0.5, 1, 3, and 6 h after the induction of taurocholate AEP in rats, as well as in sham-operated animals and the control group, which was not submitted to any operation. T-PA activity decreased significantly after 3 and 6 h of AEP; PAI activity had a time course reverse to t-PA and was parallel to α1 PI activity. ELT was slightly prolonged after 0.5, 1, and 3 h, whereas α2 AP activity and plasminogen levels increased significantly; AT III activity was increased after 1 h in comparison to control group. Sham operation caused nonsignificant changes in fibrinolysis. Increase of PAI activity and decrease of t-PA could be a reasonable explanation for inhibited plasma euglobulin fibrinolytic activity noted in the early period of AEP.

The functional and ultrastructural changes of hepatic mitochondria in acute experimental pancreatitis in dogs treated with prostacyclin.

Summary Acute pancreatitis affects the subcellular status of the liver both by enzymatic toxemia and by the impairment of the protective function exerted by a normal pancreas on the liver. The aim of this study was to investigate the effect of the cytoprotective agent prostacyclin (PGI2) on hepatic mitochondria during acute experimental pancreatitis (AEP) in dogs. AEP was induced by the injection of a bile and trypsin mixture into the pancreatic duct, and experiments were terminated after 12 hrs with the exception of dogs with AEP 24 hrs (N = 5). The hepatic mitochondria from the group with AEP 12 hrs without treatment (N = 5) showed a significant impairment of succinate oxidation in St. 3 (with ADP) to 20.1 nanoatoms O×min−1× mg−1 of mitochondrial protein in comparison to healthy dogs (N = 4) −37.6 units. The respiratory control ratio (RCR) in the former group was half as great as in the control group (1.56 and 3.13 respectively). The ADP: 0 ratio in this group was impossible to calculate. DNP-stimulated ATP-ase activity was lowered to 50.6 nM Pi min−1mg−1 of protein in comparison to the healthy animals (642.7 units). In dogs AEP 12 hrs treated with PGI2 in the dose of 20 ng/kg · min for 12 hrs (N = 5) and in the group additionally pretreated with PGI2 for 1 hr before AEP (N = 5), the respective values of succinate oxidation in St. 3 were 22.2 and 26.5 units; RCR with ADP 2.05 and 2.07; ADP: O ratio 1.10 and 1.19 (in healthy dogs ADP: O = 1.90). DNP-stimulated ATP-ase activity was also augmented in comparison with the untreated group, 94.0 and 125.6 units, respectively. In the group with AEP 12 hrs without treatment, the ultrastructural examination of the liver showed severely damaged, mitochondria with swelling, destruction of limiting membranes and cristae. In the group with AEP 24 hrs without treatment the functional and morphological picture of mitochondria was improved in comparison to AEP 12 hrs and was comparable to treated groups. Prostacyclin in investigated dosage exerts a protective effect on hepatic mitochondria, damaged during acute experimental pancreatitis in dogs.

The role of nitric oxide in caerulein-induced acute pancreatitis and the recovery process after inflammatory damage.

OBJECTIVES Nitric oxide (NO) is involved in the control of pancreatic blood flow and secretion, and its role in the maintenance of pancreatic tissue has been suggested. The aim of our study was to evaluate the influence of NO on pancreatic trophic parameters during acute pancreatitis induction and the pancreas recovery process. DESIGN/METHODS Acute pancreatitis was induced in rats by a supramaximal dose of caerulein. During acute pancreatitis induction, rats were treated with L-arginine (a substrate for NO synthesis), glyceryl trinitrate (NTG, NO donor), glycine, N(G)-nitro-L-arginine (L-NNA, NO synthase inhibitor), L-arginine + L-NNA, or saline. Pancreatic weight, total contents of RNA, DNA, protein, amylase and chymotrypsin as well as pancreas histology and the number of proliferating acinar cells were evaluated after pancreatitis induction in all groups and additionally after 7 and 14 days of recovery in untreated acute pancreatitis rats or those injected with L-NNA and/or L-arginine. RESULTS Pancreas destruction after acute pancreatitis was evidenced by similar decreases of all parameters in untreated acute pancreatitis rats or those treated with L-NNA or glycine when compared to control healthy animals. The recovery process after acute pancreatitis was not affected by L-NNA injections; however, the increased cell proliferation occurred later than in untreated rats. NTG and especially L-arginine treatment resulted in significant attenuation of pancreas damage (partially prevented by L-NNA treatment) as evidenced by biochemical data with a slight improvement in morphology. Treatment with L-arginine alone or in combination with L-NNA resulted in augmented cell proliferation after acute pancreatitis induction followed by earlier recovery in comparison to untreated acute pancreatitis rats or the L-NNA-injected group. CONCLUSION The present study suggests the involvement of NO in mild acute pancreatitis and in the recovery process after inflammatory damage.