Anna Andrzejewska

Nitric oxide protects the ultrastructure of pancreatic acinar cells in the course of caerulein‐induced acute pancreatitis

Nitric oxide (NO) as a unique biologicalmediator that has been implicated in many physiological and pathophysiological processes may have a significant influence on the course of acute pancreatitis and the recovery process. The aim of the study was to evaluate the effect of a NO synthase inhibitor or a substrate for NO endogenous production on the ultrastructural features of the acinar cells in the course of caerulein‐induced acute pancreatitis. Acute pancreatitis was induced in the rats by a supramaximal dose of caerulein. During acute pancreatitis induction, the rats were treated with l‐arginine (the substrate for NO synthesis), NG‐nitro‐ l‐arginine (L‐NNA, NO synthase inhibitor), l‐arginine + L‐NNA or saline. Light and electron microscopy examinations were performed in all groups after pancreatitis induction and additionally after 7 and 14 days of recovery. The study demonstrated that the NO synthase inhibitor given during pancreatitis induction in rats enhances the damage to the acinar cells, detected ultrastructurally, and increases the cellular inflammatory infiltration. In the later period, the considerable damage to the mitochondria and the changes in secretory compartment were observed, including dilated cisternae of Golgi apparatus, focal degranulation of rough endoplasmic reticulum, and reduced number of zymogen granules and condensing vacuoles. l‐arginine reversed to some extent the deleterious effect of L‐NNA, although when administered alone it had no apparent effect on the ultrastructure of pancreatic acinar cells compared with untreated animals. The obtained results indicate that the NO synthase inhibitor enhances the ultrastructural degenerative alterations in the pancreatic acinar cells in the course of caerulein‐induced acute pancreatitis and confirm the protective role of endogenous nitric oxide in this disease.

The endothelin‐1 receptor antagonists ameliorate histology and ultrastructural alterations in the pancreas and decrease trypsinogen activation in severe taurocholate pancreatitis in rats

Summary.  The role of potent vasoconstrictor endothelin‐1 (ET‐1) in acute pancreatitis (AP) remains controversial. The aim was to compare the effect of nonselective ET RA/B (LU‐302872) and selective ET RA (LU‐302146) antagonists on pancreatic histology, ultrastructure and trypsinogen activation in severe taurocholate AP in rats. Male Wistar rats with AP were treated with an intraperitoneous injection of 1, 5 and 10 mg/kg of body weight of each antagonist at 0, 6, 12 and 18 h after taurocholate administration. After 24 h, the samples of pancreases were taken for histological and ultrastructural examinations and for assessment of trypsinogen activation. Both antagonists, at all investigated doses, decreased the damage to the acinar cells detected in the light microscope and ultrastructurally. Trypsinogen activation increased to 29.7 ± 3.9% in the AP untreated in comparison to the control group [12.7 ± 1.4% (P < 0.001)]. This increase was attenuated to 13.8 ± 2.2% in AP treated with a high dose of the nonselective antagonist and to 8.4 ± 1.7% with low dose of selective antagonist. The obtained results indicate that ET‐1 could participate in the damage to the pancreas during AP. Both antagonists of ET‐1 receptors exerted a similar beneficial effect on the morphological changes of the pancreas in AP. One of the probable mechanism could be the attenuation of trypsinogen activation.

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.

Beneficial effect of iloprost on the course of acute taurocholate pancreatitis in rats and its limitation by antecedent acute ethanol intake.

The effects of stable prostacyclin analogue iloprost on the trypsinogen activation, labilization of lysosomal membranes, lipolytic enzymes activities, histopathological and ultrastructural changes in the pancreas of rats with severe, taurocholate acute pancreatitis (AP), preceded for 6 h by acute ethanol intake have been investigated. Iloprost (1 microg/kg b.w., i.p.) was applied every 6 hours after inducing of taurocholate AP. The antecedent intragastric 40% ethanol intake (5 g/kg b.w.) increased an index of trypsinogen activation in AP lasting 18 h. Treatment with iloprost prevented this increase in the rats with AP given earlier alcohol, and limited the labilization of lysosomal membranes in nonalcoholized rats with AP. Phospholipase A2 and lipase activities were reduced by iloprost only in the rats not given ethanol. The additional damaging effect of acute ethanol abuse prior to AP could be dependent on augmented activation of trypsinogen. The protective effect of iloprost in AP seems to be dependent on the attenuation of trypsinogen activation, decrease of total potential trypsin and the decrease of lysosomal membranes labilization. Its protective effect could be limited in taurocholate acute pancreatitis preceded by acute ethanol intake as evidenced by the differences in the cathepsin B, phospholipase A2 and lipase activities and by histopathological and ultrastructural examination.

Does Antecedent Ethanol Intake Affect Course of Taurocholate Pancreatitis in Rats

The pathogenic role of acute ethanol abuse inacute pancreatitis (AP) is still obscure. The aim of thestudy was to evaluate the effect of antecedent intake ofa high dose of 40% ethanol (5 g/kg body wt.), on trypsinogen activation, pancreatic lysosomalmembrane labilization, and activities of phospholipaseA2 and lipase in taurocholate AP in rats. In80 male Wistar rats, AP or sham operation (SO) wasproduced 6 hr after intragastric saline (S) or ethanol(E) administration, and animals were sacrificed after 6,12, and 18 hr. Free active trypsin (FAT) and totalpotential trypsin (TPT) were assayed in the pancreatic homogenate. Percentage free activity (%F/T) ofcathepsin B was determined as an index of lysosomalmembrane fragility. The most evident activation oftrypsin occured at 6 hr AP (11.6% of TPT in S group and 16.4% in E group). Antecedent ethanolincreased FAT 18 hr after SO from 0.105 0.048 μg/gprotein to 0.258 ± 0.054 and AP lasting 18 hrfrom 0.331 ± 0.072 to 0.695 ± 0.110. The%F/T of cathepsin B was highest at 18 hr of AP, suggesting maximal labilizationof lysosomal membranes at this time. This labilizationoccurred earlier (at 12 hr of AP) in E group. Theincreasing effect of antecedent E on lipolytic enzymes was evident after 6 hr of AP. In conclusion,the antecedent intake of high dose of ethanolsignificantly promoted the conversion of trypsinogen totrypsin in taurocholate acute pancreatitis, whereas its additional effect toward labilization ofpancreatic lysosomal membranes and the increase oflipolytic enzymes activities was less evident.Therefore, the promoting impact of acute ethanol intakein the development of acute pancreatitis could bemainly dependent on its increasing effect on trypsinogenactivation.

The lung lysosomal hydrolases and phospholipase A in acute experimental pancreatitis with reference to heparin treatment

The pulmonary complications are severe sequeles of acute pancreatitis. The pathogenesis of these complications is unsolved. The purpose of this work was to evaluate the status of lung lysosomes and phospholipase A activity in acute experimental pancreatitis (AEP) and the effect of heparin as a potentially protective agent. Taurocholate-induced AEP in rats lasting 24 and 48 hours was treated with heparin intraperitoneally (2 mg/kg every 8 hours). The total activity of cathepsins and B-glucuronidase in lysosomal enriched subfraction increased markedly during 48 hours of AEP in untreated animals, but the relative free activity was maximal after 24 hours. Free activity of cathepsins and acid phosphatase in supernatant was maximal after 24 hours. The phospholipase A activity was maximally elevated (more than twofold) after 48 hours. Heparin prevented the increase of activity of B-glucuronidase, depressed the relative free activity of all investigated lysosomal hydrolases and inhibited the phospholipase A activity in the lung homogenate. Our results indicate the significance of labilization of lung lysosomes and increment of phospholipase A activity in the lungs in the damage of this organ during AEP in the rats, and suggest the beneficial effect of heparin on these factors.

The ultrastructure of the liver in acute experimental pancreatitis in dogs

The ultrastructure of the liver in acute experimental pancreatitis (AEP) in 18 dogs in three groups was investigated after 6, 12 and 24 h of the pathological process. AEP was induced by injection of an incubated mixture of bile and trypsin into the pancreatic duct. Swelling of mitochondria, depletion of glycogen, formation of autophagosomes more pronounced in later stages of AEP were evident. Complete destruction of some hepatocytes after 24 h was observed. Dilatation of Disses spaces, activation of Kupffer cells and degranulation of mastocytes especially after 24 h were stated. Ultrastructural changes in the liver were present in 100% of the material. The severity of liver pathology was parallel to the intensity of pancreatic necrosis. The crucial stage of the damage to the liver during AEP seems to be the impairment of mitochondria.

Exocrine cell mitochondria of the rat pancreas after lead intoxication.

The aim of the study was to evaluate alterations in exocrine cell mitochondria of the rat pancreas after lead acetate intoxication. The experiment used 45 rats divided into 2 experimental groups receiving lead acetate to drink, of lead concentration 50 and 500 mg/dm3 (ppm), and a control group given tap water. The animals from the experimental group were decapitated after 2, 4, 6 and 8 weeks, 5 rats from the control group after 8 weeks of the experiment. Rats from experimental groups decapitated after 8 weeks had lead administration stopped after six weeks and then, for two weeks tap water was given. Pancreatic sections were examined with biochemical methods for the activity of cytochrome oxidase and succinic dehydrogenase. Ultrastructural and morphometric examinations were also performed. It was demonstrated that: a) exocrine cell mitochondria are particularly predisposed to lead effect, b) intoxication of rats with lower lead doses (50 ppm) causes reversible adaptative or compensatory changes in these organelles, c) intoxication of rats with higher lead doses (500 ppm) induces irreversible ultrastructural alterations in numerous mitochondria, including damage to inner and to outer mitochondrial membranes, d) structural changes in the mitochondria in the course of lead intoxication are the morphological expression of the impairment of metabolic processes, associated with the inhibited activity of the respiratory enzymes: succinic dehydrogenase and cytochrome oxidase.

Hepatic mitochondrial and lysosomal alterations in acute experimental pancreatitis with ethanolic coetiology in rats

In order to assess the cumulative effects of antecedent acute ethanol intake and acute pancreatitis on the liver, the mitochondrial respiratory functions and lysosomal membrane integrity of the liver were evaluated in taurocholate pancreatitis (AP) in rats, induced 6 hr after intragastric ethanol 40% (5 g/kg body wt). The oxygen consumption rate. RCR (respiratory control ratio), and ADP/O ratio were measured according to Estabrook. Fractional free activity of lysosomal hydrolases was assayed. RCR with glutamate+malate was most decreased at 12 hr of AP with partial improvement after 18 hr. The ADP/O ratio dropped maximally after 18 hr of AP. The fragility of lysosomal membranes increased significantly at 18 hr of AP. The antecedent ethanol intake abolished the partial restoration of RCR after 18 hr; however, it did not affect the ADP/O ratio or the integrity of lysosomal membranes impaired in AP at this time. In conclusion, the antecedent acute ethanol abuse could aggravate the liver mitochondrial deterioration, but not the lysosomal membrane labilization seen in AP.

Ultrastructure of liver in acute experimental pancreatitis in dogs treated with prostacyclin

The ultrastructure of the liver was studied in 3 groups of dogs (5 in each) with acute experimental pancreatitis (AEP) induced by injection of bile-trypsin mixture into the pancreatic duct. Experiments were terminated after 12 h. In the group of untreated animals, severe degeneration of mitochondria, extensive autophagocytosis, hyperactivity of Kupffer cells, depletion of glycogen granules, dilatation of endoplasmic spaces and vacuolisation of cytoplasm were found. In the group treated with prostacyclin PGI2 (20 ng/kg/min) in continuous i.v. infusion for 12 h, ultrastructural alterations were less advanced. Even better results were obtained in the group additionally pretreated for 1 h with the same rate of prostacyclin infusion. The results indicate a protective effect of prostacyclin against damage to the liver in the course of AEP.