Éva Morschl

Importance of cytokines, nitric oxide, and apoptosis in the pathological process of necrotizing pancreatitis in rats

Objectives: Ischemia-reperfusion injury can be involved in the pathophysiology of acute necrotizing pancreatitis. The aim of our study was to determine the production of cytokines, tumor necrosis factor (TNF) and interleukin-6 (IL-6), the activation of the inducible nitric oxide synthase (iNOS), and the development of apoptosis during this pathologic process. Methods: Acute pancreatitis was produced in male Wistar rats by injection of 200 μL of 6% taurocholic acid into the main pancreatic duct in combination with the temporary (15 minutes) occlusion of the inferior splenic artery. Six and 24 hours later, the histologic damage was evaluated, and serum amylase, TNF, IL-6 levels, and iNOS and apoptotic activity from pancreatic and pulmonary tissues were determined. Results: Twenty-four hours after the induction of pancreatitis, the mortality rate was 63%. During this period, the serum TNF and IL-6 levels were permanently high (50 ± 12 and 58 ± 10 U/mL and 7083 ± 1610 and 6790 ± 850 U/mL after 6 and 24 hours, respectively). The iNOS activity showed an increasing tendency in the pancreas, and a decrease following an initial increase in the lung (from 4.2 ± 0.6 to 5 ± 0.4 and from 6.8 ± 0.6 to 3.8 ± 0.5 pmol/min/mg protein after 6 and 24 hours, respectively). Histologic examination confirmed severe necrotizing pancreatitis. In the pancreas, the apoptotic activity increased significantly (from 4 ± 4 to 27 ± 5/mm2 at 6 and 24 hours), while in the lungs, following an initial increase it declined during the course of necrotizing pancreatitis (from 49 ± 4 to 11 ± 6/mm2 at 6 and 24 hours). Conclusion: Our results indicate that intraductal taurocholic acid and ischemia-reperfusion provokes severe acute necrotizing pancreatitis with a high mortality rate and leads to systemic inflammatory reaction, which appears to be the consequence of the activation of the cytokine cascade and iNOS. The degree of NO overproduction by iNOS corresponds with the apoptotic process in the pancreas and the lung.

The role of nitric oxide in edema formation in L-arginine-induced acute pancreatitis.

Introduction Nitric oxide (NO) has been implicated in the regulation of the pancreatic circulation, the promotion of the capillary integrity, and the inhibition of leukocyte adhesion. Aims To investigate the rates of changes in the pancreatic constitutive NO synthase (cNOS) and inducible NOS (iNOS) activities and the role of NO in the vascular permeability changes during the development of L-arginine (Arg)–induced acute pancreatitis. Methodology Acute pancreatitis was induced in male Wistar rats by injecting 250 mg/100 g body weight of Arg i.p. twice at an interval of 1 hour, as a 20% solution in 0.15 M NaCl (group I). The control rats received the same quantity of glycine (group II). In group III, 30 mg/kg NG-nitro-L-arginine methyl ester (L-NAME) was injected i.p. 19 hours after the first Arg injection. The rats were killed at 6, 12, 24, or 48 hours following Arg administration, and the plasma amylase concentration and the pancreatic weight/body weight (pw/bw) ratios were evaluated. NOS activity was determined via the conversion of L-14C-Arg monohydrochloride to 14C-citrulline. The vascular permeability was examined by means of the extravasation of Evans blue dye (20 mg/kg bw) into the pancreatic tissue. Results The serum amylase level was already increased at 6 hours in group I animals, peaked at 12 hours after the Arg injection (11.800 ± 590 versus 6.618 ± 252 U/L in group II), and returned to the control level at 48 hours. The pw/bw ratio peaked at 24 hours in group I (6.63 ± 0.52 versus 4.02 ± 0.22 mg/g in group II) and returned to the control level at 48 hours. The cNOS activity was depleted at 6 hours in group I (0.02 ± 0.003 versus 0.23 ± 0.02 pmol/min/mg protein in group II); it then gradually increased to a level significantly higher than that in group II and decreased thereafter (0.45 ± 0.03 and 0.13 ± 0.01 pmol/min/mg protein at 24 and 48 hours). The iNOS activity was significantly increased at 24 and 48 hours versus that in group II (0.15 ± 0.05 and 0.07 ± 0.01 versus 0.04 ± 0.01 pmol/min/mg protein). The pancreatic concentration of Evans blue dye was significantly higher in group I than in group II (138.59 ± 11.04 versus 43.57 ± 2.67 (g/dry weight). Treatment with L-NAME significantly reduced the amylase activity, pw/bw, Evans blue concentration, and cNOS activity of the pancreas but did not exert any beneficial effect on the histologic score at 24 hours after the onset of pancreatitis, as compared with those values in group I (6.528 ± 673 U/L, 4.56 ± 0.65 mg/g, 86.84 ± 3.9 (g/dry weight, 0.14 ± 0.04 pmol/min/mg protein). Conclusion Endogenous NO is involved in the formation of pancreatic edema in Arg-induced acute pancreatitis by increasing the vascular permeability and protein extravasation. L-NAME treatment decreased the cNOS activity and edema formation but did not prevent the histologic damage in Arg-induced acute pancreatitis.

Raloxifene, an oestrogen–receptor modulator, prevents decreased constitutive nitric oxide and vasoconstriction in ovariectomized rats

Administration of graded doses of [Arg(8)]vasopressin (0.06-0.18 microg kg(-1), i.v.) induced a dose-dependent increase in arterial blood pressure in the catecholamine-depleted (phentolamine; 10 mg kg(-1), i.p.) intact and ovariectomized female rat, with the elevation of blood pressure more marked following ovariectomy. In addition, ovariectomy caused the down-regulation of aortic Ca(2+)-dependent constitutive nitric oxide synthase (assessed by the citrulline assay). The down-regulation of the Ca(2+)-dependent constitutive nitric oxide synthase and augmentation of vasopressin-induced blood pressure responses were prevented by the therapy (1 month, p.o.) with the selective oestrogen receptor modulator, raloxifene (0.3-1.0 mg kg(-1) day(-1)), or with 17beta-oestradiol (0.3 mg kg(-1) day(-1)) in ovariectomized rats. Thus, oestrogen deficiency down-regulates vascular constitutive nitric oxide synthase, which appears to be involved in the increased sensitivity of the vasculature to vasopressin, since both effects can be reversed by the exogenous administration of the natural oestrogen 17beta-oestradiol or the selective oestrogen-receptor modulator raloxifene.

Helicobacter pylori lipopolysaccharide provokes iNOS-mediated acute systemic microvascular inflammatory responses in rat cardiac, hepatic, renal and pulmonary tissues

We have examined the effects of intravenous administration of a purified lipopolysaccharide (LPS) from Helicobacter pylori (3 mg kg(-1), i.v.) on rat vascular permeability, assessed by the radiolabelled human serum albumin leakage technique in the heart, kidney, liver and lung 4 h after challenge. An increased vascular permeability in cardiac, renal, hepatic and pulmonary tissues after challenge was determined. The albumin leakage observed in all these organs could be prevented by the selective inducible nitric oxide synthase inhibitor, N-(8-(aminomethyl)benzyl)-acetamidine (1400W; 0.2-1 mg kg(-1), s.c.) administered concurrently with LPS. Thus, H. pylori LPS can provoke a microvascular inflammatory response in the rat cardiac, renal, hepatic and pulmonary tissues, actions mediated through the activation of the inducible nitric oxide synthase isoenzyme.

Estrogen-mediated up-regulation of the Ca-dependent constitutive nitric oxide synthase in the rat aorta and heart.

The role of endogenous estrogens has been studied in the regulation of the Ca-dependent constitutive nitric oxide synthase (cNOS) enzyme activity in aortic and cardiac tissues of the rat. The activity of cNOS enzyme was measured by the citrulline assay in the abdominal aorta and in the left ventricle of the heart obtained from male, sham-operated female and ovariectomized female Wistar rats. Estrogen replacement therapy (17-beta-estradiol, 20-100 microg/kg/day, s.c.) has been performed in ovariectomized rats over two weeks. We found that cNOS activity was higher in the aorta and heart of female rats compared to males. Ovariectomy decreased cNOS activity in both tissues to that level what could be observed in males. Estrogen supplementation caused a dose-dependent elevation of cNOS enzyme activity in cardiac and aortic tissues, where the higher dose (100 microg) completely restored cNOS enzyme activity to the levels found in females. We concluded that endogenous estrogens up-regulate the activity of the cNOS isoenzymes in the rat aorta and heart.

Vasopressin deficiency decreases the frequency of gastroduodenal ulceration in humans

Vasopressin is a stress hormone released from the posterior pituitary. In humans suffering from central diabetes insipidus, this release of vasopressin is diminished. It was shown previously that the congenitally vasopressin-deficient Brattleboro homozygous rat is less sensitive to various ulcerogenic stimuli. In this study, we investigated the incidence of gastroduodenal ulceration in vasopressin deficient patients. Data on patients aged 20-70, hospitalized in Hungary between 1992 and 1995 were compared with those on the total population in this age group (6,681,020 in 1994). Subjects with central diabetes insipidus were selected separately (815 cases). Gastroduodenal ulceration was compared in subjects with an intact vasopressin release and vasopressin-deficient patients. The frequencies of gastroduodenal ulceration were also examined separately in male and female subjects. In the total population, the frequency of gastroduodenal ulceration was lower in vasopressin-deficient cases (2.22% versus 0.61%; P < 0.005). Among normal-vasopressin subjects, males have a higher risk of gastroduodenal ulceration than females (3.04% versus 1.46%, respectively; P < 0.001). Among vasopressin-deficient subjects, a similar male:female ratio was observed, but it was not significant (P = 0.36). In comparison to the normal-vasopressin population, the incidence of gastroduodenal ulceration was reduced among vasopressin-deficient males and females by 77% (P < 0.01) and by 82% (P < 0.05), respectively. In conclusion, endogenous vasopressin has a significant harmful action towards the human gastroduodenal mucosa. Peptide and non-peptide vasopressin receptor antagonists might have a potential therapeutic benefit in the treatment (as an adjuvant) and prevention of gastroduodenal ulceration.

Helicobacter pylori lipopolysaccharide-provoked injury to rat gastroduodenal microvasculature involves inducible nitric oxide synthase

The actions of a purified Helicobacter pylori lipopolysaccharide (3 mg x kg(-1), i.v.) on rat gastric antral and duodenal microvascular integrity (determined as radiolabelled albumin leakage) and the expression of the inducible nitric oxide (NO) synthase (iNOS; assessed by the citrulline assay) were investigated 4 h after challenge. Significant increases of albumin leakage and expression of iNOS in both antral and duodenal tissues were observed following challenge. Concurrent administration of the selective iNOS inhibitor, 1400W (N-(8-(aminomethyl)benzyl)-acetamidine; 0.2-1 mg x kg(-1), s.c.), with lipopolysaccharide, caused a dose-dependent attenuation of the gastric and duodenal albumin leakage. Thus, H. pylori lipopolysaccharide can initiate the expression of iNOS in the stomach and duodenum following systemic challenge, which can provoke gastroduodenal microvascular dysfunction.

Nitric oxide modulates the gastrointestinal plasma extravasation following intraabdominal surgical manipulation in rats

The actions of nitric oxide (NO) on gastrointestinal plasma loss, assessed by the leakage of [125I]human serum albumin, provoked by intraabdominal surgery and organ manipulation has been investigated in pentobarbitone-anaesthesized rats. Gentle manipulation (3 min) of the stomach or the small intestine following laparotomy leads to an increase in albumin extravasation in the stomach, duodenum, jejunum and colon over 1 h. Administration of the NO synthase inhibitors, N(G)-nitro-L-arginine methyl ester (1-5 mg kg(-1), s.c.) and N(G)-monomethyl-L-arginine (12.5-50 mg kg(-1), s.c.), provoked a further substantial elevation of gastrointestinal albumin extravasation in the surgically manipulated rat, but not in control rats. This effect could be prevented by the pretreatment (15 min) with L-arginine (300 mg kg(-1), s.c.) or by the concurrent infusion of the NO donor, S-nitroso-glutathione (5 microg kg(-1) min(-1), i.v.). Endogenous NO, most likely formed by endothelial NO synthase, thus appears to maintain microvascular integrity during surgery and organ manipulation of the gastrointestinal tract.

Endogenous bacteria-triggered inducible nitric oxide synthase activation protects the ovariectomized rat stomach.

Under experimental circumstances, ovariectomy attenuates gastric mucosal injury where nitric oxide (NO)-mediated pathways are involved. In this study, we have examined the changes in constitutive (cNOS) and inducible NO synthase (iNOS) enzyme activities (assessed by the citrulline assay), and the role of endogenous bacteria in ovariectomy-provoked mucosal defence. Gastric lesions were induced by indomethacin (50 mg/kg, s.c.) over a 4 h period in sham-operated and ovariectomized female Wistar rats. Groups of animals received the wide-spectrum antibiotic ampicillin (800 mg/kg/day, p.o., for 3 days), and others were injected with bacterial endotoxin (E. coli, 3 mg/kg, i.v., 5 h before autopsy). We found that ovariectomy increased iNOS and decreased cNOS activity (resulting an elevated total gastric NOS level), and protected the stomach, effects reversed by ampicillin treatment. In ovary-intact rats, administration of bacterial endotoxin enhanced gastric iNOS activity and reduced lesion-formation. These results suggest that ovariectomy improves gastric mucosal defence perhaps by endogenous bacteria-triggered induction of iNOS.

Detrimental effects of oestradiol on cysteamine-induced gastroduodenal ulceration in the female rat

The actions of the female sex steroid, oestradiol on cysteamine-induced mucosal ulceration has been evaluated in female Wistar rats. Administration of cysteamine (400 mg x kg(-1), s.c.) provoked macroscopic gastroduodenal mucosal injury (assessed planimetrically) and an increase in microvascular permeability (assessed by the extravasation of radiolabeled albumin) in the stomach and duodenum, determined 24 h later. Ovariectomy (2 weeks before cysteamine) reduced gastroduodenal macroscopic injury, and albumin extravasation following cysteamine challenge. Administration of oestradiol (1-5 mg x kg(-1), as an i.m. depot 1 week before cysteamine) dose-dependently augmented gastric and duodenal macroscopic mucosal lesions and microvascular permeability provoked by cysteamine. These findings indicate that oestradiol can exacerbate gastroduodenal ulceration and microvascular injury.