Search

Atopic dermatitis (AD) is the most common chronic inflammatory skin disease in the world. It is characterized by recurrent eczematous skin lesions, fluctuating course and chronic pruritus. Increasing evidence suggest that AD is more common in adults than previously thought. The disease is characterized by an impaired skin barrier, aberrant Th2-type cytokine production and intensive pruritus. Epithelial keratinocytes constitute the first physical, chemical and immunological barrier, classified as a part of the innate defense system. These keratinocytes secrete various factors, e.g. alarmins such as thymic stromal lymphopoietin (TSLP) and interleukin 25 (IL-25). Serum levels of substance P (SP) have been reported to be increased in patients with AD and correlated with itch intensity. Several previous studies reported a positive association between AD severity and house dust mites (HDM) sensitization. The aim of the study was to analyze IL-25, TSLP and SP concentrations in blood serum of adult patients with severe AD, depending on the degree of allergy to HDM. There were 31 adult AD patients enrolled into the study and a control group that consisted of 20 healthy subjects. AD was diagnosed on the basis of Hanifin and Rajka criteria. SCORing Atopic Dermatitis (SCORAD) and visual analogue (VAS) scores were used to assess the intensity of pruritus and blood content of specific IgE to HDM, as well as TSLP, IL-25 cytokines and SP was measured. Our study presents the evidence that IL-25 serum concentration is increased in patients with atopic dermatitis and this cytokine plays an important role in pathogenesis of this disease. HDM could stimulate the release of IL-25 which aggravates the disease severity. Our results corroborate previous findings on the role of TSLP in atopic dermatitis.

It has been demonstrated that several aspects of adipose-related physiology including adipokine release, exhibit daily oscillations. Physical exercise exerts a strong influence on adipokine release and a possible reverse disruption of peripheral circadian clocks. The aim of this study was to establish the effects of time of day and the Wingate test on appetite perception, food intake and plasma levels of adipokines. Twenty-four moderately active non-smoking males (mean ± S.D. age: 27.1 ± 3.1 years; height: 1.79 ± 0.1 m; weight: 76.1 ± 11.7 kg) were recruited for this study and divided in two groups; one fed with an ad libitum test meal and another one without an ad libitum test meal. Each subject participated in the following studies performed at 11:00 and 23:00 hours on separate days: 1) Exercise study (ES): a 30-second Wingate Anaerobic Test (WAnT), and 2) sedentary study (SS). Subjects rated their appetite perceptions (hunger and prospective food consumption) on a 100-milimeter visual analogue scale (VAS) at baseline, after exercise, after test meal and during the postprandial/control period. At those time points blood samples were obtained for the measurement of plasma leptin, visfatin and apelin concentrations. Appetite perception and energy intake results at test meal decreased in response to WAnT in comparison with sedentary subjects. Time of day had no statistically significant effect on energy intake but the appetite perception score after test meal at 24:00 hours was statistically higher than that after test meal at 12:00 hours. No significant differences in the tested plasma adipokine concentrations between the trials existed at baseline, however, all plasma adipokine levels at 24:00 hours were higher than those at 12:00 hours. Plasma apelin concentrations after WAnT were significantly higher than its pre-exercise value at 12:00 hours, unlike those at 24:00 hours. Sedentary experiments showed a modest, yet significant, rise in plasma apelin levels after the test meal at 12:00 hours but not after the one at 24:00 hours. There were no significant changes in plasma leptin concentrations after exercise or test meal but a significant decrease in plasma visfatin concentrations after exercise intervention both at the 12:00 hours test and the 24:00 hours test has been observed. Test meals caused a significant rise in visfatin concentrations in sedentary, but not exercise series, in the daytime and nighttime tests. We conclude that time of day is an important aspect to consider in the relationships between exercise, metabolism and appetite. Further studies are needed to explain the specific mechanisms underlying the effects of acute exercise on postprandial physiology at different times of the day.

Melatonin protects the pancreas from inflammation and free radical damage but the effect of the melatonin metabolite: N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) on acute pancreatitis is unknown. This study assessed the effects of AFMK on acute pancreatitis (AP) in the rats in vivo and on pancreatic cell line AR42J in vitro. AFMK (5, 10 or 20 mg/kg) was given intraperitoneally to the rats 30 min prior to the induction of AP by subcutaneous caerulein infusion (25 μg/kg). Lipid peroxidation products (MDA + 4-HNE) and the activity of an antioxidant enzyme glutathione peroxidase (GPx) were measured in pancreatic tissue. Blood samples were taken for evaluation of amylase activity and TNF-α concentration. GPx, TNF-α, proapoptotic Bax protein, antiapoptotic Bcl-2 protein and the executor of apoptosis, caspase-3, were determined by Western blot in AR42J cells subjected to AFMK or to melatonin (both used at 10(-12), 10(-10), or 10(-8)M), without or with addition of caerulein (10(-8)M). AP was confirmed by histological examination and by serum increases of amylase and TNF-α (by 800% and 300%, respectively). In AP rats, pancreatic MDA + 4-HNE levels were increased by 300%, whereas GPx was reduced by 50%. AFMK significantly diminished histological manifestations of AP, decreased serum amylase activity and TNF-α concentrations, reduced MDA + 4-HNE levels and augmented GPx in the pancreas of AP rats. In AR42J cells, AFMK combined with caerulein markedly increased protein signals for GPx, Bax, caspase-3 and reduced these for TNF-α and Bcl-2. In conclusion, AFMK significantly attenuated acute pancreatitis in the rat. This may relate to the antioxidative and anti-inflammatory effects of this molecule and possibly to the stimulation of proapoptotic signal transduction pathway.

Oxidative DNA damage may be a risk factor for development of various pathologies, including malignancy. We studied inflammation triggered modulation of repair activity in the intestines of three weeks old rats injected i.p. with E.coli or S. typhimurium lipopolysaccharides (LPS) at doses of 1, 5 or 10 mg/kg. Subsequent formation in these animals of colonic preneoplastic lesions, aberrant crypt foci (ACF) was also investigated. Five days after LPS administration no differences were observed in repair rate of 1,N(6)-ethenoadenine (εA), 3,N(4)-ethenocytosine (εC) and 8-oxoguanine (8-oxoG) in intestines of these rats, as measured by the nicking assay. However a significant increase in all three repair activities was found within one and two months after S. typhimurium LPS treatment. E. coli LPS significantly increased only the 8-oxoG repair. S. typhimurium LPS stimulated mRNA transcription of pro-inflammatory proteins, lipooxygenase-12 and cyclooxygenase-2, as well as some DNA repair enzymes like AP-endonuclease (Ape1) and εC-glycosylase (Tdg). mRNA level of DNA glycosylases excising εA (MPG) and 8-oxoG (OGG1) was also increased by LPS treatment, but only at the highest dose. Transcription of all enzymes increased for up to 30 days after LPS, and subsequently decreased to the level observed before treatment, with the exception of APE1, which remained elevated even two months after LPS administration. Thus, the repair efficiency of εA, εC and 8-oxoG depends on the availability of APE1, which increases OGG1 and TDG turnover on damaged DNA, and presumably stimulates MPG. One and two months after administration of E. coli or S. typhimurium LPS, the number of aberrant crypt foci in rat colons increased in a dose and time dependent manner. Thus, inflammation stimulates the repair capacity for εA, εC and 8-oxoG, but simultaneously triggers the appearance of preneoplastic changes in the colons. This may be due to increased oxidative stress and imbalance in DNA repair.

Kynuramines, metabolites of melatonin and L-tryptophan, are synthesized endogenously by oxygenases or in spontaneous reaction by an interaction with free radicals. We have reported previously that melatonin stimulates expression and phosphorylation of heat shock protein (HSP) 27, as well as production of HSP70 and HSP90αβ in pancreatic carcinoma cells (PANC-1). Based on those results, we hypothesized that above processes could have been involved in the interruption of intrinsic proapoptotic pathway. Herein, we report that incubation of PANC-1 cells with N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) or with L-kynurenine (L-KYN) lead to the overexpression of heat shock protein synthesis and these effects are partially reversed by 5-HT3 or MT1/MT2 receptor antagonists. PANC-1 cells in culture were treated with AFMK or L-KYN, with non selective MT1/MT2 receptor antagonist (luzindole), with 5-HT2 and 5-HT3 receptor antagonists (ketanserin and MDL72222), or combination of these substances. Both AFMK and L-KYN significantly decreased cytoplasmic HSP27 and this effect was presumably due to increased of its phosphorylation and consequent nuclear translocation, confirmed by immunoprecipitation of phosphorylated form of HSP27. These changes were accompanied by marked augmentation of HSP70 and HSP90αβ in the cytosolic fraction. Pretreatment of cell cultures with luzindole or MDL72222 followed by the addition of AFMK or L-KYN reversed the stimulatory effects of these substances on HSP expression in PANC-1 cells, whereas ketanserin failed to influence mentioned above phenomenon. We conclude that activation of HSPs in pancreatic carcinoma cells seems to be dependent on an interaction of AFMK or L-KYN with MT1/MT2 or/and 5-HT3 receptors.

Immune system cells, particularly phagocytes, are exposed to direct contact with pathogens. Because of its nature - elimination of pathogenes - their cytoprotective systems supposed to be quick and forceful. Physiological consequence of phagocytosis for the phagocyte is the apoptotic death to prevent the eventual survival of bacteria as intracellular parasites. However, in some cases, defense systems used by the bacteria force the immune cells to prolong the contact with the pathogen for its effective elimination. Experiments were performed on Monomac-6 cells exposed to live CagA, VacA expressing Helicobacter pylori (H. pylori) over different period of time. Total cellular RNA, cytoplasmic and nuclear proteins were isolated for polymerase chain reaction, Western-blot and electrophoretic mobility shift assay, respectively. We found that Monomac-6 cells infection with H. pylori resulted in the translocation of the entire cellular content of the heat shock protein 70 (HSP70) into the cytoplasm, where its presence could protect cell against toxic products of engulfed bacteria and premature apoptosis. At the same time the nuclear translocation of heat shock factor 1 (HSF-1) and activation of HSP70 gene transcription was noticed. Action of HSP70 might to postpone monocyte apoptosis through protecting cytoplasmic and nuclear proteins from damaging effect of bacterial products, what could be the defending mechanism against the toxic stress caused by engulfed bacteria and provide the immune cell with the sufficient amount of time required for neutralization of the bacteria from phagosomes, even at the expense of temporary lack of the protection of nuclear proteins.

Ulcerative colitis (UC) is a chronic disease characterized by the variable clinical picture with the inflammatory changes which can involve the whole colon or its distal part. The current treatments for UC are mostly nonspecific, not always effective, and often accompanied by serious side effects. Therefore, there is a considerable interest in finding alternative and more tolerable treatments for this serious disease. Several lines of experimental studies have shown that melatonin (MEL) regulates the extensive gut immune system and exerts antiinflammatory and immunomodulatory effects suggesting its beneficial action in UC by reducing and controlling inflammation. The study aimed at evaluating the effect of MEL on the activity of inflammatory process and sustaining the remission in patients with UC. It comprised 60 patients with left-sided UC, divided in two equal groups of 30 patients each (38 women and 22 men, aged 26-49 years), similar in both groups, who were in clinical remission for the last 12 months. Patients, during a next period of 12 months, were given mesalazine in daily doses 2 x 1.0 g and melatonin 5 mg daily at bedtime (group I) or placebo (group II). All the patients on MEL adjuvant treatment remained in remission during 12 months of observation with The Mayo Clinic Disease Activity Index (MCDAI) values 1.50±0.51 at the beginning and 2.75±1.86 points after 12 months. In the placebo group significantly higher MCDAI values were observed than in patients on MEL after 6, 9 and 12 months. At the inclusion MCDAI was 1.61±0.68 points and at the end of observation it reached the value of 5.10±2.22 points. In MEL group CRP level remained within the normal range during the course of the study (from 3.49±1.40 to 4.17±2.10 mg/dl). Whereas in the placebo group from the end of the third month the steady rise in CRP blood concentration was noted from 3.85±1.29 to 13.13±6.08 mg/dl. Parallelly to CRP rise a significant decrease in hemoglobin concentration in blood from 12.05±0.69 to 10.93±0.81 g/dl was observed in patients receiving placebo and the values significantly differed between the groups after 3 (p<0.05), 6, 9 and 12 months (p<0.01). The level of anxiety and the intensity of depression in patients on adjuvant MEL decreased during the study but there were no statistical differences noted between the groups. The results of the study allowed drawing the conclusion that adjuvant melatonin therapy may help in sustaining remission in patients with UC.

Pancreatic enzyme secretion is controlled by complex of neurohormonal mechanisms, activated by nutrients. Food components in the duodenum acts as the signals for activation of intestinal phase of pancreatic secretion. Direct stimulation of pancreatic exocrine function involves several hormones, which bind to the receptors on pancreatic acinar cell. Indirect mechanism depends on the activation of autonomic nervous reflexes. Brain is also implicated in the regulation of pancreatic exocrine function. Dorsal vagal complex of the brainstem (DVC) appears the center of long vago-vagal cholinergic entero-pancreatic reflex. Mucosal terminals, which initiates entro-pancreatic reflex could be stimulated by CCK, serotonin and perhaps others peptides, which are released into duodenum from the enteroendocrine (EE) cells of the gastrointestinal mucosa. Melatonin, leptin and ghrelin are released from the EE cells into the gastrointestinal lumen. These substances given intraduodenally to the rats produced dose-dependent stimulation of pancreatic enzyme secretion, but they failed to affect directly amylase release from isolated pancreatic acini. Intraluminal application of melatonin, its precursor: L-tryptophan, leptin or ghrelin dose-dependently increased plasma CCK level. Above stimulatory effects of investigated substances on CCK release were completely abolished by bilateral, subdiaphragmatic vagotomy, capsaicin-deactivation of afferent nerves as well as blockade of CCK receptors. We conclude that melatonin, leptin or ghrelin, which are released into duodenal lumen by nutrients, stimulate pancreatic enzyme secretion by activation of CCK release and activation of duodeno-pancreatic reflex.

Physiological process of cell death, apoptosis, plays a beneficial role in organism survival, but in some pathologies, like gastric Helicobacter pylori (Hp) infection, this process may turn against the host organism causing tissue damage. Knowledge of the mechanisms controlling apoptosis may have potential significance in treatment of these pathologic states. Therefore, we sought to determine whether apoptosis induced in the gastric epithelial cells exposed to live Hp involves the alteration in heat shock protein 70 (HSP70) expression and activation of caspase-3 in peroxisome proliferator-activated receptors (PPARgamma dependent manner). Experiments were performed with KATO III, gastric epithelial cells, exposed to CagA and Vac A positive live Hp, water Hp extracts or Hp culture supernatant over different time periods. Total cellular RNA and proteins were isolated for PCR, western-blot and EMSA studies. Genomic DNA was isolated to analyze apoptosis status. We propose new model of Hp induced HSP70 dependent, caspase-3 executed apoptosis in human gastric epithelium. KATO III cells exposed to Hp, showed an increase in caspase-3 activity accompanied and preceeded by activation of nuclear translocation of PPARg peaking at 48 h of culture. Moreover, heat shock factor 1 (HSF-1) bound up with phosphorylated STAT-3 was unable to activate HSP70 protein synthesis in KATO III exposed to Hp. Lack of protective effect of HSP70, activation of caspase-3--dependent apoptosis pathway caused by Hp and alteration of the bax/bcl-2 cellular equilibrium led to gastric epithelial cell death. The observed phenomenon might be helpful in understanding of the mechanism of Hp related gastrointestinal tract diseasess, especially gastric cancer.

Unlabelled: Bacterial endotoxin (lipopolysaccharide, LPS), is the component of the cellular wall of Gram negative bacteria. Endotoxemia (sepsis) could produce multiorgan failure and could be particularly danger in the early period of life. The effects of endotoxemia induced in the neonatal period of life on the pancreatic secretory function and on pancreatic defense of adult organism have not been investigated yet. To induce endotoxemia suckling rats (30 g) have been injected intraperitoneally with LPS from E. coli (5, 10 or 15 mg/kg-day) during 5 consecutive days. Three months later in these animals (300 g) the studies on pancreatic secretion and acute pancreatitis were carried out. In the adult rats, which have been subjected in infancy to endotoxemia, basal pancreatic secretion was unaffected, whereas amylase secretions stimulated by caerulein or by diversion of pancreatic-biliary juice to the exterior were significantly, and dose-dependently reduced as compared to the untreated control. In the rats pretreated with LPS in the suckling period of life caerulein-induced amylase release from isolated pancreatic acini was significantly decreased, and dose-dependent reduction of mRNA signal for CCK1 receptor on pancreatic acini have been observed. Caerulein infusion (25 microg/kg) produced caerulein induced pancreatitis (AP) in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS (10 or 15 mg/kg-day x 5 days) all manifestations of AP have been reduced. In these animals acute inflammatory changes of pancreatic tissue have been significantly diminished. Pancreatic weight and plasma lipase activity, have been markedly decreased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in an antioxidative enzyme; SOD concentration was reversed and accompanied by significant reduction of lipid peroxidation products; MDA+ 4 HNE in the pancreatic tissue., Conclusions: 1/ neonatal endotoxemia reduces gene expression for CCK1 receptor and could produce impairment of the exocrine pancreatic function at adult age; 2/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age and this effect could be related to the increased concentration of antioxidative enzyme SOD in the pancreatic tissue.

The aim of the study were to answer the question 1.) Whether circulating pro-inflammatory markers of endothelial dysfunction and due to chronic low-grade inflammation of obesity, are altered in untreated lean, young relatively healthy polycystic ovary syndrome (PCOS) patients in comparison with healthy controls; 2.) Whether postprandial plasma concentration pattern of ghrelin and PYY can be predictable as risk factors for atherosclerosis and depend of obesity. Forty young women with PCOS were divided in two groups: 19 lean and 21 obese. The control group included 20 lean, healthy volunteers. Plasma total and active ghrelin, total PYY and PYY(3-36), serum adiponectin and insulin were measured using RIA technique, serum sCD40L, visfatin, sP-, sE-selectins, resistin by EIA. Composition of test meal was: 527 kcal total and consisted of 24.1% fat, 54.4% carbohydrate and 21.5% protein. Total and active ghrelin and total PYY were significantly lower in obese PCOS women, whereas active ghrelin was also significantly lower in lean PCOS women compared to controls. Postprandial plasma total ghrelin levels decrease were blunted in lean and obese compared to controls (12.8 % and 18.2% vs 28.2 %). Postprandial plasma active ghrelin decreased in lean and obese PCOS groups (49.9 % and 44.1 %) and controls (63.8 %). PCOS subjects exhibited smaller rises in postprandial levels of total PYY. Postprandial plasma PYY(3-36) levels increased in obese PCOS women (30.9 %) and controls (41%), whereas lean PCOS women exhibited blunted increase (11.5%). sCD40L levels increased, whereas adiponectin decreased in PCOS groups independently, whereas rise in visfatin, sE- and sP-selectin and the fall in adiponectin was associated with obesity. sP- and sE -selectins correlated positively with obesity. In summary, our study provides the first evidence that lean untreated young PCOS women contribute to the so called "pancreatic islet adaptation to insulin resistance" because of ghrelin and PYY profiles. We confirmed existing of low-grade chronic inflammation in early stage of visceral obesity in lean PCOS patients. The lost endogenous "islet adaptation to insulin resistance" may lead to endothelial dysfunction and promote acceleration of atherosclerosis.

Melatonin, the main product of the pineal gland, is also released from the gastrointestinal endocrine-neurocrine (EE) cells. The concentrations of melatonin produced in the gut exceeds that originating from central nervous system. In spite of the presence of melatonin receptors in the pancreatic tissue little is known about the role of this indole in the pancreas. Our experimental studies have shown that exogenous melatonin, as well as this produced endogenously from its precursor; L-tryptophan, strongly stimulates pancreatic amylase secretion when given intraperitoneally, or into the gut lumen. This was accompanied by significant increases of CCK plasma level. Above pancreatostimulatory effects of luminal administration of melatonin, were completely reversed by bilateral vagotomy, capsaicin deactivation of sensory nerves or pretreatment of the rats with CCK1 receptor antagonist; tarazepide as well as serotonin antagonist; ketanserin. Melatonin, as well as its precursor; L-tryptophan, effectively protects the pancreas against the damage induced by caerulein overstimulation or ischemia/reperfusion. The beneficial effects of melatonin or L-tryptophan on acute pancreatitis could be related to the ability of melatonin to scavenge the free radicals, to activate antioxidative enzymes and to modulate the cytokine production.

Unlabelled: Melatonin, known as a product of pineal gland is also produced in the digestive system. Melatonin receptors have been detected on pancreatic beta cells and this indoloamine influences the endocrine pancreatic function but the role of melatonin on pancreatic exocrine secretion is not known., Aim: To evaluate the effects of intraduodenal administration of melatonin or its precursor L-tryptophan on pancreatic protein output under basal conditions or following the stimulation of exocrine pancreas with diversion of pancreato-bliliary juice (DBPJ) and to assess the involvement of vagal nerves, and CCK in this process., Methods: Under pentobarbiturate anesthesia the Wistar rats weighting 300g were surgically equipped with silicone catheters, one of them was inserted into pancreato-biliary duct, the other one--into duodenum. Melatonin (1, 5 or 25 mg/kg) or L-tryptophan (10, 50 or 250 mg/kg) were administered to the rats as intraduodenal (i.d.) bolus injection. Bilateral vagotomy was performed in the group of animals 7 days before the experiment. To assess the role of CCK in the melatonin or L-tryptophan-induced pancreatic secretory functions, lorglumide, the CCK(1) receptor antagonist was administered at dose of 1 mg/kg i.d. 15 minutes before the application of examine substances. During the study samples of pancreato-biliary juice were collected in 15 minutes aliquots to measure the protein outputs., Results: Melatonin (1, 5, or 25 mg/kg ) or L-tryptophan (10, 50 or 250 mg/kg) produced significant and dose-dependent increases in pancreatic protein secretion under basal conditions or following the stimulation of this secretion by DBPJ. This was accompanied by a dose-dependent rise in CCK plasma level. Stimulation of pancreatic protein outputs caused by melatonin or L-tryptophan was completely abolished by vagotomy, or pretreatment with lorglumide. We conclude that melatonin as well as its precursor L-tryptophan, stimulates pancreatic exocrine function via mechanisms involving enteropancreatic reflexes and CCK.

Unlabelled: Heat shock protein 27 (HSP27) is a cytoprotective chaperone, activated by stressful stimuli. HSP27 modulates aggregation and degradation of many proteins. Recent evidence suggests that HSP27 could be involved in the progression of tumor growth and in the development of resistance of various tumors to chemo- and radiotherapy. It has been reported that melatonin protects pancreatic cells and various tissues against inflammatory damage. Previous experimental studies have shown that melatonin stimulates pancreatic enzyme secretion and improves the outcome of experimental pancreatitis. To investigate whether melatonin could affect HSP27 protein level in human pancreatic carcinoma cells (PANC-1). PANC-1 cells were incubated in the standard medium DMEM supplemented with 10% fetal bovine serum at 37 degrees C with 5% CO2 and humidified atmosphere under basal conditions or in the presence of decreasing doses of melatonin (10(-6) - 10(-12)M). Control experiments were performed with the vehicle only (0,1% DMSO) without melatonin. After 24 h and 48 h the cells were harvested, the cytoplasmic and nuclear proteins were isolated for western blot and immunoblotting studies. Incubation of the PANC-1 cells with melatonin resulted in the stimulation both cytoplasmic and nuclear nonphosphorylated HSP27 protein levels after 24 h of incubation, however, above pools of nonphosphorylated chaperone protein levels were strongly diminished after subsequent 24 h. These changes were accompanied by marked rise of nuclear phosphorylated HSP27. The significant increase of this nuclear protein was observed after 48h of incubation., Conclusion: Melatonin stimulates phosphorylation of HSP27 in human pancreatic carcinoma cells (PANC-1).

Lipopolysaccharide (endotoxin, LPS) is responsible for septic shock and multiorgan failure, but pretreatment of the rats with low doses of LPS reduced pancreatic damage produced by caerulein-induced pancreatitis (CIP). In spite of this observations the effects of LPS and caerulein on pro-apoptotic HSP60 and Bax protein expression in the pancreatic acinar cells has not been examined yet. The aim of this study was to assess the effects of endotoxemia induced in the early period of life on the pro-apoptotic nuclear HSP60 and mitochondrial Bax protein expressions detected in the pancreas of adult animals. Newborn rats (25 g) were injected with endotoxin (Escherichia coli) for 5 consecutive days, at the total doses of 25, 50 or 75 mg/kg. Control animals received injections of physiological saline. Two months later the pancreatic acinar cells were isolated from all above groups of rats and subjected to caerulein over stimulation (10(-8)M). Total nuclear HSP60 and mitochondrial Bax protein expression were isolated for Western blot and co-immunoprecipitation studies. High levels of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein has been observed in the pancreatic acinar cells under basal conditions. Pretreatment of newborn rats with LPS failed to affect significantly the HSP60 and Bax protein levels in the pancreatic acini isolated from the same animals 2 months later, as compared to the control group. Caerulein stimulation significantly reduced the level of these proteins. Pretreatment of suckling rats with LPS (at the total doses of 25, 50 or 75 mg/kg) reversed above caerulein-induced suppression of pro-apoptotic nuclear HSP60 and mitochondrial Bax protein levels in the pancreatic acini obtained from adult rats. We conclude that pretreatment of suckling rats with LPS reversed the suppression of pro-apoptotic HSP60 and Bax protein levels produced by caerulein overstimulation in the pancreatic acini. This mechanism could take a part in the LPS-induced protection of the pancreatic tissue against acute damage.

Lipopolysaccharide (LPS, endotoxin) is the component of the cellular wall of Gram negative bacteria. Endotoxemia (sepsis) could produce multiorgan failure and in the early period of life LPS are responsible for the changes of metabolism and for the reduction of protein synthesis. The influence of neonatal endotoxemia on the pancreas at adults has not been investigated yet. The aim of this study was to assess the pancreatic exocrine function in the adult rats which have been subjected, in the neonatal period of life, to chronic LPS pretreatment. LPS from E. coli or S. typhi at doses of 5, 10 or 15 mg/kg-day was administered intraperitoneally (i.p.) to the suckling rats (30 g) during 5 consecutive days. Three months later these animals (300 g) were equipped with pancreato-biliary fistulae for the in vivo secretory study. Amylase release from isolated pancreatic acini obtained from these rats was also assessed. Pancreatic tissue samples were taken for histological assessment and for the determination of gene expression for CCK1 receptor by RT-PCR. Pancreatic amylase secretions stimulated by caerulein or by diversion of pancreatic-biliary juice to the exterior (DBPJ) was significantly, and dose-dependently reduced in the adult rats which have been subjected in infancy to chronic pretreatment with LPS from E. coli or S. typhi, as compared to the untreated control. In these animals basal secretion was unaffected. In the rats pretreated with LPS in the suckling period of life caerulein-induced amylase release from isolated pancreatic acini was significantly decreased, as compared to the untreated with LPS control. This was accompanied by dose-dependent reduction of mRNA signal for CCK1 receptor on pancreatic acini. Neonatal endotoxemia failed to affect significantly pancreatic morphology as well as plasma amylase level in the adult rats. We conclude that neonatal endotoxemia reduces gene expression for CCK1 receptor and could produce impairment of the exocrine pancreatic function at adult age.

Unlabelled: Previous studies have shown that ischemic preconditioning protects several organs, including the pancreas, from ischemia/reperfusion-induced injury. The aim of the investigation was to determine whether ischemic preconditioning affects the course edematous pancreatitis., Methods: In rats, ischemic preconditioning was performed by short-term clamping the celiac artery. Acute pancreatitis was induced by caerulein. The severity of acute pancreatitis was evaluated between the first and tenth day of inflammation., Results: Ischemic preconditioning applied alone caused a mild pancreatic damage. Combination of ischemic preconditioning with caerulein attenuated the severity of pancreatitis in histological examination and reduced the pancreatitis-evoked increase in plasma lipase and pro-inflammatory interleukin-1beta. This effect was associated with an increase in plasma level of anti-inflammatory interleukin-10 and partial reversion of the pancreatitis-evoked drop in pancreatic DNA synthesis and pancreatic blood flow. In secretory studies, ischemic preconditioning in combination with induction of acute pancreatitis attenuated the pancreatitis-evoked decrease in secretory reactivity of isolated pancreatic acini to stimulation by caerulein. In the initial period of acute pancreatitis, ischemic preconditioning alone and in combination with caerulein-induced acute pancreatitis prolonged the activated partial thromboplastin time (APTT), increased plasma level of D-dimer and shortened the euglobulin clot lysis time. The protective effect of ischemic preconditioning was observed during entire time of experiment and led to acceleration of pancreatic regeneration., Conclusions: Ischemic preconditioning reduces the severity of caerulein-induced pancreatitis and accelerates pancreatic repair; and this effect is related to the activation of fibrinolysis and reduction of inflammatory process.

Bacterial endotoxin (lipopolysaccharide, LPS), at high concentration is responsible for sepsis, and neonatal mortality, however low concentration of LPS protected the pancreas against acute damage. The aim of this study was to investigate the effect of exposition of suckling rats to LPS on the course of acute pancreatitis at adult age. Suckling rat (30-40g) received intraperitoneal (i.p.) injection of saline (control) or LPS from Escherichia coli or Salmonella typhi (5, 10 or 15 mg/kg-day) during 5 consecutive days. Two months later these rats have been subjected to i.p. cearulein infusion (25 microg/kg) to produce caerulein-induced pancreatitis (CIP). The following parameters were tested: pancreatic weight and morphology, plasma amylase and lipase activities, interleukin 1beta (IL-1 beta), interleukin 6 (IL-6), and interleukin 10 (IL-10) plasma concentrations. Pancreatic concentration of superoxide dismutase (SOD) and lipid peroxidation products; malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) have been also measured. Caerulein infusion produced CIP in all animals tested, that was confirmed by histological examination. In the rats, which have been subjected in the neonatal period of life to LPS at doses 10 or 15 mg/kg-day x 5 days, all manifestations of CIP have been reduced. In these animals acute inflammatory infiltration of pancreatic tissue and pancreatic cell vacuolization have been significantly diminished. Also pancreatic weight, plasma lipase and alpha-amylase activities, as well as plasma concentrations of IL-1beta and IL-6 have been markedly decreased, whereas plasma anti-inflammatory IL-10 concentration was significantly increased in these animals as compared to the control rats, subjected in the infancy to saline injection instead of LPS. Caerulein-induced fall in pancreatic SOD concentration was reversed and accompanied by significant reduction of MDA + 4 HNE in the pancreatic tissue. The effects of LPS derived from E. coli or S. typhi were similar. Pretreatment of suckling rats with LPS at dose of 10 mg/kg-day x 5 days resulted in the most prominent attenuation of acute pancreatitis at adult age, whereas LPS at dose of 5 mg/kg-day x 5 days given to the neonatal rats failed to affect significantly acute pancreatitis induced in these animals 2 months later. We conclude that: 1/ Prolonged exposition of suckling rats to bacterial endotoxin attenuated acute pancreatitis induced in these animals at adult age. 2/ This effect could be related to the increased concentration of antioxidative enzyme SO in the pancreatic tissue and to the modulation of cytokines production in these animals.

Ghrelin and melatonin are produced in the central nervous system and in the gastrointestinal tissues; ghrelin in the stomach, and melatonin - in the liver and in the intestine. Both ghrelin and melatonin have been reported to protect the gastric mucosa against acute lesions and to influence gastrointestinal motility and secretions, however the physiological significance of these peptides in the gastrointestinal tissues remains unknown. In spite of the presence of ghrelin and melatonin receptors in the pancreatic tissue little is known about the role of these peptides in the pancreas. It is very likely that both ghrelin and melatonin, which are released from the gastrointestinal tract in relation to food ingestion, could be implicated in the postprandial stimulation of pancreatic enzyme secretion though the activation of cholinergic entero-pancreatic reflex and CCK release. Our experimental studies have shown that exogenous melatonin, as well as this produced endogenously from its precursor; L-tryptophan, strongly stimulates pancreatic amylase secretion when given intraperitoneally, or into the gut lumen. Intraduodenal administration of ghrelin also increases pancreatic enzyme secretion. This was accompanied by significant increases of CCK plasma levels. Above pancreatostimulatory effects of luminal administration of melatonin or ghrelin were completely reversed by bilateral vagotomy, capsaicin deactivation of sensory nerves or pretreatment of the rats with CCK1 receptor antagonist; tarazepide. Our previous findings have revealed that melatonin, as well as its precursor; L-tryptophan, effectively protects the pancreas against the damage induced by caerulein overstimulation. The beneficial effects of melatonin and L-tryptophan on the pancreas have been related to the ability of melatonin to scavenge the radical oxygen species (ROS), to activate antioxidative enzymes and to modulate the cytokine production. It has been previously shown that systemic application of ghrelin attenuated acute pancreatitis activating the immune defense mechanisms. Our recent data demonstrate that ghrelin is able to prevent pancreatic inflammatory damage though the activation of central nervous mechanisms leading to the improvement of antioxidative properties of pancreatic tissue. The results of experimental studies indicated that melatonin and ghrelin could take a part in the protection of pancreatic tissue against the damage under physiological conditions.

Leptin, circulating protein involved in the control of body weight and energy expenditure received attention as a modulator of immune response of the organism. Leptin receptors have been detected in the pancreas and experimental studies have shown that leptin protects the pancreas against the damage induced by caerulein overstimulation. Heat shock proteins (HSP) are endogenous proteins produced by various cells exposed to high temperature or to the noxious agents. HSP protect the cells against various environmental and endogenous stressors. The implication of HSP60 in the leptin-induced pancreatic protection has not been examined yet. The aim of this study was: to investigate the changes of HSP60 mRNA signal in the pancreatic AR42J cells subjected to caerulein and leptin. AR42J cells were incubated in standart medium at 37 degrees C for: 0, 1, 3, 5, 12 or 24 h, under basal conditions. Incubation time of 3 h was selected for the next experiments. AR42J cells were incubated in presence of caerulein (10(-11), 10(-9) or 10(-7) M), leptin (10(-8) or 10(-6) M), or combination of above. Gene expression for HSP60 was determined by RT-PCR. The mRNA signal for HSP60 has been observed in AR42J pancreatic cells under basal conditions. Incubation of AR42J cells in presence of leptin (10(-8) or 10(-6) M) resulted in the significant increase of gene expression for HSP60 in both groups of AR42J cells. Caerulein stimulation reduced mRNA signal for HSP60. The strongest mRNA signal for HSP60 has been observed after the exposition of AR42J cells to combination of leptin and caerulein. We conclude that: 1. Gene expression for HSP60 has been detected in pancreatic AR42J cells under basal conditions. 2. HSP60 gene expression was significantly increased in pancreatic AR42J cells stimulated by leptin whereas caerulein reduced this signal. 3. The strongest gene expression for HSP60 has been detected in the cells incubated with combination of caerulein and leptin.

Heat shock proteins (HSPs) have been reported to protect the pancreatic cells from the acute damage produced by caerulein overstimulation. However the effects of caerulein, melatonin or hyperthermia preconditioning on mRNA signal for HSP60 in the pancreatic acinar cells has not been examined yet. The aims of this study were: 1. To investigate the gene expression for HSP60 in the pancreatic AR42J cells stimulated by melatonin, caerulein or combination of both these substances. 2. To compare above changes with mRNA signal for HSP60 in pancreatic AR42J cells subjected to hyperthermia preconditioning. AR42J cells were incubated in standard medium at 37 degrees C for: 0, 1, 3, 5, 12 or 24 h, under basal conditions. Above cells were then subjected to heat shock (42 degrees C) for 0, 1 or 3 h. In the next part of the study AR42J cells were incubated in presence of caerulein (10(-11), 10(-9) or 10( -7) M), melatonin (10(-8) or 10(-6) M), or combination of above under basal conditions or following heat shock pretreatment. Gene expression for HSP60 was determined by RT-PCR. The mRNA signal for HSP60 has been observed in AR42J cells under basal conditions, and this signal was markedly and time-dependently increased in these cells subjected to hyperthermia preconditioning. Incubation of AR42J cells in presence of melatonin (10(-8) or 10(-6) M) resulted in the significant and dose-dependent increase of gene expression for HSP60 in both groups of AR42J cells: preconditioned and in those, which were not subjected to hyperthermia. Caerulein stimulation reduced mRNA signal for HSP60. The strongest signal has been observed after the exposition of AR42J cells to hyperthermia preconditioning, combined with melatonin and caerulein. We conclude that: 1. Gene expression for HSP60 has been detected in pancreatic AR42J cells under basal conditions. 2. Hyperthermia preconditioning resulted in a significant and time-dependent increase of HSP60 signal in pancreatic AR42J cells. 3. HSP60 gene expression was significantly increased in pancreatic AR42J cells stimulated by melatonin whereas caerulein reduced this signal. 4. The strongest gene expression for HSP60 has been found in the cells subjected to the combination of hyperthermia preconditioning, caerulein and melatonin.

Melatonin, a pineal hormone, is also produced in the gastrointestinal tract. Melatonin receptors have been detected in the stomach, intestine and pancreas. This indole inhibits insulin secretion but its role in the physiological modulation of exocrine pancreatic function is yet unknown. The aim of this study was to evaluate the pancreatic secretory effect of melatonin and its precursor; L-tryptophan given intraduodenally (i.d.) to the conscious rats with intact or capsaicin deactivated sensory nerves. CCK(1) receptor antagonist; tarazepide, was used in the part of the study to determine the involvement of CCK in the secretory effects of melatonin. The secretory studies were performed on awaken rats surgically equipped with silicone catheters, one of them was inserted into pancreato-biliary duct, the other one--into duodenum. Melatonin (1, 5 or 25 mg/kg) or L-tryptophan (10, 50 or 250 mg/kg) were administered i.d. Samples of pancreatic juice were collected in 15 minutes aliquots. Tarazepide (2,5 mg/kg i.p.) was given to the rats 15 min prior to the administration of melatonin or L-tryptophan. Neurotoxic dose of capsaicin (100 mg/kg s.c.) was used to deactivate afferent nerves and thus to assess the role of these nerves in the melatonin-induced pancreatic enzyme secretion. Administration of melatonin (1, 5 or 25 mg/kg i.d.) or L-tryptophan (10, 50 or 250 mg/kg i.d.) significantly increased pancreatic amylase outputs. Deactivation of sensory nerves by capsaicin or administration of CCK(1) - receptor antagonist; tarazepide, reversed the stimulatory effects of melatonin or L-tryptophan on pancreatic secretory function. Administration of melatonin or its amino-acid precursor to the rats resulted in the significant and dose-dependent rises of melatonin and CCK plasma levels. We conclude that melatonin or its precursor; L-tryptophan stimulates pancreatic enzyme secretion via stimulation of CCK release and activation of duodeno-pancreatic reflexes.

Leptin, 16- kDa protein produced and secreted from white adipocytes is known to regulate food intake and energy expenditure. Leptin receptors have been detected in the pancreas and it has been shown that systemic application of this protein diminished postprandial pancreatic secretion. Leptin is also produced in the stomach and released into the gastrointestinal lumen but the implication of luminal leptin in the regulation of pancreatic enzyme secretion has not been elucidated. The aim of our study was to evaluate the effects of intraduodenal (i.d.) leptin administration on pancreatic enzyme secretion and to assess the involvement of afferent nerves and CCK in above effects. The secretory studies were carried out on anaesthetized Wistar rats with acute pancreatic fistulae. Leptin was administered to the animals at doses of 0.1 1.0 or 10.0 microg/kg i.d. Tarazepide (2.5 mg/kg i.d.), a CCK(1) receptor antagonist, was given to the rats prior to the application of leptin. Rats with capsaicin deactivated sensory nerves were used in part of the study. Samples of pancreatic juice were taken at 15 min intervals to measure the volume flow and protein and amylase concentrations. CCK plasma level was measured by radioimmunoassay (RIA) following administration of leptin to the rats. Intraduodenal administration of leptin (1.0 or 10.0 microg/kg) to the fasted rats significantly and dose-dependently increased pancreatic protein and amylase outputs. Pancreatic secretory responses to leptin were totally abolished by prior capsaicin deactivation of sensory nerves or by pretreatment of the rats with tarazepide. Under basal conditions plasma CCK level averaged about 15.46 +/- 1,4 pg/ml. Exogenous leptin, given i.d. at doses of 0.1 1.0 or 10.0 microg/kg i.d. to the rats with intact or capsaicin-deactivated sensory nerves resulted in dose-dependent rise of plasma CCK level, reaching the highest value at the dose of 10.0 microg/kg i.d. We conclude that leptin given i.d. stimulates pancreatic enzyme secretion and this effect could be related to the stimulation of CCK release and activation of duodeno-pancreatic reflexes.

Melatonin, produced from L-tryptophan, protects the pancreas against acute damage by improving the antioxidative status of tissue. Melatonin receptors have been detected in the brain, but the contribution of these receptors to the pancreatic protection is unknown. The aim of our study was to compare the effects of melatonin precursor; L-tryptophan given intracerebroventricularly (i.c.v.) or intraperitoneally (i.p.) on the course of acute pancreatitis. Acute pancreatitis was induced by subcutaneous infusion of caerulein (5 microg/kg-h x 5 h). L-tryptophan was given i.p. (2.5, 25 or 250 mg/kg) or administered into right cerebral ventricle (0.02, 0.2 or 2.0 mg/rat) 30 min prior to the start of caerulein infusion. Plasma amylase, lipase and TNF alpha activities were measured to determine the severity of caerulein-induced pancreatitis (CIP). The lipid peroxidation products: malonylodialdehyde and 4-hydroksynonenal (MDA + 4-HNE) and activity of superoxide dismutase (SOD) were measured in the pancreas of intact or CIP rats with or without L-tryptophan pretreatment. Melatonin blood level was measured by RIA. CIP was confirmed by histological examination and manifested as an edema and rises of plasma levels of amylase, lipase and TNF alpha (by 550%, 1000% and 600%). MDA + 4-HNE was increased by 600%, whereas SOD activity was reduced by 75% in the pancreas of CIP rats. All manifestations of CIP were significantly reduced by pretreatment of the rats with L-tryptophan given i.c.v. at doses of 0.2 or 2.0 mg/rat, or by peripheral administration of this amino acid used at dose of 250 mg/kg i.p. In control rats plasma level of melatonin averaged about 40 +/- 2 pg/ml and was not significantly affected by CIP, by central application of L-tryptophan (0.02, 0.2 or 2.0 mg/rat) or by peripheral administration of this melatonin precursor used at doses of 2.5 or 25 mg/kg i.p. Plasma melatonin level was markedly increased by pretreatment of the rats with L-tryptophan given i.p. at dose of 250 mg/kg. We conclude that central administration of melatonin precursor; L-tryptophan, as well as peripheral application of high dose of this melatonin precursor prevented the pancreatic damage produced by CIP. The favorable effect of peripherally administered L-tryptophan could be related to the rise of melatonin plasma level and to pancreatoprotective action of this indoleamine. The beneficial effect of centrally administered L-tryptophan could be mediated through activation of central receptors for locally produced melatonin.

Leptin released by adipocytes has been implicated in the control of food intake but recent detection of specific leptin receptors in the pancreas suggests that this peptide may also play some role in the modulation of pancreatic function. This study was undertaken to examine the effect of exogenous leptin on pancreatic enzyme secretion in vitro using isolated pancreatic acini, or in vivo in conscious rats with chronic pancreatic fistulae. Leptin plasma level was measured by radioimmunoassay following leptin administration to the animals. Intraperitoneal (i.p.) administration of leptin (0.1, 1, 5, 10, 20 or 50 microg/kg), failed to affect significantly basal secretion of pancreatic protein, but markedly reduced that stimulated by feeding. The strongest inhibition has been observed at dose of 10 microg/kg of leptin. Under basal conditions plasma leptin level averaged about 0.15 +/- 0.04 ng/ml and was increased by feeding up to 1.8 +/- 0.4 ng/ml. Administration of leptin dose-dependently augmented this plasma leptin level, reaching about 0.65 +/- 0.04 ng/ml at dose of 10 microg/kg of leptin. This dose of leptin completely abolished increase of pancreatic protein output produced by ordinary feeding, sham feeding or by diversion of pancreatic juice to the exterior. Leptin (10(-10)-10(-7) M) also dose-dependently attenuated caerulein-induced amylase release from isolated pancreatic acini, whereas basal enzyme secretion was unaffected. We conclude that leptin could take a part in the inhibition of postprandial pancreatic secretion and this effect could be related, at least in part, to the direct action of this peptide on pancreatic acini.

The present study investigated the involvement of endogenous melatonin in the prevention of pancreatic damage provoked by caerulein-induced pancreatitis (CIP) by using the luzindole, the antagonist of melatonin MT2 receptors. CIP was produced by subcutaneous infusion of caerulein to conscious rats (25 microg/kg). Luzindole (1, 2 or 4 mg/kg) was given as an intraperitoneal bolus injection 30 min prior to the start of CIP. Lipid peroxidation products, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were measured in the pancreas by LPO-584 commercial kit. CIP was confirmed by histological examination and manifested by significant increases of plasma activities of amylase, lipase and tumor necrosis factor alpha (TNFalpha) (by 500%, 1000% and 600%, respectively) comparing to the control values. This was accompanied by a 40% limitation in pancreatic blood flow (PBF) and by 200% increase of MDA+4-HNE in the pancreas of CIP rats. Administration of luzindole to the CIP rats reduced PBF, aggravated the histological manifestations of pancreatitis, resulted in the significant augmentation of pancreatic MDA + 4-HNE content, and produced the marked increases of plasma levels of lipase, amylase and TNFalpha, comparing to the values observes in the rats with CIP alone. These results suggest that endogenous melatonin through its receptor MT2 plays an important role in the attenuation of pancreatic damage produced by overstimulation with caerulein.

Leptin is involved in the regulation of food intake and previous studies have shown that leptin affects the inflammatory response in various tissues. The objective of this study was to examine the influence of leptin administration on the development and the course of acute ischemic pancreatitis. Acute pancreatitis was induced by limitation of pancreatic blood flow by clamping of inferior splenic artery for 30 min, followed by reperfusion. Leptin was administered three times daily at the dose 10 or 50 microg/kg. Animals were sacrificed 1, 3, 5, 10 and 21 days after removal of vascular clips. Administration of leptin reduced development of pancreatic damage and accelerated pancreatic regeneration what was manifested by the improvement of pancreatic histology, the decrease in serum lipase and amylase activity, and the reduction in serum interleukin-1beta concentration. Also, treatment with leptin caused the increase in the pancreatic blood flow and pancreatic DNA synthesis. Leptin administration was without effect on serum interleukin-10 concentration. Leptin at the dose 50 microg/kg was more effective than 10 microg/kg. We conclude that leptin reduces the pancreatic damage in the course of ischemic pancreatitis and accelerates the pancreatic tissue repair. The beneficial effects of leptin appear to be dependent on the improvement of pancreatic blood flow, the increase in pancreatic cell growth, and the limitation of pro-inflammatory interleukin-1beta release.

Central nervous system affects pancreatic secretion of enzymes however, the neural modulation of acute pancreatitis has not been investigated. Leptin and melatonin have been recently reported to affect the inflammatory response of various tissues. The identification of specific receptors for both peptides in the pancreas suggests that leptin and melatonin could contribute to the pancreatic protection against inflammation. The aim of this study was: 1/ to compare the effect of intracerebroventricular (i.c.v.) or intraperitoneal (i.p.) administration of leptin or melatonin on the course of caerulein-induced pancreatitis (CIP) in the rat, 2/ to examine the involvement of sensory nerves (SN) and calcitonin gene-related peptide (CGRP) in pancreatic protection afforded by leptin or melatonin, 3/ to assess the effect of tested peptides on lipid peroxidation products (MDA + 4-HNE) in the pancreas of CIP rats, 4/ to investigate the influence of leptin or melatonin on nitric oxide (NO) release from isolated pancreatic acini and 5/ to determine the effects of caerulein and leptin on leptin receptor gene expression in these acini by RT-PCR. CIP was induced by subcutaneous (s.c.) infusion of caerulein (25 microg/kg) to the conscious rats, confirmed by the significant increases of pancreatic weight and plasma amylase and by histological examination. This was accompanied in marked reduction of pancreatic blood flow and significant rise of MDA + 4-HNE in the pancreas. Leptin or melatonin were administered i.p. or i.c.v. 30 min prior to the start of CIP. Deactivation of SN was produced by s.c. capsaicin (100 mg/kg). An antagonist of CGRP, CGRP 8-37 (100 microg/kg i.p.), was given together with leptin or melatonin to the CIP rats. MDA + 4-HNE was measured using LPO commercial kit. NO was determined using the Griess reaction. Pretreatment of CIP rats with i.p. leptin (2 or 10 microg/kg) or melatonin (10 or 50 mg/kg) significantly attenuated the severity of CIP. Similar protective effects were observed following i.c.v. application of leptin (0.4 or 2 microg/rat) but not melatonin (10 or 40 microg/rat) to the CIP rats. Capsaicin deactivation of SN oradministration of CGRP 8-37 abolished above beneficial effects of leptin on CIP, whereas melatonin-induced protection of pancreas was unaffected. Pretreatment with i.p. melatonin (10 or 50 mg/kg), but not leptin, significantly reduced MDA + 4-HNE in the pancreas of CIP rats. Leptin (10(-10) - 10(-6) M) but not melatonin (10(-8) - 10(-5) M) significantly stimulated NO release from isolated pancreatic acini. Leptin receptor gene expression in these acini was significantly increased by caerulein and leptin. We conclude that 1/ central or peripheral pretreatment with leptin protects the pancreas against its damage induced by CIP, whereas melatonin exerts its protective effect only when given i.p., but not following its i.c.v. adminstration, 2/ activation of leptin receptor in the pancreatic acini appears to be involved in the beneficial effects of leptin on acute pancreatitis, 3/ the protective effects of leptin involve sensory nerves, CGRP and increased generation of NO whereas melatonin-induced protection of the pancreas depends mainly on the antioxidant local effect of this indole, and scavenging of the radical oxygen species in the pancreatic tissue.

Prostaglandins (PG), the products of arachidonate metabolism through cyclooxygenase (COX) pathway, protect the pancreas from the acute damage. The existence of two isoforms of COX was documented including: COX-1, present in normal tissues and COX-2, expressed at the site of inflammation, such as induced by bacterial lipopolysaccharide (LPS). Pretreatment with low dose of LPS and activation of nitric oxide (NO) synthase (NOS) has been shown to prevent the injury caused by caerulein-induced pancreatitis (CIP) in the rat. The aim of this study was to investigate the role of COX-1 and COX-2 in the LPS-induced protection of the pancreas against CIP and the involvement of NOS in the activation of COX-PG system in the rats with CIP. CIP was produced by subcutaneous (s.c.) infusion of caerulein (5 microg/kg-h for 5 h) to the conscious rats. Protective dose of LPS, from Escherichia coli, (1 mg/kg) was given intraperitoneally (i.p.) 15 min prior to the start of CIP. Nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg), selective inhibitor of COX-1: resveratrol, or a highly selective inhibitors of COX-2: rofecoxib or NS-398 (2 or 10 mg/kg) were injected i.p. 15 min prior to the administration of LPS. COX-1 or COX-2 mRNA was determined by reverse transcription-polimerase chain reaction (RT-PCR) in the pancreatic tissue. Pancreatic blood flow (PBF) was measured by a laser Doppler flowmetry. PGE2 content in the pancreas was measured by radioimmunoassay. CIP was manifested by an increase of pancreatic weight and plasma amylase activity (by 500% and 700%, respectively) and it was confirmed by histological examination. CIP slightly increased pancreatic PGE2 generation (by 12%) and diminished PBF (by about 40%). LPS (1 mg/kg i.p.), given prior to the start of CIP, increased PGE2 generation in the pancreas (by 45%), reversed the histological manifestations of pancreatitis, reduced the rise in amylase blood level and improved PBF. Administration of nonselective inhibitor of COX; indomethacin (5 or 10 mg/kg i.p.) prior to the injection of LPS abolished its protective effects on CIP and reduced pancreatic PGE2 generation. Selective inhibitor of COX-1; resveratrol (10 mg/kg i.p.) given prior to the injection of LPS reversed its protective effects against CIP. Pretreatment with a selective inhibitors of COX-2: rofecoxib or NS-398 (10 mg/kg) attenuated LPS-induced pancreatic protection in the CIP rats. COX-1 expression was detected in the intact pancreas and was not significantly changed by CIP, LPS, indomethacin, NS-389 and their combination, while COX-2 mRNA expression appeared in the pancreas of ratssubjected to CIP and was significantly increased after LPS injection to these rats. Addition of selective COX-2 inhibitor; NS-389, or nonselective inhibitor of COX; indomethacin, enhanced COX-2 mRNA expression in the rats with CIP pretreated with LPS. Pretreatment of the rats with inhibitor of NOS; L-NNA (20 mg/kg i.p.), given together with LPS, 15 min prior to the start of caerulein overstimulation, resulted in complete reversion of LPS-induced pancreatic protection and decreased PGE2 generation stimulated by LPS. Addition to L-NNA of the substrate for NOS; L-arginine (100 mg/kg i.p.), restored pancreatic protection afforded by low dose of LPS and increased pancreatic PGE2 level in the rats with CIP. We conclude that: 1. increased pancreatic PGE2 generation, induced by low dose LPS pretreatment, contributes to the pancreatic resistance to acute damage produced by caerulein overstimulation and 2. the NO-system is involved in above stimulation of PGE2 generation and pancreatic protection against acute damage.

Calcitonin gene related peptide (CGRP) is widely distributed in the myenteric neurons along GI tract. Biological effects of CGRP on gastrointestinal tract include increase in the intestinal blood flow, relaxation of the smoth muscle and slight increase in the slow wave amplitude (SWA) with no effect on frequency (SWF) of intestinal myoelectric activity (IMA). The aim of this study was to evaluate the possible protective effects of endo- and exogenous CGRP against ischemia/reperfusion (I/R) induced bowel injury in rats. Experiments were performed on 30 fasted anesthetized Wistar rats. Systemic arterial blood pressure (AP), mesenteric blood flow (MBF) and microcirculatory intestinal blood flow (LDBF) were measured. Oxygen consumption (VO2) was estimated from MBF and mesenteric AVO2 difference. IMA was recorded by 4 monopolar electrodes in proximal jejunum and analyzed using computer program with Fourrier analysis of SWF. Control ischemia induced by 30 min total occlusion of anterior mesenteric artery (AMA) reduced SWA by 25+/-5% and SWF by 24+/-4%. At the end of 60 minute reperfusion period SWA and SWF values were restored to control values but SWF showed instability. At the 15th minute of reperfusion period MBF, LDBF and VO2 increased to 109+/-6, 119+/-11 and 120+/-7% of control values respectively. Infusion of exogenous CGRP (0,16 microg/kg/min i.a.) increased MBF, LDBF and VO2 by 26+/-6, 31+/-9 and 20+/-4% respectively in comparison to control values. In the same experimental group SWA increase of 14% was observed with not significant changes in SWF. In the group where CGRP was administrated before and during 30 min period of intestinal ischemia MBF, LDBF and VO2 values at 15th minute of reperfusion were significantly higher by 24+/-6, 32+/-7 and 17+/-5% respectively in comparison to the values observed in the control reperfusion. In that group SWA values were restored to preocclusion values at 15th minute of reperfusion yet and SWF showed much more stability. Infusion of CGRP receptor antagonist (CGRP 8-37) reduced MBF, LDBF and VO2 by 12+/-7, 14+/-8 and 11+/-6% respectively (differences not significant versus control). In I/R group when CGRP 8-37 was given hemodynamic parameters (during reperfusion) were significantly lower and IMA parameters were not restored to preocclusion values. We conclude, that endogenous and exogenous CGRP attenuate circulatory parameters of the small intestine during ischemia and reperfusion. A direct correlation exists between hemodynamic, metabolic and myoelectric effects of CGRP.

Background: Recent studies clearly demonstrate that Helicobacter pylori (H. pylori) infection of the stomach causes persistent elevation of ammonia (NH3) in gastric juice leading to hypergastrinemia and enhanced pancreatic enzyme secretion., Methods: The aim of this study is to evaluate the influence of NH4OH on plasma gastrin level and exocrine pancreatic secretion in vivo in conscious dogs equipped with chronic pancreatic fistulas and on secretory activity of in vitro isolated acini obtained from the rat pancreas by collagenase digestion. The effects of NH4OH on amylase release from pancreatic acini were compared with those produced by simple alkalization of these acini with NaOH., Results: NH4OH given intraduodenally (i.d.) in increasing concentrations (0.5, 1.0, 2.0, 4.0, or 8.0 mM/L) resulted in an increase of pancreatic protein output, reaching respectively 9%, 10%, 19%, 16% and 17% of caerulein maximum in these animals and in a marked increase in plasma gastrin level. NH4OH (8 x 0 mM/L, i.d.) given during intravenous (i.v.) infusion of secretin (50 pmol/kg-h) and cholecystokinin (50 pmol/kg-h) reduced the HCO3 and protein outputs by 35% and 37% respectively, as compared to control obtained with infusion of secretin plus cholecystokinin alone. When pancreatic secretion was stimulated by ordinary feeding the same amount of NH4OH administered i.d. decreased the HCO3- and protein responses by 78% and 47% respectively, and had no significant effect on postprandial plasma gastrin. In isolated pancreatic acini, increasing concentrations of NH4OH (10(-7)-10(-4) M) produced a concentration-dependent stimulation of amylase release, reaching about 43% of caerulein-induced maximum. When various concentrations of NH4OH were added to submaximal concentration of caerulein (10(-12) M) or urecholine (10(-5) M), the enzyme secretion was reduced at a dose 10(-5) M of NH4OH by 38% or 40%, respectively. Simple alkalization with NaOH of the incubation medium up to pH 8.5 markedly stimulated basal amylase secretion from isolated pancreatic acini, whereas the secretory response of these acini to pancreatic secretagogues was significantly diminished by about 30%. LDH release into the incubation medium was not significantly changed in all tests indicating that NH4OH did not produce any apparent damage of pancreatic acini and this was confirmed by histological examination of these acini., Conclusions: 1. NH4OH affects basal and stimulated pancreatic secretion. 2. The excessive release of gastrin may be responsible for the stimulation of basal pancreatic enzyme secretion in conscious animals, and 3. The inhibitory effects of NH4OH on stimulated secretion might be mediated, at least in part, by its direct action on the isolated pancreatic acini possibly due to the alkalization of these acini.

Lipopolysaccharide (LPS) derived from the bacterial cell wall activates the inflammatory response in the tissue but the role of LPS in the pathogenesis of pancreatic damage and in the activation of NO system in the pancreas has not been fully explained. The aim of this study was to investigate the effect of repeated administration of LPS to the rats on the integrity of the pancreas, on the ability of isolated pancreatic acini to secrete the amylase and on the plasma level of tumor necrosis factor alpha (TNFalpha). The role of NO in the pancreatic resistance to the damage was assessed in animals subjected to repeated administration of LPS. To induce pancreatic damage one group of rats received intraperitoneal (i.p.) injection of LPS (from E. coli) every day during 5 consecutive days (10 mg/kg--day). Another groups of animals were given N(G)-nitro-L-arginine (L-NNA), an inhibitor of NO synthase (NOS) (20 mg/kg i.p.) alone or in combination with L-arginine (100 mg/kg i.p.), 30 min prior to each LPS injection. Plasma level of TNFalpha was determined by ELISA kit. Repeated administration of LPS produced mild pancreatic inflammation that was most pronounced at day 5 of LPS treatment and manifested as edema, neutrophil infiltration and hemorrhage of the pancreas. The survival rate after 5 days treatment with LPS was 87.5%. Pancreatic weight, plasma levels of TNFalpha and amylase, pancreatic blood flow (PBF) and NO generation by pancreatic acini were markedly increased in rats subjected to repeated administration of LPS whereas the amylase response of isolated pancreatic acini to pancreatic secretagogues was significantly attenuated. Suppression of NOS by L-NNA resulted in a dramatic increase in the mortality of the animals reaching 50% and significantly increased inflammatory changes in the pancreatic tissue, decreased PBF, abolished the ability of pancreatic acini to release NO and to secrete amylase. Pancreatic weight and plasma levels of amylase and TNFalpha significantly increased in the group of rats treated with combination of LPS+L-NNA as compared to the animals received LPS alone. Addition of L-arginine to L-NNA+LPS administration reversed all harmful effects produced by L-NNA in the pancreas. We conclude that repeated administration of high doses of bacterial LPS to the rats could induce pancreatic tissue damage by itself, however, it is not able to produce severe pancreatitis. Suppression of NO generation significantly aggravates the pancreatic lesion produced by LPS leading to the dramatic mortality in treated rats. The rise of plasma level of TNFalpha corresponds to the severity of pancreatic inflammation.

Sensory nerves are implicated in gastroprotection and regulation of visceral circulation but their role in exocrine secretion and pancreatic circulation in intact pancreas and in acute pancreatitis has not been established. We investigated the role of sensory fibers in pancreatic secretion in vivo and amylase release from pancreatic slices (containing nerve fibers) or isolated pancreatic acini, and in caerulein-induced pancreatitis. In conscious rats, the stimulation of sensory nerves by low dose of capsaicin given intraduodenally (0.25-0.5 mg/kg) reduced basal pancreatic secretion, whereas dose of 1 mg/kg increased this secretion. Deactivation of sensory nerves by neurotoxic dose of capsaicin (100 mg/kg over 3 days s.c.) 10 days before tests failed to affect basal secretion but diminished the secretion induced by feeding or the diversion of pancreatic juice. In pancreatic slices, capsaicin (10(-10)-10(-6) M) increased enzyme secretion and this response was abolished by atropine (10(-6) M) or previous deactivation of sensory nerves. In pancreatic acini, capsaicin failed to affect basal and stimulated amylase secretion in response to caerulein or urecholine. In intact rats, stimulatory dose of capsaicin (0.5 mg/kg i.g.) caused about 32% increase of pancreatic blood flow and it was without any effect on the pancreatic DNA synthesis, weight, RNA, DNA and protein content. In contrast, neurotoxic dose of capsaicin caused a reduction (by 27%) in pancreatic blood flow followed by a significant decrease in RNA content and DNA synthesis in pancreatic tissue. Infusion of caerulein (10 g/kg-h) for 5 h produced acute edematous pancreatitis accompanied by over 60% decrease in DNA synthesis, nearly 50% inhibition of pancreatic blood flow, and a significant increase in pancreatic weight, protein content and plasma amylase concentration. Stimulatory dose of capsaicin attenuated the pancreatic tissue damage in caerulein induced pancreatitis, as manifested by a significant reversal of pancreatic blood flow and DNA synthesis decrease. Capsaicin induced inactivation of sensory nerves prior to pancreatitis caused an increase of all parameters of pancreatic damage; pancreatic blood flow dropped by 68%, DNA synthesis decreased by 70%; pancreatic weight, protein content and plasma amylase were also significantly enhanced. We conclude that sensory neurons are involved in the regulation of pancreatic secretion by an indirect mechanism and exhibit a beneficial effect on the pancreatic integrity, mainly due to improving the pancreatic blood flow.

Exogenous eicosanoids were reported to affect pancreatic secretion, but it is unknown whether this effects is mediated by the changes in pancreatic circulation or by direct action on pancreatic secretory cells. In this study the effects of prostaglandins (PG), leukotriene C4 (LTC4) and platelet activating factor (PAF) and the blockers of their biosynthesis or receptor antagonists on amylase release from the isolated rat pancreatic acini were examined. The acini were incubated with pancreatic secretagogues, such as caerulein or urecholine in the presence or absence of various concentrations (10(-9)-10(-5) M) of PGE2, Nocloprost (stable analog of PGE2), PGI2, PGF1 alpha, LTC4, PAF, indomethacin (inhibitor of endogenous PG formation) and A-63162 (blocker of endogenous LT biosynthesis). PGE2, PGI2 and PGF1 alpha inhibited secretagogues-stimulated enzyme secretion from isolated pancreatic acini but their inhibitory potency was about 1000 times lower, on molar basis, than that of Nocloprost. PAF and LTC4 produced concentration-dependent stimulation of amylase release from the unstimulated acini reaching about 50% of caerulein-induced maximal response and enhanced amylase release induced by caerulein or urecholine. The effects of PAF and LTC4 were reversed by the addition of respective receptor antagonist for PAF (TCV-309) and for LTC4 (FPL-55712). These results indicate that PG inhibit, while PAF and LTC4 stimulate pancreatic enzyme secretion and thus could be implicated in the control of this secretion.

Tachykinins (TK) are family of peptides including substance P (SP), substance K (SK) and neuromedin K (NK) that have been found in the nerves of the gastrointestinal tract and proposed to act as neurotransmitters to affect the motor, secretory and circulatory functions of the gut, but little is known about their action on the pancreas. In this study three series of tests were carried out to determine the action of SP, SK and NK on pancreatic secretion in conscious dogs and amylase release from the dispersed rat pancreatic acini and to correlate the alterations in pancreatic secretory and circulatory effects of TK in anesthetized dogs. SP, SK and NK infused i.v. in graded doses (0.12-1.0 microgram/kg per h) in conscious dogs stimulated pancreatic protein outputs reaching, respectively, 38% and 23% of the maximal response to CCK (40 pmol/kg per h). HCO3- outputs were also significantly increased but the highest response did not exceed about 5% of secretin (328 pmol/kg per h) maximum. Cholinergic blockade by atropine abolished the pancreatic responses to tachykinins. When added at various concentrations (10(-11)-10(-7) M) to the incubation medium of rat dispersed pancreatic acini, SK, SP and NK increased in concentration-dependent manner the release of amylase from the resting pancreatic acini and augmented the enzyme release induced by CCK-8 and by urecholine. In anesthetized dogs infused with a background dose of secretin (82 pmol/kg per h), addition of SP, SK and NK caused an immediate and dose-dependent increase in the pancreatic blood flow, oxygen consumption and pancreatic secretion accompanied by a dose-dependent decrease in arterial blood pressure. This study shows that TK are potent pancreatic circulatory stimulants and moderate secretagogues both in vivo and in vitro, acting, at least in part, via cholinergic pathway.