Kynuramines induce overexpression of heat shock proteins in pancreatic cancer cells via 5-hydroxytryptamine and MT1/MT2 receptors.

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 HSP90αß 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 HSP90αß 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.

The dynamics of heat shock system activation in Monomac-6 cells upon Helicobacter pylori infection.

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.

Brain-gut axis in the modulation of pancreatic enzyme secretion.

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.

Effect of neonatal endotoxemia on the pancreas of adult rats.

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.

Melatonin stimulates HSP27 phosphorylation in human pancreatic carcinoma cells (PANC-1).

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).

Exposition of newborn rats to bacterial endotoxin impairs pancreatic enzyme secretion at adult age.

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.

Endotoxemia in newborn rats attenuates acute pancreatitis at adult age.

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.

Melatonin as modulator of pancreatic enzyme secretion and pancreatoprotector.

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.

Involvement of vagal nerves in the pancreatostimulatory effects of luminal melatonin, or its precursor L-tryptophan. Study in the rats.

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.

Modulation of pancreatic enzyme secretion by melatonin and its precursor; L-tryptophan. Role of CCK and afferent nerves.

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 is able to stimulate pancreatic enzyme secretion via activation of duodeno-pancreatic reflex and CCK release.

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 precursor; L-tryptophan protects the pancreas from development of acute pancreatitis through the central site of action.

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.

Role of endogenous melatonin and its MT2 receptor in the modulation of caerulein-induced pancreatitis in the rat.

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.