Ethacrynic acid inhibits pancreatic exocrine secretion.

AIM: The effect of ethacrynic acid on pancreatic exocrine secretion function and potential mechanisms of interference with the secretory process in pancreatic acinar cells were investigated. METHODS: After incubation with ethacrynic acid for 30 min, caerulein-stimulated amylase release and cholecystokinin (CCK) receptor binding characteristics were assessed in isolated rat pancreatic acini. The level of thiol groups (glutathione and protein thiols) and cytosolic free calcium were measured in pancreatic acinar cells. RESULTS: Ethacrynic acid decreased caerulein (0.1 nmol/L)-stimulated amylase release and the level of pancreatic acinar glutathione in a concentration-dependent fashion without a marked increase in cell damage. Ethacrynic acid also inhibited the caerulein (1 nmol/L)-induced Ca2+ mobilization in pancreatic acinar cells. But neither protein thiol nor CCK-receptor binding characteristics was altered by ethacrynic acid. CONCLUSION: Ethacrynic acid inhibit pancreatic exocrine secretion by depletion of glutathione and down-regulation of caerulein-induced Ca2+ mobilization. Glutathione might play a potential role in the secretory process in pancreatic acinar cells and in the secretory blockade observed in acute pancreatitis.

Menadione-induced oxidative stress inhibits cholecystokinin-stimulated secretion of pancreatic acini by cell dehydration.

The present study evaluated the effects of free radicals generated by menadione on morphology and function of pancreatic acinar cells focusing on enzyme secretion, stimulus-secretion coupling, and cell hydration. Various experiments evaluated morphology and function of isolated rat pancreatic acinar cells exposed to menadione. Menadione instantaneously generated free radicals (luminol and deoxyribose assays) followed by a time-dependent cell injury (uptake of trypan blue). Early ultrastructural changes included vacuolization and alterations of mitochondria, endoplasmic reticulum, and nucleus. Menadione caused a rapid glutathione oxidation followed by a depletion in reduced glutathione. An increase in lipid peroxides and a depletion of adenosine triphosphate were seen only after 30-60 minutes. Menadione markedly inhibited amylase release stimulated by cholecystokinin (CCK) and carbachol and simultaneously caused cell shrinkage after a few minutes. Similar degrees of cell shrinkage induced by hyperosmolar incubation and by menadione inhibited amylase secretion to a similar extent. CCK binding and its effect on calcium and inositol 1,4,5-trisphosphate (IP3) were not affected by menadione. Menadione (without CCK) induced an instantaneous increase of intracellular calcium followed by a slow constant increase. In single cells, menadione induced calcium oscillations with a frequency lower than that seen after CCK stimulation. Some morphologic and functional alterations owing to menadione-induced oxidative stress may be caused by adenosine triphosphate and glutathione depletion, lipid peroxidation, and changes in cytosolic calcium. The marked inhibition of secretagogue-stimulated enzyme secretion owing to menadione may be mediated to a large part by cell dehydration, whereas classical steps of stimulus-secretion coupling like receptor binding, calcium release, and IP3 generation remained unchanged.

Oxidative stress in acute pancreatitis.

The present work critically reviews the evidence for an involvement of free radicals in the pathophysiology of acute pancreatitis and the potential of treatment with antioxidants and scavenger substances. Data originating from clinical trials, experimental pancreatitis studies and in vitro investigations are included. Enhanced free radical activities and increased concentrations of lipid peroxides in plasma and tissue have been found in both patients and experimental animals with acute pancreatitis. The individual contribution of possible sources of free radicals (e.g., invading inflammatory cells, xanthine oxidase, cytochromes P450, nitric oxide synthase) is not yet clear, however. Since prophylactic administration of antioxidants diminished, in particular, pancreatic edema formation, free radicals seem to play an important role in the genesis of edema in acute pancreatitis. An involvement of free radicals in the pathogenesis of pancreatic necrosis could not yet be proven. Thus, no antioxidant treatment has proven useful for therapy of fulminant pancreatitis in animals to date. However, in severe acute pancreatitis characterized by death occurring after 12-18 hours, the seleno-organic compound Ebselen, which has a glutathione peroxidase-like activity, and the membrane permeable ascorbic acid derivative CV-3611 have been demonstrated to be effective. To date, controlled clinical studies have failed to demonstrate the therapeutic efficacy of antioxidant selenium or glutathione precursor supplementation. Therefore, further controlled clinical trials are needed to determine whether supplements of antioxidants can alter the clinical course of acute pancreatitis. Since the nitric oxide radical may even protect the pancreas, a purely negative discussion of the role of free radicals on the pancreas is not justified. The actual role of free radicals in acute pancreatitis, i.e. serving the body's defense against infection, being an epiphenomenon of the inflammatory process without pathophysiological relevance, or having true pathogenic significance, is not yet clear. Lipid peroxidation may perhaps not be the cause but rather the sequel of pancreatic inflammation and may likely reflect the severity of the systemic inflammatory response rather than that of pancreatic parenchyma damage. In vitro, exposure of isolated pancreatic acinar cells to oxidative stress caused rapid cell damage and death. Such knowledge from cellular studies might help to plan therapeutical trials to evaluate potentially effective therapies in the experimental animal, as well as in patients suffering from pancreatitis. Thus, to further clarify the role of oxidative stress in acute pancreatitis, an integrated approach is needed, including investigations at various biological levels, from isolated cells or even organelles to laboratory animals and, finally, clinical studies in man.

The role of calcium in pancreatitis.

BACKGROUND/AIMS: A large, sustained increase in acinar [Ca2+]i may play a key role in the pathogenesis of acute pancreatitis. Many mechanisms which lead to cell damage in vitro and pancreatitis in vivo, such as free radicals or supraphysiological cerulein concentrations, cause a rapid increase in [Ca2+]i in pancreatic acinar cells. Little is known about why [Ca2+]i increases in some instances stimulate secretion and in other instances initiate cell death. So far, [Ca2+]i increases were thought to represent physiological signals when they occurred as oscillations at the single cell level. METHODOLOGY: This paper reviews recent literature and our own original research about the role of calcium in the function of pancreatic acinar cells and the development of pancreatitis. RESULTS: Recent studies showed that exposure of acinar cells to free radicals not only caused a bulk increase in [Ca2+]i but also resulted in calcium oscillations which had a lower frequency than, but similar amplitude to oscillations occurring after physiological stimuli. The absolute increase in [Ca2+]i did not definitely determine the cellular response. Instead, the duration of [Ca2+]i increase may have been more important. In contrast to previous belief of a direct relationship between [Ca2+]i oscillations and exocytosis, recent results show that radicals can induce [Ca2+]i oscillations which do not exert exocytosis but inhibit the secretory response to physiological stimuli. Further experiments showed that the [Ca2+]i release caused by radicals originates from thapsigargin-insensitive, ryanodine-sensitive stores. CONCLUSIONS: The origin and duration of [Ca2+]i increases rather than their extent or oscillatory nature, determine whether the cell will secrete or die. An abnormal [Ca2+]i increase can trigger trypsin activation, acinar cell damage and acute pancreatitis. This hypothesis is supported by studies which show that calcium chelators inhibit radical-induced trypsin activation as well as cell necrosis and apoptosis. Thus, an inhibition of pathological [Ca2+]i release may have a therapeutic potential.

Supraphysiologic concentrations of cerulein induce apoptosis in the rat pancreatic acinar cell line AR4-2J.

Little is known as yet about the role of apoptosis in pancreatic damage. This study evaluated the effects of supraphysiologic concentrations of the cholecystokinin (CCK) analog, cerulein, which causes cell damage in vitro and acute pancreatitis in vivo, on cell proliferation and DNA fragmentation in the rat pancreatic acinar cell line AR4-2J. Cerulein inhibited the cell proliferation of AR4-2J time- and dose-dependently to approximately 60% of the control level at 10(-6) M after 72 h. DNA fragmentation, as assessed by both electrophoresis and enzyme-linked immunosorbent assay (ELISA), occurred at cerulein concentrations > or = 10(-8) M. The maximal DNA fragmentation as measured by ELISA was reached after 24 h. Cerulein at concentrations > or = 10(-9) M induced wild-type p53. Glutathione (1 mM) diminished the effects of cerulein on both cell proliferation and DNA fragmentation, whereas spermine (100 microM), which partially attenuated DNA fragmentation, did not have an effect on cell proliferation. The CCK-A-receptor antagonist loxiglumide completely abolished the effect of cerulein on DNA fragmentation. The serine-protease inhibitor FUT-175 (10 microM), the cysteine-protease inhibitor NCO-700 (5 mM), and ethylene glycol tetraacetic acid (EGTA; 500 microM) all had no effects on the changes in cell proliferation and DNA fragmentation induced by cerulein. The data suggest that supraphysiologic concentrations of cerulein rapidly induce apoptosis in AR4-2J cells and only later inhibit cell proliferation. These effects are mediated by CCK-A receptors. Cerulein-induced apoptosis may involve the induction of wild-type p53 or glutathione depletion or both.

Long-term effects of CCK-agonist and -antagonist on food intake and body weight in Zucker lean and obese rats.

The present study evaluates long-term effects of the CCK-agonist caerulein and the CCK-A antagonist loxiglumide in obese and lean Zucker rats. Caerulein and loxiglumide altered food intake neither in obese nor in lean rats. By as yet unknown mechanisms, however, weight increase was accelerated by loxiglumide and reduced by caerulein in obese and lean rats. Caerulein increased pancreatic weight only in lean but not in obese rats. Thus, obese rats show a resistance of pancreatic CCK-A receptors. The failure of CCK-agonist and -antagonist to alter food intake suggests that this CCK-resistance is not responsible for obesity in the genetically altered rats.

Inhibition of cathepsin B does not affect the intracellular activation of trypsinogen by cerulein hyperstimulation in isolated rat pancreatic acinar cells.

Activation of trypsinogen is thought to trigger the autodigestive process in acute pancreatitis. The lysosomal enzyme cathepsin B was suggested to cause the activation of trypsinogen because it is known that cathepsin B is able to activate trypsinogen in special circumstances and that lysosomal and digestive enzymes are colocalized within intracellular vacuoles in the early stage of pancreatitis. As yet this hypothesis has been difficult to prove because activated trypsin is difficult to quantify in pancreatitis by conventional enzymatic measurements. We therefore employed an ELISA for trypsin activating peptide (TAP), which is a small peptide cleaved during the activation of trypsinogen and can be determined reliably. Supraphysiological concentrations of cerulein (1 nM-1 microM) resulted in a marked increase in TAP in freshly isolated pancreatic acinar cells, indicating activation of trypsinogen. This activation as determined by the TAP increase was significantly reduced by the serine protease inhibitor Fut-175 but not by the cathepsin B inhibitors E-64 and NCO-700. The concentrations of NCO-700 and E-64 abolished the cathepsin B activity of pancreatic acinar cells but did not significantly reduce the trypsin activity (after enterokinase preincubation); correspondingly the concentrations of Fut-175 used abolished the trypsin activity but did not reduce the cathepsin B activity. The results indicate that an autoactivation of trypsin rather than an activation of trypsinogen by cathepsin B triggers trypsin activation by supramaximal cerulein concentrations.

Cell volume changes modulate cholecystokinin- and carbachol-stimulated amylase release in isolated rat pancreatic acini.

BACKGROUND & AIMS: Changes in cell volume have been recently identified as modulators of cell function and gene expression. This study evaluated the regulation of exocrine secretion by pancreatic acini on the basis of changes in cell hydration. METHODS: Acini were exposed to hypotonicity or hypertonicity. The effects of corresponding changes in cell volume on various cell functions were analyzed. RESULTS: Hypertonicity and hypotonicity caused a stepwise cell shrinkage and swelling, respectively. Cell shrinkage decreased and cell swelling increased amylase secretion stimulated by cholecystokinin (CCK) and carbachol but not by secretin. Changes in cell volume did not alter basal or CCK-stimulated calcium concentrations or CCK-stimulated inositol triphosphate generation. The regulation of secretion by cell volume is not mediated via changes in CCK receptor binding or protein kinase C. The increase of amylase release caused by hypotonicity was completely inhibited by cytochalasin B, colchicine, and genistein. Hypotonicity as well as CCK caused activation of mitogen-activated protein kinases. CONCLUSIONS: Changes in cell volume regulate exocrine secretion of pancreatic acini. The effects were found only for secretagogues that act via the calcium/inositol-trisphosphate pathway. However, the mechanisms involved are located at luminal parts of the signal-transduction cascade and involve the cytoskeleton, protein phosphorylation, and activation of mitogen-activated protein kinases.

Cytotoxicity of peroxynitrite in rat pancreatic acinar AR4-2J cells.

Peroxynitrite (0.5-50 microM) induced dose-dependent cytotoxic effects in rat pancreatic acinar AR4-2J cells. Glutathione (2 mM) and ebselen (10 microM) partially reduced the cytotoxicity caused by 1-10 microM concentrations of peroxynitrite. Higher concentrations (10-50 microM) of peroxynitrite induced DNA smear suggestive of necrosis, while lower concentrations (2-5 microM) induced DNA fragmentations suggestive of apoptosis. The effects of peroxynitrite on [Ca2+]i showed a similar dose dependency. Peroxynitrite concentrations > 10 microM rapidly increased [Ca2+]i in a dose-dependent manner, while concentrations < 5 microM did not affect [Ca2+]i. In contrast, the presentation of wild-type P53 was accelerated at lower concentrations of peroxynitrite (< or = 10 microM) but not at higher concentrations (50 microM). The present study suggests that peroxynitrite at lower concentrations (2-5 microM) induces wildtype P53 and apoptosis, which is potentially a protective response toward the DNA damage caused by peroxynitrite. On the other hand, higher concentrations of peroxynitrite (10-50 microM) rapidly increase [Ca2+]i and eventually induce necrosis.

Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells.

The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.

CCK-resistance in Zucker obese versus lean rats.

Obese Zucker rats are less sensitive to the satiety effect of CCK than lean litter mates. The present studies further characterised this CCK resistance. Subcutaneous injection of the CCK agonist caerulein dose-dependently decreased food intake in Zucker obese and lean rats whereas the CCK-B agonist gastrin-17 did not. Caerulein at 4 microg/kg, which resulted in CCK plasma bioactivity slightly above postprandial levels, decreased food intake in lean rats but not in obese rats. The decrease in food intake was also more marked at higher caerulein doses (20-100 microg/kg) in lean versus obese rats. In lean animals the satiety effects of the "near physiological" 4 microg/kg caerulein dose was abolished after blockade of vagal afferents with capsaicin, whereas the effects of higher caerulein doses were not. CCK-stimulated amylase secretion from pancreatic acini and binding capacity of 125I-labelled CCK-8 were decreased in obese versus lean rats. The CCK-A antagonist loxiglumide at 20 mg/kg, a dose which abolished the action of all caerulein doses on food intake, failed to alter the food intake either in obese or in lean rats when given without an agonist. The results suggest that the satiety effects of "near physiological" doses of caerulein in lean rats are mediated by vagal afferents whereas pharmacological doses act via non-vagal mechanisms. The differences in CCK's satiety effect between lean and obese rats may be due to differences in CCK-receptor binding and action at peripheral vagal sites. However, the failure of the CCK-A antagonist to increase food intake questions whether any of the effects of exogenous CCK are of physiological relevance.

Menadione induces both necrosis and apoptosis in rat pancreatic acinar AR4-2J cells.

This study evaluated the action of menadione on cell proliferation and integrity of the rat pancreatic acinar cell line, AR4-2J. Menadione at 1-20 microM dose- and time-dependently inhibited cell proliferation of AR4-2J cells. In contrast, a high concentration of menadione (100 microM) caused rapid cell death (> 90% of cells took up trypan blue within 4-h). While the high concentration of menadione (100 microM) induced DNA smear in electrophoresis indicative of necrosis, lower concentrations (10-20 microM) induced a DNA ladder indicative of apoptosis. Similar results were obtained using a DNA fragmentation ELISA. Glutathione (1 mM), the calcium chelator EGTA (500 microM), and the cysteine protease inhibitor NCO-700 (5 mM) partly inhibited the effect of 1-10 microM menadione on cell proliferation and DNA fragmentation. Menadione at 1-20 microM induced wild-type P53, whereas the 100 microM menadione had a minor effect on wild-type P53. It is concluded that menadione induced necrosis at high concentrations and apoptosis at low concentrations in AR4-2J cells. Apoptosis induced by lower concentrations of menadione may be mediated by wild-type P53, intracellular calcium, and mechanisms which decrease the intracellular concentration of reduced glutathione.