Pancreatic Homogenates Research Articles

Evidence of increased flux to n-6 docosapentaenoic acid in phospholipids of pancreas from cftr −/− knockout mice

An association has been reported between alterations in fatty acid metabolism and cystic fibrosis (CF). We hypothesized that these alterations are specific for a particular lipid component(s) and are the result of a specific metabolic defect. The different lipid classes were examined for fatty acid changes by using pancreatic homogenates and primary cultures of pancreatic acini from cftr −/− (CF) and wild-type mice. Lipid classes and phospholipids were separated by aminopropyl column chromatography and high-performance liquid chromatography, and fatty acid methyl esters were analyzed. The results indicate that in CF mice (1) linoleate was decreased in phospholipids but not in neutral lipids; (2) there was an increase in dihomo- γ-linolenate and in docosapentaenoate, the terminal fatty acid of the n-6 pathway, in total lipids and total phospholipids, but not in the neutral lipid class; and (3) the docosapentaenoate (n-6)/docosahexaenoate (n-3) ratio was significantly elevated in neutral phospholipids. This suggests an enhanced flux through the n-6 pathway beyond arachidonate. This study provides a more in-depth understanding of the fatty acid alterations found in CF, as reflected by the cftr −/− mouse model.

Bile-Pancreatic Juice Exclusion Promotes Akt/NF-κB Activation and Chemokine Production in Ligation-Induced Acute Pancreatitis

Using a unique surgical model (the donor rat model), we showed previously that duodenal replacement of bile-pancreatic juice, obtained fresh from a donor rat, ameliorates ligation-induced acute pancreatitis. We hypothesize that bile-pancreatic juice exclusion from gut exacerbates Akt/nuclear factor-kappaB (NF-kappaB) pathway activation and induces chemokine production in ligation-induced acute pancreatitis. We compared rats with bile-pancreatic duct ligation to those with duodenal bile-pancreatic juice replacement fresh from a donor rat beginning immediately before duct ligation. Sham control rats had ducts dissected but not ligated. Rats were killed 1 or 3 hours after operation (n = 7/group). Akt activation (immunoblotting, immune-complex kinase assay, and ELISA), inhibitory protein I-kappaB (IkappaB) activation (immunoblotting), and production of chemokines MCP-1 and RANTES (ELISA) were measured in pancreatic homogenates. NF-kappaB was quantitated in nuclear fractions using electrophoretic mobility shift assay. Duct ligation produced significant increases in pancreatic Akt, IkappaB, and NF-kappaB activation and production of MCP-1 and RANTES. Activation of the Akt/NF-kappaB pathway and increased MCP-1 and RANTES production in response to duct ligation were significantly reduced by bile-pancreatic juice replacement (ANOVA, P < 0.05). Bile-pancreatic juice exclusion stimulates Akt/NF-kappaB pathway activation and increases chemokine production in ligation-induced acute pancreatitis.

Lipid cytotoxicity in shock may by mediated by lipid‐binding proteins

Shock is accompanied by an increase in mucosal intestinal permeability, allowing digestive enzymes from the lumen to enter the intestinal wall. Incubating individual pancreatic proteases or homogenates of the luminal content with intestinal homogenates results in the generation of cytotoxic activity. Our goal is to characterize the factors in these intestinal digests that cause cytotoxicity. We hypothesize that in intestinal homogenates, lipases digest lipids into cytotoxic mediators [free fatty acids, etc.], which are adsorbed by lipid-binding proteins like albumin. Adding proteases would digest the proteins, releasing the mediators. Intestinal homogenates that had been exposed to chymotrypsin, the luminal content homogenate, or PBS were separated into hydrophilic, hydrophobic, and precipitated fractions using chloroform/methanol extraction. These fractions were tested for cytotoxicity. The hydrophilic and precipitated fractions caused little cell death, but the hydrophobic fractions from both digested and undigested intestinal homogenates caused significant cell death. A recombination of the hydrophilic and precipitated fractions with the hydrophobic fraction reduced cytotoxicity compared to hydrophobic fraction alone. We also show intestinal homogenates contain lipase activity and cell death from intestinal homogenate exposed to luminal homogenate is prevented by adding albumin. These results suggest that lipid-binding proteins may control lipid cytotoxicity, but during shock these protective proteins may be destroyed. Supported by HL67825.

Fructokinase activity in rat liver, ileum, parotid gland, pancreas, pancreatic islet, B and non-B islet cell homogenates

The presence of fructokinase (ketohexokinase) in rat pancreatic islet homogenates was previously documented. However, no information was so far available on the activity of this enzyme in islets relative to that in other tissues and on the respective contribution of insulin-producing B cells and non-B islet cells. The present study provides such an information. The activity of fructokinase, as assessed by the phosphorylation of 1.0 mM D-fructose, was compared to that of hexokinase isoenzyme(s), as measured in the presence of 1.0 mM D-glucose, and further characterized by its heat-resistance, K+ dependency and resistance to the inhibitory action of D-mannoheptulose. As judged from the results obtained in heated homogenates, the activity of fructokinase, expressed relative to protein content (nmol/min per mg protein) was highest in liver (21.5 +/- 2.5; n = 11) and lowest in parotid gland (0.16 +/- 0.09; n = 3), with in-between values in ileum (2.45 +/- 0.53; n = 3), pancreas (0.82 +/- 0.11; n = 11) and pancreatic islets (0.46 +/- 0.07; n = 6). The paired ratio between fructokinase and hexokinase isoenzyme activity was also highest in liver (548 +/- 45%; n = 8) and lowest in parotid gland (0.93 +/- 0.52%; n = 3). Such a ratio was not significantly different in pancreas, islets and purified B or non-B islet cells, with an overall mean value of 2.57 +/- 0.46% (n = 12). The present findings thus unambiguously document the presence of fructokinase activity in all cell types under consideration, except possibly parotid cells, with the following hierarchy: liver > ileum > pancreas. Relative to paired hexokinase activity, no obvious difference was found for fructokinase activity in B versus non-B islet cells.

BCL-XL IS UPREGULATED IN DIFFERENT MODELS OF ACUTE PANCREATITIS AND PROTECTS PANCREAS FROM CELL DEATH

Background and Aims: Very little is known about the mechanisms of parenchymal cell death in pancreatitis. Bcl-2 proteins are key regulators of cell death; however, their roles in pancreatitis are not known. The aim of this study was to determine the expression of key pro- and anti-apoptotic Bcl-2 proteins in pancreas and their role in different models of acute pancreatitis. Methods: Pancreatitis was induced in mice and rats by i.p. injections of 50 μg/kg cerulein (CR); by i.p. injections of L-arginine (2.5 g/kg) in rats; and by feeding a choline deficient, ethionine supplemented (CDE) diet to mice. Bcl-2 protein levels and cytochrome c release were measured by Western blot in both pancreatic tissue homogenates and mitochondrial fractions. Membrane potential of isolated pancreatic mitochondria (ΔΨm) was measured using TPP electrode and fluorescent probe TMRM. Results: Key anti-apoptotic protein Bcl-xL was greatly (up to 7-fold) upregulated in all 4 models of pancreatitis under study, both in tissue homogenates and mitochondrial fractions. Changes in pro-apoptotic Bax, Bak and Bim and the anti-apoptotic Bcl-2 were not pronounced and varied between the models. CR-induced Bcl-xL upregulation was prevented in p50−/−p65+/− mice deficient in key NF-κB proteins, indicating that Bcl-xL stimulation in pancreatitis is controlled by NF-κB. We next evaluated the role of Bcl-xL in death responses of pancreatic mitochondria by applying Bcl-xL inhibitors BH3I-2′ and HA14-1. In mitochondria isolated from normal pancreas, the inhibitors induced dose-dependent depolarization and cytochrome c release, indicating an important protective role for Bcl-xL. Mitochondria isolated from pancreas of rats and mice with CR pancreatitis were more sensitive to the deleterious effects of Bcl-xL inhibition, as compared with normal pancreas. Conclusions: Significant upregulation of Bcl-xL is a common event in models of acute pancreatitis. Bcl-xL is stimulated via NF-κB and protects the pancreas from mitochondrial damage in pancreatitis. The results suggest that increasing the level of Bcl-xL may have a therapeutic benefit in pancreatitis.

Bile-pancreatic juice (BPJ) exclusion exacerbates Akt/NF-kB pathway activation and increases chemokine production in ligation-induced acute pancreatitis

Abstract Introduction: Using a unique surgical model (The Donor Rat Model) we showed that duodenal replacement of BPJ, obtained fresh from a Donor Rat, ameliorates pancreatic morphologic changes, hyperamylasemia and hypercholecystokininemia in ligation-induced acute pancreatitis. We hypothesize that BPJ exclusion from gut exacerbates Akt/NF-kB pathway activation and induces proinflammatory chemokine production in ligation-induced acute pancreatitis. The cytosolic IkB/NF-kB complex dissociates when IkB is phosphorylated following Akt activation, allowing NF-kB to translocate to the nucleus and induce transcription of various inflammatory mediators including chemokines. Methods: We studied rats as follows: Diseased-controls had bile-pancreatic duct ligation. Diseased-treated rats had duodenal BPJ replacement fresh from a Donor Rat beginning immediately before duct ligation. Sham controls had ducts dissected only. Rats were killed after 1 or 3 hours (n = 7/group). Pancreatic homogenates were used to determine Akt activation (immunoblotting, immunecomplex kinase assay and ELISA), IkB activation (immunoblotting), and production of chemokines MCP-1 and RANTES (ELISA). NF-kB was quantitated in pancreatic nuclear fractions using EMSA. Results: Compared to sham, duct ligation was associated with marked increases in pancreatic Akt and IkB activation, NF-kB nuclear translocation, and MCP-1 and RANTES production. Akt/NF-kB pathway activation and increased MCP-1 and RANTES production after duct ligation were substantially ameliorated by BPJ replacement (ANOVA, p Conclusions: BPJ exclusion from gut exacerbates Akt/NF-kB pathway activation and increases chemokine production in ligation-induced acute pancreatitis. These findings support our central hypothesis that BPJ exclusion-induced acinar cell hyperstimulation exacerbates acute pancreatic inflammation in this experimental corollary of gallstone-induced acute pancreatitis. (Support: ACS Faculty Research Fellowship, NIH-K08 Award#DK062805)

Pulmonary injuries and cytokine levels after the intraperitoneal administration of pancreatic homogenates in rats.

Our objective was to investigate the effects of the administration of pancreatic homogenates, with or without enzymatic activation, to healthy animals regarding cytokine serum levels and the development of pulmonary distress. 106 male Wistar rats, divided into three groups, were studied: group A, intraperitoneal administration of homogenates activated with enterokinase; group B, homogenates without enterokinase; and group C, control group with administration of physiological saline solution. Each group was divided into 4 subgroups according to the time of sacrifice: 0, 2, 6 and 24 hours. We studied the pulmonary and pancreatic histology, serum parameters of renal and hepatic function, and serum levels of IL-1beta, IL-6 and TNFalpha. There was no mortality in any group. Pancreatic disorders in A and B groups were noted at 24 hours. These two groups had statistically significant higher transaminase serum levels than those of the control group, as well as statistically significant higher creatinine levels in group A. IL-1beta showed a statistically significant higher level at 6 h in both groups, A and B, but was higher in group A, which also exhibited significant pulmonary histologic damage with respect to controls at 6 h. The higher IL-1beta level in group A may result from production by peritoneal macrophages under the influence of homogenate enzymatic activation. This may be the reason for lung damage.

Quercetin, a flavonoid antioxidant, prevents and protects streptozotocin-induced oxidative stress and β-cell damage in rat pancreas

The aim of the present study was the evaluation of possible protective effects of quercetin (QE) against β-cell damage in experimental streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg kg −1 for diabetes induction. QE (15 mg kg −1 day, intraperitoneal (i.p.) injection) was injected for 3 days prior to STZ administration; these injections were continued to the end of the study (for 4 weeks). It has been believed that oxidative stress plays a role in the pathogenesis of diabetes mellitus (DM). In order to determine the changes of cellular antioxidant defense system, antioxidant enzymes such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) activities were measured in pancreatic homogenates. Moreover we also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, if there is an imbalance between oxidant and antioxidant status. Pancreatic β-cells were examined by immunohistochemical methods. STZ induced a significant increase lipid peroxidation, serum NO concentrations and decreased the antioxidant enzyme activity. Erythrocyte MDA, serum NO and pancreatic tissue MDA significantly increased ( P < 0.05) and also the antioxidant levels significantly decreased ( P < 0.05) in diabetic group. QE treatment significantly decreased the elevated MDA and NO ( P < 0.05), and also increased the antioxidant enzyme activities ( P < 0.05). QE treatment has shown protective effect possibly through decreasing lipid peroxidation, NO production and increasing antioxidant enzyme activity. Islet cells degeneration and weak insulin immunohistochemical staining was observed in STZ induced diabetic rats. Increased staining of insulin and preservation of islet cells were apparent in the QE-treated diabetic rats. These findings suggest that QE treatment has protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic β-cell integrity.

Effects of Nigella sativa on oxidative stress and beta-cell damage in streptozotocin-induced diabetic rats.

The aim of the present study was to evaluate the possible protective effects of Nigella sativa L. (NS) against beta-cell damage from streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg/kg to induce diabetes. NS (0.2 ml/kg/day, i.p.) was injected for 3 days prior to STZ administration, and these injections were continued throughout the 4-week study. Oxidative stress is believed to play a role in the pathogenesis of diabetes mellitus (DM). To assess changes in the cellular antioxidant defense system, we measured the activities of antioxidant enzymes (such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (CAT)) in pancreatic homogenates. We also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, to determine whether there is an imbalance between oxidant and antioxidant status. Pancreatic beta-cells were examined by immunohistochemical methods. STZ induced a significant increase in lipid peroxidation and serum NO concentrations, and decreased antioxidant enzyme activity. NS treatment has been shown to provide a protective effect by decreasing lipid peroxidation and serum NO, and increasing antioxidant enzyme activity. Islet cell degeneration and weak insulin immunohistochemical staining was observed in rats with STZ-induced diabetes. Increased intensity of staining for insulin, and preservation of beta-cell numbers were apparent in the NS-treated diabetic rats. These findings suggest that NS treatment exerts a therapeutic protective effect in diabetes by decreasing oxidative stress and preserving pancreatic beta-cell integrity. Consequently, NS may be clinically useful for protecting beta-cells against oxidative stress.

O0093 THE RYANODINE RECEPTOR MODULATES SECRETAGOGUE STIMULATED PANCREATIC ZYMOGEN ACTIVATION

Introduction: Premature activation of intra-acinar digestive proenzymes (zymogens) is an early and critical event in the pathogenesis of acute pancreatitis. A rise in cytosolic calcium is required for this activation. Two endoplasmic reticulum release channels contribute to the rise in cytosolic calcium, the 1,4,5-inositol trisphosphate receptor and the ryanodine receptor (RYR). In this study we examined the role of calcium release from the RYR in secretagogue stimulated zymogen activation using both in vitro and in vivo models. Methods: In the in vitro model, rat pancreatic acini were isolated by collagenase digestion and then preincubated with the RYR antagonist, dantrolene (100uM). Acini were subsequently stimulated with agonists that cause zymogen activation (chole-cystokinin (CCK) or carbachol +/− the cAMP analogue, 8-bromo-cAMP (100uM)). Chymotrypsin and trypsin activities were assayed in homogenates and percent amylase secretion was measured. To determine the relevance of the results, the effects of dantrolene on zymogen activation were examined in an in vivo model of pancreatitis using supraphysiologic CCK. Rats were pretreated (1 hr) with intravenous dantrolene (5mg/kg). Pancreatitis was induced by administering hourly intraperitoneal injections of the CCK analogue caerulein (40ug/kg/dose). Rats were sacrificed one hour after the first caerulein injection and pancreatic homogenates were used to assay chymotrypsin and trypsin activities. Results: Preincubation of isolated acinar cells with dan-trolene reduced supraphysiologic CCK (100nM)-induced and cAMP-enhanced zymogen activation by 30% and 70%, respectively. Dantrolene also reduced physiologic CCK (100pM) and cAMP-induced zymogen activation by 35%. Supraphysiologic carbachol (1mM)-induced and cAMP-enhanced activation were reduced by 30% and 50%, respectively. In contrast, amylase secretion was unaffected by the RYR antagonist in the above conditions. In the in vivo model, dantrolene pretreatment reduced supraphysiologic CCK-induced generation of chymotrypsin activity by 62% and trypsin activity by 45%. Conclusion: Together, these in vitro and in vivo studies suggest that initial zymogen activation, but not digestive enzyme secretion, is primarily mediated by calcium release from a pool that is regulated by the RYR. In addition, the RYR may be the major target of cAMP-dependent sensitization to secretagogue stimulated zymogen activation.

Large doses of zinc oxide increases the activity of hydrolases in rats

The effect of pharmacological doses of zinc oxide (1000; 2500; 5000 mg per kg diet) and two levels of dietary protein on pancreatic and intestinal hydrolase activity in rats were studied. It was hypothesized that ZnO would increase intestinal and pancreatic hydrolase enzyme activity. Male Wistar rats, averaging 64 g body weight, were randomly allocated to dietary treatments (chow diets- meeting all NRC requirements) containing 10% or 15% protein supplemented with additional ZnO (above 100 mg/kg ZnSO 4) as follows: 0.0; 0.1; 0.25; 0.5% w/w. Water and food were provided ad libitum. Animals were fed the diets for 10 days and body weights were recorded; after decapitation blood and organ samples were collected. Amylase, lipase, trypsin, and total protease activity of pancreatic homogenates and small intestinal contents were determined. ZnO supplementation dose dependently increased the plasma Zn concentration and significantly increased amylase, lipase, trypsin and total protease activity in pancreatic homogenates and small intestinal contents. The statistical analysis showed significant protein and ZnO interaction on the activity of amylase in the pancreas, and amylase, trypsin and total-protease in the small intestinal content. Therefore ZnO at high dietary concentration may influence the digestion of nutrients via increased hydrolase activity.

Vitamin E attenuates biochemical and morphological features associated with development of chronic pancreatitis

The objective was to investigate the effects of vitamin E on collagen deposition induced by Cyclosporin A (CsA) administration in rats with caerulein (Cr) pancreatitis. CsA transforms the fully regenerative, self-limited form of Cr pancreatitis into a chroniclike disease in conjunction with increased transforming growth factor (TGF)-beta and myofibroblast proliferation. Vitamin E inhibits TGF-beta release in mesangial cells and reduces CsA cytotoxicity. Wistar rats received CsA daily (20 mg/kg), and CR pancreatitis was induced on days 1 and 8 (Cr + CsA group). In a separate group, vitamin E (600 mg.kg(-1).day(-1)) was administered starting 4 days before CsA. Three other groups received either vehicle, CsA, or Cr alone. Thiobarbituric acid-reactive substance (TBARS), 8-isoprostanes, and hyaluronic acid were measured in plasma obtained on the day the animals were killed (day 15). Pancreases were weighed and processed for light microscopy to assess connective tissue and myofibroblast number. Pancreatic homogenates were also assayed for collagen (hydroxyproline) and TBARS content. TBARS, 8-isoprostane, and TGF-beta were elevated in CsA and Cr + CsA rats. Vitamin E treatment greatly decreased these parameters. Vitamin E also decreased the fall in pancreatic weight observed in Cr + CsA pancreas. Pancreatic hydroxyproline and plasma hyaluronic acid were increased in Cr + CsA rats but were effectively reduced by vitamin E. Morphology showed improvement in fibrosis score and a decreased number of myofibroblasts in vitamin E-treated rats. Vitamin E reduces oxidative stress and collagen deposition during the development of experimental chronic pancreatitis. Adjuvant antioxidants may be of value in the treatment of chronic pancreatitis.

Exercise training prevents and protects streptozotocin-induced oxidative stress and beta-cell damage in rat pancreas.

The aim of the present study was the evaluation of possible protective effects of exercise against beta-cell damage in streptozotocin (STZ)-induced diabetes in rats. The animals were divided into five groups: the control group, the STZ-induced diabetes group, the STZ-induced diabetes and light-intensity exercise group, the STZ-induced diabetes and moderate-intensity exercise group, and the STZ-induced diabetes and heavy-intensity exercise group. Animals in the exercise groups were made to swim one of three exercise protocols once a day for 12 consecutive weeks. STZ was injected intraperitoneally at a single dose of 50 mg/kg for diabetes induction. Exercise training was continued for 4 weeks prior to STZ administration; these applications were continued end of the study (for 12 weeks). Erythrocyte and pancreatic tissue malondialdehyde (MDA) levels and serum nitric oxide (NO) concentration were measured. Moreover glutathione peroxidase (GSHPx), superoxide dismutase (SOD) and catalase (CAT) were also measured in pancreatic homogenates. Pancreatic beta-cells were examined by immunohistochemical methods. STZ increased lipid peroxidation and decreased the antioxidant enzyme activity significantly. Exercise, especially moderate-intensity exercise has shown protective effect probably through decreasing lipid peroxidation and increasing antioxidant enzyme activity. Islet cell degeneration and weak insulin immunohistochemical staining were observed in STZ induced diabetic rats. Increased intensity of staining for insulin and preservation of beta-cell numbers were apparent in the exercise-applied diabetic rats. Interestingly, the best result was obtained from moderate-intensity exercise. These findings suggest that exercise has a therapeutic and/or protective effect in diabetes by decreasing oxidative stress and preservation of pancreatic beta-cell integrity.

Cholinergic receptor induction and JNK activation in acute pancreatitis

Background Cholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38 MAPK) and cJUN N-terminal kinase (JNK), are implicated in the pathogenesis of ligation-induced acute pancreatitis in rats. As ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38 MAPK activation, and as receptor induction could amplify acinar hyperstimulation and exacerbate cell stress, we tested the hypothesis that the cholinergic M3 receptor is induced and JNK is activated in this model. Methods Cholinergic M3 receptor expression and JNK activation was compared in rats 1, 3, or 24 hours after sham operation or duct ligation. Results Immunoblot analysis of pancreatic homogenates showed a time-dependent increase in cholinergic M3 receptor protein, total JNK, and phospho-JNK after duct ligation. Conclusions There is a rapid and progressive cholinergic M3 receptor induction and JNK activation in ligation-induced acute pancreatitis in rats. These findings may have significance in the mechanism of disease pathogenesis.

Characterization of Two Classes of Pancreatic Shock Factors: Functional Differences Exhibited by Hydrophilic and Hydrophobic Shock Factors

Pancreatic tissue homogenate induces a powerful pathophysiologic response sufficient to produce lethal shock in a rat. However, limited progress has been made in the biochemical characterization of these pancreas-derived active factors or their mechanisms of action. It has been shown that the pancreas is a major source of these shock-inducing factors and that they are generated by pancreatic proteinases. Porcine pancreas was homogenized and the filtered homogenate was subjected to organic extraction both before and after incubation for 2.5 h at 37 degrees C. The aqueous and lipid extracts of pancreatic homogenates were collected and analyzed for their ability to activate human neutrophils and to induce lethal shock in the rat. Neutrophil activation, a presumed hallmark of shock, was determined by chemiluminescence and myeloperoxidase (MPO) release. Only the intact homogenate and lipid extracts stimulated the neutrophils, and the aqueous extracts proved to be inactive. Neutrophils exhibited enhanced cellular activation when exposed to substimulatory levels of either formyl-methionyl-leucyl-phenylalanine (FMLP) or platelet-activating factor (PAF) followed by substimulatory levels of the lipid extracts, but not by the aqueous extracts. Both the lipid and aqueous extracts induced dramatic decreases in heart rate and blood pressure when injected in the rat, often resulting in lethal shock. In all cases, incubation of the homogenates at 37 degrees C enhanced the potency of the extracts. Our results demonstrated that the pancreas-derived homogenate and lipid factors were capable of inducing both neutrophil activation and shock. These results support the hypothesis that shock is produced via neutrophils that have been activated by inflammatory components. However, the shock-inducing factors in the aqueous extracts (i.e., hydrophilic fraction of the homogenate) apparently function via a pathway independent of neutrophil activation. This is the first evidence that there are both hydrophobic and hydrophilic factors generated in tissue homogenates capable of inducing shock, and that these different chemical classes of factors appear to function via separate mechanisms.

Pancreatic digestive enzymes are potent generators of mediators for leukocyte activation and mortality.

Shock is associated with a dramatic rise in the level of inflammatory mediators found in plasma. The exact source of these mediators has remained uncertain. We recently examined a previously undescribed mechanism for production of inflammatory mediators in shock involving pancreatic digestive enzymes. The current in vitro study was designed to identify particular pancreatic enzymes and organs that may potentially produce inflammatory mediators. A selection of different organs from the rat (heart, liver, brain, spleen, pancreas, intestine, diaphragm, kidney, and lung) were homogenized and incubated with purified trypsin, chymotrypsin, elastase, lipase, nuclease, or amylase and the supernatant was incubated with fresh naïve leukocytes for 15 min. The level of leukocyte activation in the form of pseudopod formation and the fraction of cell death were measured. Without the addition of purified enzymes, only the homogenate of the pancreas yielded enhanced cell activation. Organs incubated with physiological concentrations of trypsin also stimulated significantly higher levels of pseudopod formation as compared with the undigested organs or enzymatic controls. Lipase and chymotrypsin were able to elicit cellular activation from selected organs such as the heart, intestine, liver and diaphragm. Undigested pancreatic homogenates were capable of producing substantial cell death, as compared with all other undigested organs. Intestinal digests with elastase, lipase, trypsin and chymotrypsin also stimulated significant cell mortality. Lipase-treated heart, liver, intestine, diaphragm, kidney, and lung stimulated cell death as well. We conclude that the intestine, as well as several other organs, may serve as a major source of inflammatory mediators during shock if exposed to digestive enzymes.

Size-exclusion chromatography of biological samples which contain extremely alkaline proteins

An improved size-exclusion chromatography (SEC) was developed to isolate extremely basic (alkaline) proteins, such as trypsin (p I=10.5), lysozyme (p I=11), and histone (p I=10.8). Develosil 300 Diol-5 (300×8 mm I.D., 30 nm average pore diameter) column was used with an eluent of 0.1 M sodium phosphate, 1.5 M sodium chloride, glycerol (40%, v/v), 2-propanol (10%, v/v), and Brij-58 (1%, v/v). Under these conditions, the final apparent pH becomes to 4.0, and pH adjustment is not necessary. Column temperature and flow rate were 15 °C and 0.2 ml/min, respectively. This elution system is stable and reliable, and applications onto human pancreatic juice, human bile, and tissue homogenates were successfully achieved. Since this system is convenient for protein analysis, it is expected to be generally applicable to clinical and biochemical research for identifying protein components in combination with microsequencing.

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.

Protective effect of melatonin on β-cell damage in streptozotocin-induced diabetes in rats

The aim of the present study was the evaluation of possible protective effects of melatonin against beta-cell damage in streptozotocin-induced diabetes in rats. Malondialdehyde levels and glutathione peroxidase activity were measured in pancreatic homogenates. Pancreatic beta-cells were examined by immunohistochemical methods. Streptozotocin was injected intraperitoneally at a single dose of 60 mg/kg for induction of diabetes. Melatonin (200 microg/kg/day, ip) was injected for 3 days prior to administration of streptozotocin; these injections were continued until the end of the study (4 weeks). Streptozotocin induced a significant increase in malondialdehyde levels (p < 0.01) and a significant decrease in glutathione peroxidase activity (p < 0.05) in pancreatic tissue. Degeneration of islet cells and weak immunohistochemical staining of insulin was observed in diabetic rats. Treatment of diabetic rats with melatonin markedly reduced malondialdehyde production (p < 0.05) and increased glutathione peroxidase activity (p < 0.01) without affecting hyperglycemia. Increased staining of insulin and preservation of islet cells were apparent in the melatonin-treated diabetic rats. These data suggest that melatonin treatment has a therapeutic effect in diabetes by reduction of oxidative stress and preservation of pancreatic beta-cell integrity.

Neutrophils and NADPH oxidase mediate intrapancreatic trypsin activation in murine experimental acute pancreatitis

Intrapancreatic activation of digestive enzymes is a key event in the parenchymal cell injury of pancreatitis. We hypothesized that neutrophils recruited to the pancreas during pancreatitis may contribute to such activation. To cause experimental pancreatitis, rats and mice were treated with high doses of cerulein. Activation of the digestive enzyme, trypsin, was measured in pancreatic homogenates using a fluorogenic assay and localized immunocytochemically with antibody to trypsin-activation peptide (TAP). Compared with controls, rats depleted of neutrophils with antineutrophil serum exhibited a marked attenuation in intrapancreatic trypsin activation and acinar cell TAP labeling induced by high-dose cerulein. To examine the mechanism, mice deficient in either nicontinamide adenine dinucleotide phosphate (NADPH) oxidase, or myeloperoxidase (MPO) were studied for trypsin activation. Mice deficient in NADPH oxidase exhibited attenuation of the cerulein-induced trypsin activation, but those deficient in MPO did not. Using measurements of Western blot analysis, generation of reactive oxygen species, and immunocytochemistry, we demonstrated the NADPH oxidase activity is in neutrophils and not pancreatic acinar tissue. The results demonstrate a novel role for neutrophils infiltrating the pancreas in pathologic activation of digestive enzymes in acute pancreatitis and indicate that this effect is mediated by products of NADPH oxidase.