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Cholinergic receptor induction and JNK activation in acute pancreatitis
The American Journal of Surgery 186 (2003) 569 –574
Scientific Paper
Cholinergic receptor induction and JNK activation in acute pancreatitis Isaac Samuel, M.D.a,*, Smita Zaheer, Ph.D.a, Rory A. Fisher, Ph.D.b, Asgar Zaheer, Ph.D.c a
Department of Surgery, Veterans Affairs Medical Center, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 200 Hawkins Dr., 4625 JCP, Iowa City, IA 52242, USA b Department of Pharmacology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA c Department of Neurology, University of Iowa Roy J. and Lucille A. Carver College of Medicine and the Veterans Affairs Medical Center, Iowa City, IA, USA Manuscript received June 2, 2003; revised manuscript July 16, 2003
Abstract Background: Cholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38MAPK) 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 p38MAPK 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. © 2003 Excerpta Medica, Inc. All rights reserved. Keywords: Acute pancreatitis; CCK; Cholinergic receptor; JNK; Stress-activated protein kinase; Signal transduction
Acute pancreatitis is a common disease and is potentially fatal [1]. However, as the mechanism of disease pathogenesis has not yet been elucidated, the initial treatment remains merely nonspecific and supportive [1,2]. The rodent model of bile-pancreatic duct ligation-induced acute pancreatitis is a useful experimental corollary of gallstoneinduced acute pancreatitis [2]. Bile-pancreatic duct ligation in rats excludes bile-pancreatic juice from gut and induces acute pancreatitis [2]. Cholecystokinin-A (CCK-A) and cholinergic receptor-mediated exocrine pancreatic hyperstimulation secondary to bile-pancreatic juice exclusion is implicated in disease pathogenesis [3]. In dispersed pancreatic acini, stimulation of these G-protein coupled receptors can activate the stressactivated protein kinases p38 mitogen-activated protein kinase (p38MAPK) and cJUN N-terminal kinase (JNK) [4,5].
We previously showed that duct ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38MAPK activation [6]. In the present study, we show that ligation-induced acute pancreatitis in rats is associated with cholinergic muscarinic (M3) receptor induction and JNK activation. As an increase in the number of G-protein coupled receptors can amplify exclusion-induced exocrine pancreatic hyperstimulation, and as activation of stress kinases can induce acinar cell proinflammatory cytokine production, our observations may have relevance in the mechanism of disease pathogenesis.
Material and methods Material
* Corresponding author. Tel.: ⫹1-319-384-7220; fax: ⫹1-319-3568378. E-mail address: [email protected]
A phospho-MAPK antibody sampler kit (Cat. No. 9910), containing rabbit polyclonal immunoglobulin G (IgG) against phospho-JNK (Thr183/Tyr185; Cat. No. 9251) and
0002-9610/03/$ – see front matter © 2003 Excerpta Medica, Inc. All rights reserved. doi:10.1016/j.amjsurg.2003.07.016
570
I. Samuel et al. / The American Journal of Surgery 186 (2003) 569 –574
HRP-conjugated anti-rabbit IgG secondary antibody (Cat. No. 7074), was purchased from New England Biolabs (Beverly, Massachusetts). The corresponding antibody against total JNK was also from New England Biolabs. The [32P]ATP (3000 Ci/mmol) was from Perkin Elmer Life Sciences/ NEN (Woodbridge, Ontario). The recombinant human cJUN protein (sc-4113; aa 1-79) and specific antibody to cholinergic M3 receptor (sc-9108) were from Santa Cruz Biotechnology (Santa Cruz, California). Enhanced chemiluminescence (ECL) immunoblot detection reagents were from Amersham Pharmacia Biotech (Piscataway, New Jersey). Specific antibody to -actin was from Sigma Chemical (St. Louis, Missouri). Animal surgery and specimen collection All protocols were approved by the University of Iowa Animal Care and Use Committees. Male Sprague-Dawley rats weighing 250 to 325 g were purchased from Harlan Sprague-Dawley (Indianapolis, Indiana). A midline laparotomy was performed under general anesthesia induced with ketamine hydrochloride (87 mg/kg) and xylazine hydrochloride (13 mg/kg). In diseased groups the distal bilepancreatic duct was ligated, while in sham-operated controls it was dissected but not ligated. Under general anesthesia induced as above, rats were killed by inferior vena caval exsanguination after 1, 3, or 24 hours (n ⫽ 6 rats at each time point in each study group). An additional group of nonoperated rats were killed (0-hour sham) to evaluate whether 1-, 3-, or 24-hour sham controls showed changes in JNK activation or M3 receptor induction after sham operation. The pancreas was quickly excised and divided into portions: a portion was fixed in 10% formaldehyde for morphologic studies; a portion was immediately snap-frozen in liquid nitrogen and stored at ⫺80°C for western blotting, immunoprecipitation, and immune complex kinase assay. Morphology One hematoxylin and eosin–stained section was prepared from each 10% formaldehyde-fixed, paraffin-embedded portion of pancreas and examined on a light microscope in a blinded fashion to be assigned an acute pancreatitis histology score (range 0 to 30) based on degree of white blood cell infiltration (range 0 to 10), acinar cell vacuolation (range 0 to 10), and focal necrosis (range 0 to 10; Table 1). Plasma amylase activity Determination of plasma amylase activity was performed at the Clinical Biochemistry Laboratory of the Iowa City VA Medical Center on a Hitachi spectrophotometer model P800 with bichromatic wavelengths 415 nm and 700 nm.
Table 1 Hyperamylasemia and morphological changes after duct ligation
Plasma amylase (⫻ 102 U/L) Sham Ligation Acute pancreatitis histology score (range 0–30) Sham Ligation
1 Hour
3 Hours
24 Hours
30 ⫾ 1.5 279 ⫾ 85*
43 ⫾ 8 900 ⫾ 233*
31 ⫾ 4 151 ⫾ 14*
0.3 ⫾ 0.2 5.0 ⫾ 0.3*
1.0 ⫾ 0.3 6.2 ⫾ 0.9*
2.8 ⫾ 1.2 19.3 ⫾ 0.5*
Six rats were studied in each experimental group at each time point. Bile-pancreatic duct ligation induced acute pancreatitis as evidenced by hyperamylasemia and pancreatic morphologic changes [WBC infiltration (0 –10), acinar vacuolation (0 –10) and focal acinar cell necrosis (0 –10)], compared with sham operation. Results are mean ⫾ SEM; Asterisk (*) indicates a statistically significant difference between the duct ligation group versus sham-operated control group at the same time point; P ⬍ 0.05, Kruskal-Wallis ANOVA was used for acute pancreatitis histology score, Student-Newman-Keuls test was used for analysis of plasma amylase levels.
Immunoblotting Immunoblot analysis was carried out essentially as described earlier [7]. In brief, portions of pancreas were snapfrozen in liquid nitrogen, stored at ⫺80°°C, homogenized in 10 mM Hepes buffer (pH 7.5), centrifuged at 500g to pellet cell debris, and then centrifuged at 15,000g to obtain a soluble fraction that was quantitated for protein using the Bradford assay. From each sample 40 g of total protein in SDS-sample buffer (62.5 mM Tris, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromophenol blue) were separated on 4% to 20% gradient gels by SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. Protein blots were probed with specific primary antibodies (1:1000 v/v) and developed using the appropriate secondary antibody conjugated to horseradish peroxidase (HRP [1:2000 v/v]), and using the enhanced chemiluminescence (ECL) method as recommended by the manufacturer. Additional immunoblots were performed using -actin antibody as the primary antibody to evaluate equal loading. Immunoprecipitation Immunoprecipitation was carried out as described earlier [8]. In brief, tissues were extracted with a lysis buffer consisting of 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 0.1 mM sodium vanadate, 1 mM benzamidine, 1 mM PMSF (phenyl methane sulfonyl flouride), and 10 g/mL each of aprotinin, leupeptin, chymostatin, pepstatin A, and antipain. Protein estimations were carried out using a commercial kit (Pierce) for the modified Lowry’s method. Tissue extracts were centrifuged at 12,000 rpm for 15 minutes to collect the supernatants. Aliquots of clear supernatants containing 500 g total protein in 1 mL lysis buffer were incubated with 2 g of
I. Samuel et al. / The American Journal of Surgery 186 (2003) 569 –574
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specific JNK antibody overnight at 4°C followed by an additional incubation with 20 L of a 50% suspension of protein G-agarose for 1 hour at room temperature. The insoluble immune complex was collected and washed three times by brief centrifugations prior to immune complex kinase assay. Immune complex kinase assay The JNK was immunoprecipitated from tissues as described above, and followed by two additional washes with a kinase assay buffer prior to immune complex kinase assay. The assays were carried out as described earlier [8], in a total reaction volume of 20 L containing 100 mM TrisHCl, pH 7.0, 0.4 mM sodium ortho vanadate, 40 mM magnesium acetate, 1 mM dithiothreitol, and 30 M calmidazolium, 10 L of JNK immune complex, 2 g of kinase substrate (cJUN), 100 M [r-32P]-ATP (200 cpm/pmol), and was incubated at 30°C for 10 minutes. The reaction was stopped by addition of SDS-PAGE sample buffer, boiled for 2 minutes, centrifuged, and the supernatant was subjected to SDS-PAGE followed by autoradiography. Statistical analysis SigmaStat software (www.spss.com) was used for statistical analysis. Kruskal-Wallis analysis of variance (ANOVA) was used to analyze non-parametric data (acute pancreatitis histology score) and the Student-NewmanKeuls test was used for analysis of plasma amylase levels. Six rats were studied in each experimental group at each time point. A P value below 0.05 was considered statistically significant.
Results Compared with sham controls, 1, 3, and 24 hours of duct ligation induced acute pancreatitis as evidenced by pancreatic morphologic changes and hyperamylasemia (Table 1). Immunoblots of pancreatic homogenates showed a timedependent increase after duct ligation in cholinergic M3 receptor protein expression (Fig. 1), whereas sham-operation was not associated with an increase. The expression and activation of JNK were determined by immunoblot analysis using specific antibodies against its total and activated (phosphorylated) forms. In all immunoblots, -actin was used as a control to confirm equal protein loading (Fig. 1). The results indicate a rapid and sustained increase in the expression as well as activation of JNK in duct ligationinduced acute pancreatitis (Fig. 2). There was no difference in JNK expression or phosphorylation in sham-operated controls. The activation of JNK by an immune-complex kinase assay using cJUN as substrate showed a dramatic time-dependent increase in JNK activation after duct liga-
Fig. 1. Representative immunoblot shows increased cholinergic M3 receptor protein expression in duct ligation-induced acute pancreatitis compared with sham-operated controls. Tissue extracts of pancreas were prepared and subjected to immunoblotting using specific antibody against cholinergic M3 receptor. Immunoblot of -actin confirmed equal loading of lanes. The position of the cholinergic M3 receptor band was verified with molecular size standards. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
tion (Fig. 3), confirming our immunoblot finding of temporally increased JNK activation.
Comments The results of the current study show that ligation-induced acute pancreatitis in rats is associated with a persistent and time-dependent increase in expression of the cholinergic muscarinic M3 receptor, with a parallel increase in activation of the stress-activated protein kinase JNK. These findings are of a similar pattern as our previous findings of CCK-A receptor induction and p38MAPK activation in the same experimental model [6]. An increase in the number of G-protein coupled receptors, such as the cholinergic M3 receptor and the CCK-A receptor, has the potential of augmenting bile-pancreatic juice exclusion-induced pancreatic acinar cell hyperstimulation. Also, as stress-activated protein kinases such as p38MAPK and JNK are activated by G-protein coupled receptor stimulation, and are capable of inducing production of proinflammatory cytokines when activated, our findings may be important in elucidating early events in acute pancreatitis pathogenesis [4,5]. The stress-activated protein kinase pathways, also called mitogen activated protein kinase pathways or MAPK pathways (p38, JNK, and ERK), are novel protein kinase cascades involved in intracellular signal transduction [4,5]. A variety of noxious stimuli are known to activate p38, JNK, and ERK5 signaling pathways including heat shock, ionizing radiation, oxidant stress (peroxide), osmotic shock (sorbitol), DNA damaging agents (topoisomerase inhibitors), and inhibitors of protein synthesis (cycloheximide, anisomycin). In addition, MAPK pathways are also activated by
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Fig. 2. Temporal increase in JNK expression and activation in duct ligation-induced acute pancreatitis compared with sham-operated controls by immunoblot analysis using specific antibodies against the phosphorylated and total forms of JNK. The relative density units were obtained for comparison of changes with digitizing densitometry of this representative immunoblot. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
stimulation of diverse receptor families such as G-protein coupled receptors (CCK receptor), receptor tyrosine kinases (insulin, epidermal growth factor), and cytokine receptors. The activated MAPKs in turn regulate cell functions ranging from gene transcription, cell differentiation, protein synthesis, cell cycle arrest, apoptosis, and cell death. Following
Fig. 3. Autoradiogram of immune complex kinase assay using cJUN as substrate after immunoprecipitation with specific JNK antibody confirms the time-dependent JNK activation seen in immunoblots in Fig. 2. The position of cJUN was verified with molecular size standards. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
activation by phosphorylation via upstream kinases, the MAPKs activate downstream protein kinases or transcription factors to modulate specific cell functions. The potential for transcription factors to induce cellular proinflammatory cytokine production emphasizes the possible role of MAPK signaling pathways in the pathogenesis of diseases such as acute pancreatitis [4,5]. Acute pancreatitis is a common disease and gallstones are the most common etiologic factor world-wide [1]. Ligation-induced acute pancreatitis in rats is a useful experimental corollary of gallstone-induced acute pancreatitis to investigate early events in disease pathogenesis [2]. As the pathogenesis of acute pancreatitis in humans remains elusive, therapeutic strategies to alter the natural history of acute pancreatitis are not specific and consist of fasting the patient to rest the pancreas, intravenous fluid resuscitation, and other general supportive measures [1,2]. The investigation of acute pancreatitis pathogenesis in humans is difficult because the pancreas is anatomically inaccessible, invasive studies of the pancreas are dangerous, and surgical intervention occurs only after the disease has progressed to late stages with complications such as infected necrosis. Therefore, investigations into the mechanisms of disease pathogenesis have to rely on experimental models of acute pancreatitis and a variety of these models have been developed [2,9]. Evidence for the role of MAPK pathways in acute pancreatitis pathogenesis has recently been reported mainly in the lethal rat model of retrograde ductal infusion of bile salts and the nonlethal model of acute edematous pancreatitis caused by supramaximal doses of a CCK analog caerulein [10 –15]. The specific JNK inhibitor CEP 1347 and also inhibitors of the ERK signaling pathway (U0126, PD98059) have been reported to ameliorate caerulein-induced acute pancreatitis in rats [13–15]. CNI-1493, an inhibitor of p38MAPK activation, attenuated acute pancreatitis and improved survival after retrograde infusion of bile salts in rats, and also decreased the severity of caerulein-induced acute pancreatitis [10]. In acute pancreatitis induced by retrograde infusion of bile salts, CNI-1493 also ameliorated pancreatitis-associated pulmonary and hepatocellular injury along with limiting the increase of proinflammatory cytokines TNF-␣ and IL-1 in the plasma, lung, and liver [10 –12]. On the other hand, there is also a report where the specific p38MAPK inhibitor SB203580 exacerbates caerulein-induced acute pancreatitis in rats, suggesting that p38MAPK may have a protective rather than detrimental role in acute pancreatitis [13]. In view of these contradictory findings, further studies—including the use of other experimental models of acute pancreatitis—are required to clarify the role of p38MAPK and other stress kinases in disease pathogenesis. In a unique in vivo study, NF-B activation was achieved in pancreatic acinar cells by overexpressing the active RelA/ p65 subunit using adenoviral-mediated gene transfer techniques and was sufficient for the induction of both a pancreatic and systemic inflammatory response [16]. Taken
I. Samuel et al. / The American Journal of Surgery 186 (2003) 569 –574
together, the above reports support the hypothesis that stress-activated protein kinases and transcription factors modulate development of acute pancreatitis. In view of these observations, we evaluated activation of JNK in ligation-induced acute pancreatitis and showed rapid activation followed by a temporal increase in activation of JNK. Ligation of the bile-pancreatic duct in rats results in duct obstruction, bile-pancreatic juice exclusion from gut, and acute pancreatitis [2]. In rats, a neurohormonal duodenal response is responsible for the observed acinar cell hyperstimulation after bile-pancreatic juice exclusion [3]. The hormonal limb is mediated mainly by CCK secreted by I-cells of the duodenal mucosa, whereas the neural component is mediated via muscarinic cholinergic (vagal) pathways [17,18]. The absence of pancreatic juice trypsin in the duodenal lumen is the predominant stimulus for the duodenal response to pancreatic juice exclusion, while the combined absence of bile salts and pancreatic enzymes results in a synergistic—rather than additive—increase in hypercholecystokininemia and acinar cell hyperstimulation compared with the absence of either alone [2]. We have previously shown that ligation-induced acute pancreatitis in rats is associated with significant hypercholecystokininemia [2]. Using a unique and original surgical model, the donor rat model, we have also shown that duodenal replacement of bile-pancreatic juice— obtained fresh from a donor rat— achieves substantial amelioration of hypercholecystokininemia and ligation-induced acute pancreatitis in rats [2]. In another study, we showed that combined CCK-A and muscarinic receptor blockade ameliorates acinar hyperstimulation in the same experimental model [3]. These observations suggest that bile-pancreatic juice exclusion-induced acinar cell hyperstimulation in the presence of duct obstruction exacerbates acute pancreatitis through CCK-A and muscarinic receptor–mediated pathways. We propose a model for the pathogenesis of ligation-induced acute pancreatitis in rats where combined duct obstruction and acinar hyperstimulation imposes undue stress upon the acinar cell and thus activates acinar cell stress-activated protein kinase pathways resulting in acinar cell production of proinflammatory cytokines. Our demonstration of persistent and progressive activation of the stress kinase JNK in ligationinduced acute pancreatitis in the present study is consistent with this hypothesis for disease pathogenesis. The cholinergic M3 receptor induction observed in this study may contribute mechanistically in the exacerbation of acute pancreatitis as an increase in receptor number may amplify the hyperstimulatory effects of cholinergic stimulation. In summary, in the present study we have shown that there is a rapid, persistent and progressive induction of cholinergic M3 receptor and JNK—with JNK activation—in duct ligation-induced acute pancreatitis in rats. These findings are consistent with our previous observations that ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38 induction and activation. In this experimental corollary of gallstone-induced
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acute pancreatitis, cholinergic and CCK-A receptor induction could amplify cholinergic- and CCK-induced exocrine pancreatic hyperstimulation. This excessive hyperstimulation in the presence of duct obstruction can intensify acinar cell stress. In consequence, activation of stress kinase pathways (JNK and p38MAPK) within the stressed acinar cell has the potential to induce acinar cell production of proinflammatory cytokines that exacerbate acute pancreatitis. Our findings in the current study are consistent with our central hypothesis that G-protein coupled receptors and stress-activated protein kinases are involved in acute pancreatitis pathogenesis. Acknowledgments Supported by a National Institutes of Health NIDDK Career Development Award (Grant 1-K08-DK062805-01 to Dr. Samuel) and internal grants from the University of Iowa and the Carver College of Medicine. References [1] Ranson J. Etiological and prognostic factors in human acute pancreatitis: a review. Am J Gastroenterol 1982;77:633– 8. [2] Samuel I, Toriumi Y, Wilcockson DP, et al. Bile and pancreatic juice replacement ameliorates early ligation- induced acute pancreatitis in rats. Am J Surg 1995;169:391–9. [3] Samuel I, Joehl RJ. Bile-pancreatic juice replacement, not cholinergic and cholecystokinin-receptor blockade, reverses acinar cell hyperstimulation after bile-pancreatic duct ligation. Am J Surg 1996;171: 207–11. [4] Williams JA. Intracellular signaling mechanisms activated by cholecystokinin- regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 2001;63:77–97. [5] Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807– 69. [6] Samuel I, Zaheer A, Nelson JJ, et al. p38 MAP kinase and CCK-A receptor protein expression increase in parallel in duct ligation-induced acute pancreatitis in rats. Pancreas 2002;25:448. [7] Kaplan R, Zaheer A, Jaye M, Lim R. Molecular cloning and expression of biologically active human glia maturation factor-beta. J Neurochem 1991;57:483–90. [8] Zaheer A, Lim R. In vitro inhibition of MAP kinase (ERK1/ERK2) activity by phosphorylated glia maturation factor (GMF). Biochemistry 1996;35:6283– 8. [9] Steer ML. Workshop on experimental pancreatitis. Dig Dis Sci 1985; 30:575– 81. [10] Yang J, Denham W, Tracey KJ, et al. The physiologic consequences of macrophage pacification during severe acute pancreatitis. Shock 1998;10:169 –75. [11] Yang J, Denham W, Carter G, et al. Macrophage pacification reduces rodent pancreatitis-induced hepatocellular injury through down-regulation of hepatic tumor necrosis factor alpha and interleukin-1beta. Hepatology 1998;28:1282– 8. [12] Yang J, Murphy C, Denham W, et al. Evidence of a central role for p38 map kinase induction of tumor necrosis factor alpha in pancreatitis-associated pulmonary injury. Surgery 1999;126:216 –22. [13] Fleischer F, Dabew R, Goke B, Wagner AC. Stress kinase inhibition modulates acute experimental pancreatitis. World J Gastroenterol 2001;7:259 – 65.
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[14] Wagner AC, Mazzucchelli L, Miller M, et al. CEP-1347 inhibits caerulein-induced rat pancreatic JNK activation and ameliorates caerulein pancreatitis. Am J Physiol Gastrointest Liver Physiol 2000; 278:G165–72. [15] Clemons AP, Holstein DM, Galli A, Saunders C. Cerulein-induced acute pancreatitis in the rat is significantly ameliorated by treatment with MEK1/2 inhibitors U0126 and PD98059. Pancreas 2002;25:251–9. [16] Chen X, Ji B, Han B, et al. NF-kappaB activation in pancreas induces
pancreatic and systemic inflammatory response. Gastroenterology 2002;122:448 –57. [17] Miyasaka K, Green GM. Effect of atropine on rat basal pancreatic secretion during return or diversion of bilepancreatic juice. Proc Soc Exp Biol Med 1983;174:187–92. [18] Chariot J, Nagain C, Hugonet F, et al. Control of interdigestive and intraduodenal meal-stimulated pancreatic secretion in rats. Am J Physiol 1990;259:G198 –204.
Scientific Paper
Cholinergic receptor induction and JNK activation in acute pancreatitis Isaac Samuel, M.D.a,*, Smita Zaheer, Ph.D.a, Rory A. Fisher, Ph.D.b, Asgar Zaheer, Ph.D.c a
Department of Surgery, Veterans Affairs Medical Center, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 200 Hawkins Dr., 4625 JCP, Iowa City, IA 52242, USA b Department of Pharmacology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA, USA c Department of Neurology, University of Iowa Roy J. and Lucille A. Carver College of Medicine and the Veterans Affairs Medical Center, Iowa City, IA, USA Manuscript received June 2, 2003; revised manuscript July 16, 2003
Abstract Background: Cholecystokinin-A (CCK-A) and cholinergic receptor pathways, capable of activating stress kinases p38 mitogen-activated protein kinase (p38MAPK) 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 p38MAPK 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. © 2003 Excerpta Medica, Inc. All rights reserved. Keywords: Acute pancreatitis; CCK; Cholinergic receptor; JNK; Stress-activated protein kinase; Signal transduction
Acute pancreatitis is a common disease and is potentially fatal [1]. However, as the mechanism of disease pathogenesis has not yet been elucidated, the initial treatment remains merely nonspecific and supportive [1,2]. The rodent model of bile-pancreatic duct ligation-induced acute pancreatitis is a useful experimental corollary of gallstoneinduced acute pancreatitis [2]. Bile-pancreatic duct ligation in rats excludes bile-pancreatic juice from gut and induces acute pancreatitis [2]. Cholecystokinin-A (CCK-A) and cholinergic receptor-mediated exocrine pancreatic hyperstimulation secondary to bile-pancreatic juice exclusion is implicated in disease pathogenesis [3]. In dispersed pancreatic acini, stimulation of these G-protein coupled receptors can activate the stressactivated protein kinases p38 mitogen-activated protein kinase (p38MAPK) and cJUN N-terminal kinase (JNK) [4,5].
We previously showed that duct ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38MAPK activation [6]. In the present study, we show that ligation-induced acute pancreatitis in rats is associated with cholinergic muscarinic (M3) receptor induction and JNK activation. As an increase in the number of G-protein coupled receptors can amplify exclusion-induced exocrine pancreatic hyperstimulation, and as activation of stress kinases can induce acinar cell proinflammatory cytokine production, our observations may have relevance in the mechanism of disease pathogenesis.
Material and methods Material
* Corresponding author. Tel.: ⫹1-319-384-7220; fax: ⫹1-319-3568378. E-mail address: [email protected]
A phospho-MAPK antibody sampler kit (Cat. No. 9910), containing rabbit polyclonal immunoglobulin G (IgG) against phospho-JNK (Thr183/Tyr185; Cat. No. 9251) and
0002-9610/03/$ – see front matter © 2003 Excerpta Medica, Inc. All rights reserved. doi:10.1016/j.amjsurg.2003.07.016
570
I. Samuel et al. / The American Journal of Surgery 186 (2003) 569 –574
HRP-conjugated anti-rabbit IgG secondary antibody (Cat. No. 7074), was purchased from New England Biolabs (Beverly, Massachusetts). The corresponding antibody against total JNK was also from New England Biolabs. The [32P]ATP (3000 Ci/mmol) was from Perkin Elmer Life Sciences/ NEN (Woodbridge, Ontario). The recombinant human cJUN protein (sc-4113; aa 1-79) and specific antibody to cholinergic M3 receptor (sc-9108) were from Santa Cruz Biotechnology (Santa Cruz, California). Enhanced chemiluminescence (ECL) immunoblot detection reagents were from Amersham Pharmacia Biotech (Piscataway, New Jersey). Specific antibody to -actin was from Sigma Chemical (St. Louis, Missouri). Animal surgery and specimen collection All protocols were approved by the University of Iowa Animal Care and Use Committees. Male Sprague-Dawley rats weighing 250 to 325 g were purchased from Harlan Sprague-Dawley (Indianapolis, Indiana). A midline laparotomy was performed under general anesthesia induced with ketamine hydrochloride (87 mg/kg) and xylazine hydrochloride (13 mg/kg). In diseased groups the distal bilepancreatic duct was ligated, while in sham-operated controls it was dissected but not ligated. Under general anesthesia induced as above, rats were killed by inferior vena caval exsanguination after 1, 3, or 24 hours (n ⫽ 6 rats at each time point in each study group). An additional group of nonoperated rats were killed (0-hour sham) to evaluate whether 1-, 3-, or 24-hour sham controls showed changes in JNK activation or M3 receptor induction after sham operation. The pancreas was quickly excised and divided into portions: a portion was fixed in 10% formaldehyde for morphologic studies; a portion was immediately snap-frozen in liquid nitrogen and stored at ⫺80°C for western blotting, immunoprecipitation, and immune complex kinase assay. Morphology One hematoxylin and eosin–stained section was prepared from each 10% formaldehyde-fixed, paraffin-embedded portion of pancreas and examined on a light microscope in a blinded fashion to be assigned an acute pancreatitis histology score (range 0 to 30) based on degree of white blood cell infiltration (range 0 to 10), acinar cell vacuolation (range 0 to 10), and focal necrosis (range 0 to 10; Table 1). Plasma amylase activity Determination of plasma amylase activity was performed at the Clinical Biochemistry Laboratory of the Iowa City VA Medical Center on a Hitachi spectrophotometer model P800 with bichromatic wavelengths 415 nm and 700 nm.
Table 1 Hyperamylasemia and morphological changes after duct ligation
Plasma amylase (⫻ 102 U/L) Sham Ligation Acute pancreatitis histology score (range 0–30) Sham Ligation
1 Hour
3 Hours
24 Hours
30 ⫾ 1.5 279 ⫾ 85*
43 ⫾ 8 900 ⫾ 233*
31 ⫾ 4 151 ⫾ 14*
0.3 ⫾ 0.2 5.0 ⫾ 0.3*
1.0 ⫾ 0.3 6.2 ⫾ 0.9*
2.8 ⫾ 1.2 19.3 ⫾ 0.5*
Six rats were studied in each experimental group at each time point. Bile-pancreatic duct ligation induced acute pancreatitis as evidenced by hyperamylasemia and pancreatic morphologic changes [WBC infiltration (0 –10), acinar vacuolation (0 –10) and focal acinar cell necrosis (0 –10)], compared with sham operation. Results are mean ⫾ SEM; Asterisk (*) indicates a statistically significant difference between the duct ligation group versus sham-operated control group at the same time point; P ⬍ 0.05, Kruskal-Wallis ANOVA was used for acute pancreatitis histology score, Student-Newman-Keuls test was used for analysis of plasma amylase levels.
Immunoblotting Immunoblot analysis was carried out essentially as described earlier [7]. In brief, portions of pancreas were snapfrozen in liquid nitrogen, stored at ⫺80°°C, homogenized in 10 mM Hepes buffer (pH 7.5), centrifuged at 500g to pellet cell debris, and then centrifuged at 15,000g to obtain a soluble fraction that was quantitated for protein using the Bradford assay. From each sample 40 g of total protein in SDS-sample buffer (62.5 mM Tris, pH 6.8, 2% w/v SDS, 10% glycerol, 50 mM DTT, 0.1% w/v bromophenol blue) were separated on 4% to 20% gradient gels by SDS-polyacrylamide gel electrophoresis and electroblotted onto nitrocellulose membranes. Protein blots were probed with specific primary antibodies (1:1000 v/v) and developed using the appropriate secondary antibody conjugated to horseradish peroxidase (HRP [1:2000 v/v]), and using the enhanced chemiluminescence (ECL) method as recommended by the manufacturer. Additional immunoblots were performed using -actin antibody as the primary antibody to evaluate equal loading. Immunoprecipitation Immunoprecipitation was carried out as described earlier [8]. In brief, tissues were extracted with a lysis buffer consisting of 1% Triton X-100, 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 50 mM NaF, 0.1 mM sodium vanadate, 1 mM benzamidine, 1 mM PMSF (phenyl methane sulfonyl flouride), and 10 g/mL each of aprotinin, leupeptin, chymostatin, pepstatin A, and antipain. Protein estimations were carried out using a commercial kit (Pierce) for the modified Lowry’s method. Tissue extracts were centrifuged at 12,000 rpm for 15 minutes to collect the supernatants. Aliquots of clear supernatants containing 500 g total protein in 1 mL lysis buffer were incubated with 2 g of
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specific JNK antibody overnight at 4°C followed by an additional incubation with 20 L of a 50% suspension of protein G-agarose for 1 hour at room temperature. The insoluble immune complex was collected and washed three times by brief centrifugations prior to immune complex kinase assay. Immune complex kinase assay The JNK was immunoprecipitated from tissues as described above, and followed by two additional washes with a kinase assay buffer prior to immune complex kinase assay. The assays were carried out as described earlier [8], in a total reaction volume of 20 L containing 100 mM TrisHCl, pH 7.0, 0.4 mM sodium ortho vanadate, 40 mM magnesium acetate, 1 mM dithiothreitol, and 30 M calmidazolium, 10 L of JNK immune complex, 2 g of kinase substrate (cJUN), 100 M [r-32P]-ATP (200 cpm/pmol), and was incubated at 30°C for 10 minutes. The reaction was stopped by addition of SDS-PAGE sample buffer, boiled for 2 minutes, centrifuged, and the supernatant was subjected to SDS-PAGE followed by autoradiography. Statistical analysis SigmaStat software (www.spss.com) was used for statistical analysis. Kruskal-Wallis analysis of variance (ANOVA) was used to analyze non-parametric data (acute pancreatitis histology score) and the Student-NewmanKeuls test was used for analysis of plasma amylase levels. Six rats were studied in each experimental group at each time point. A P value below 0.05 was considered statistically significant.
Results Compared with sham controls, 1, 3, and 24 hours of duct ligation induced acute pancreatitis as evidenced by pancreatic morphologic changes and hyperamylasemia (Table 1). Immunoblots of pancreatic homogenates showed a timedependent increase after duct ligation in cholinergic M3 receptor protein expression (Fig. 1), whereas sham-operation was not associated with an increase. The expression and activation of JNK were determined by immunoblot analysis using specific antibodies against its total and activated (phosphorylated) forms. In all immunoblots, -actin was used as a control to confirm equal protein loading (Fig. 1). The results indicate a rapid and sustained increase in the expression as well as activation of JNK in duct ligationinduced acute pancreatitis (Fig. 2). There was no difference in JNK expression or phosphorylation in sham-operated controls. The activation of JNK by an immune-complex kinase assay using cJUN as substrate showed a dramatic time-dependent increase in JNK activation after duct liga-
Fig. 1. Representative immunoblot shows increased cholinergic M3 receptor protein expression in duct ligation-induced acute pancreatitis compared with sham-operated controls. Tissue extracts of pancreas were prepared and subjected to immunoblotting using specific antibody against cholinergic M3 receptor. Immunoblot of -actin confirmed equal loading of lanes. The position of the cholinergic M3 receptor band was verified with molecular size standards. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
tion (Fig. 3), confirming our immunoblot finding of temporally increased JNK activation.
Comments The results of the current study show that ligation-induced acute pancreatitis in rats is associated with a persistent and time-dependent increase in expression of the cholinergic muscarinic M3 receptor, with a parallel increase in activation of the stress-activated protein kinase JNK. These findings are of a similar pattern as our previous findings of CCK-A receptor induction and p38MAPK activation in the same experimental model [6]. An increase in the number of G-protein coupled receptors, such as the cholinergic M3 receptor and the CCK-A receptor, has the potential of augmenting bile-pancreatic juice exclusion-induced pancreatic acinar cell hyperstimulation. Also, as stress-activated protein kinases such as p38MAPK and JNK are activated by G-protein coupled receptor stimulation, and are capable of inducing production of proinflammatory cytokines when activated, our findings may be important in elucidating early events in acute pancreatitis pathogenesis [4,5]. The stress-activated protein kinase pathways, also called mitogen activated protein kinase pathways or MAPK pathways (p38, JNK, and ERK), are novel protein kinase cascades involved in intracellular signal transduction [4,5]. A variety of noxious stimuli are known to activate p38, JNK, and ERK5 signaling pathways including heat shock, ionizing radiation, oxidant stress (peroxide), osmotic shock (sorbitol), DNA damaging agents (topoisomerase inhibitors), and inhibitors of protein synthesis (cycloheximide, anisomycin). In addition, MAPK pathways are also activated by
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Fig. 2. Temporal increase in JNK expression and activation in duct ligation-induced acute pancreatitis compared with sham-operated controls by immunoblot analysis using specific antibodies against the phosphorylated and total forms of JNK. The relative density units were obtained for comparison of changes with digitizing densitometry of this representative immunoblot. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
stimulation of diverse receptor families such as G-protein coupled receptors (CCK receptor), receptor tyrosine kinases (insulin, epidermal growth factor), and cytokine receptors. The activated MAPKs in turn regulate cell functions ranging from gene transcription, cell differentiation, protein synthesis, cell cycle arrest, apoptosis, and cell death. Following
Fig. 3. Autoradiogram of immune complex kinase assay using cJUN as substrate after immunoprecipitation with specific JNK antibody confirms the time-dependent JNK activation seen in immunoblots in Fig. 2. The position of cJUN was verified with molecular size standards. 0 ⫽ 0-hour sham; S1 ⫽ 1-hour sham; S3 ⫽ 3-hour sham; S24 ⫽ 24-hour sham; L1 ⫽ 1-hour ligation; L3 ⫽ 3-hour ligation; L24 ⫽ 24-hour ligation.
activation by phosphorylation via upstream kinases, the MAPKs activate downstream protein kinases or transcription factors to modulate specific cell functions. The potential for transcription factors to induce cellular proinflammatory cytokine production emphasizes the possible role of MAPK signaling pathways in the pathogenesis of diseases such as acute pancreatitis [4,5]. Acute pancreatitis is a common disease and gallstones are the most common etiologic factor world-wide [1]. Ligation-induced acute pancreatitis in rats is a useful experimental corollary of gallstone-induced acute pancreatitis to investigate early events in disease pathogenesis [2]. As the pathogenesis of acute pancreatitis in humans remains elusive, therapeutic strategies to alter the natural history of acute pancreatitis are not specific and consist of fasting the patient to rest the pancreas, intravenous fluid resuscitation, and other general supportive measures [1,2]. The investigation of acute pancreatitis pathogenesis in humans is difficult because the pancreas is anatomically inaccessible, invasive studies of the pancreas are dangerous, and surgical intervention occurs only after the disease has progressed to late stages with complications such as infected necrosis. Therefore, investigations into the mechanisms of disease pathogenesis have to rely on experimental models of acute pancreatitis and a variety of these models have been developed [2,9]. Evidence for the role of MAPK pathways in acute pancreatitis pathogenesis has recently been reported mainly in the lethal rat model of retrograde ductal infusion of bile salts and the nonlethal model of acute edematous pancreatitis caused by supramaximal doses of a CCK analog caerulein [10 –15]. The specific JNK inhibitor CEP 1347 and also inhibitors of the ERK signaling pathway (U0126, PD98059) have been reported to ameliorate caerulein-induced acute pancreatitis in rats [13–15]. CNI-1493, an inhibitor of p38MAPK activation, attenuated acute pancreatitis and improved survival after retrograde infusion of bile salts in rats, and also decreased the severity of caerulein-induced acute pancreatitis [10]. In acute pancreatitis induced by retrograde infusion of bile salts, CNI-1493 also ameliorated pancreatitis-associated pulmonary and hepatocellular injury along with limiting the increase of proinflammatory cytokines TNF-␣ and IL-1 in the plasma, lung, and liver [10 –12]. On the other hand, there is also a report where the specific p38MAPK inhibitor SB203580 exacerbates caerulein-induced acute pancreatitis in rats, suggesting that p38MAPK may have a protective rather than detrimental role in acute pancreatitis [13]. In view of these contradictory findings, further studies—including the use of other experimental models of acute pancreatitis—are required to clarify the role of p38MAPK and other stress kinases in disease pathogenesis. In a unique in vivo study, NF-B activation was achieved in pancreatic acinar cells by overexpressing the active RelA/ p65 subunit using adenoviral-mediated gene transfer techniques and was sufficient for the induction of both a pancreatic and systemic inflammatory response [16]. Taken
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together, the above reports support the hypothesis that stress-activated protein kinases and transcription factors modulate development of acute pancreatitis. In view of these observations, we evaluated activation of JNK in ligation-induced acute pancreatitis and showed rapid activation followed by a temporal increase in activation of JNK. Ligation of the bile-pancreatic duct in rats results in duct obstruction, bile-pancreatic juice exclusion from gut, and acute pancreatitis [2]. In rats, a neurohormonal duodenal response is responsible for the observed acinar cell hyperstimulation after bile-pancreatic juice exclusion [3]. The hormonal limb is mediated mainly by CCK secreted by I-cells of the duodenal mucosa, whereas the neural component is mediated via muscarinic cholinergic (vagal) pathways [17,18]. The absence of pancreatic juice trypsin in the duodenal lumen is the predominant stimulus for the duodenal response to pancreatic juice exclusion, while the combined absence of bile salts and pancreatic enzymes results in a synergistic—rather than additive—increase in hypercholecystokininemia and acinar cell hyperstimulation compared with the absence of either alone [2]. We have previously shown that ligation-induced acute pancreatitis in rats is associated with significant hypercholecystokininemia [2]. Using a unique and original surgical model, the donor rat model, we have also shown that duodenal replacement of bile-pancreatic juice— obtained fresh from a donor rat— achieves substantial amelioration of hypercholecystokininemia and ligation-induced acute pancreatitis in rats [2]. In another study, we showed that combined CCK-A and muscarinic receptor blockade ameliorates acinar hyperstimulation in the same experimental model [3]. These observations suggest that bile-pancreatic juice exclusion-induced acinar cell hyperstimulation in the presence of duct obstruction exacerbates acute pancreatitis through CCK-A and muscarinic receptor–mediated pathways. We propose a model for the pathogenesis of ligation-induced acute pancreatitis in rats where combined duct obstruction and acinar hyperstimulation imposes undue stress upon the acinar cell and thus activates acinar cell stress-activated protein kinase pathways resulting in acinar cell production of proinflammatory cytokines. Our demonstration of persistent and progressive activation of the stress kinase JNK in ligationinduced acute pancreatitis in the present study is consistent with this hypothesis for disease pathogenesis. The cholinergic M3 receptor induction observed in this study may contribute mechanistically in the exacerbation of acute pancreatitis as an increase in receptor number may amplify the hyperstimulatory effects of cholinergic stimulation. In summary, in the present study we have shown that there is a rapid, persistent and progressive induction of cholinergic M3 receptor and JNK—with JNK activation—in duct ligation-induced acute pancreatitis in rats. These findings are consistent with our previous observations that ligation-induced acute pancreatitis in rats is associated with CCK-A receptor induction and p38 induction and activation. In this experimental corollary of gallstone-induced
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acute pancreatitis, cholinergic and CCK-A receptor induction could amplify cholinergic- and CCK-induced exocrine pancreatic hyperstimulation. This excessive hyperstimulation in the presence of duct obstruction can intensify acinar cell stress. In consequence, activation of stress kinase pathways (JNK and p38MAPK) within the stressed acinar cell has the potential to induce acinar cell production of proinflammatory cytokines that exacerbate acute pancreatitis. Our findings in the current study are consistent with our central hypothesis that G-protein coupled receptors and stress-activated protein kinases are involved in acute pancreatitis pathogenesis. Acknowledgments Supported by a National Institutes of Health NIDDK Career Development Award (Grant 1-K08-DK062805-01 to Dr. Samuel) and internal grants from the University of Iowa and the Carver College of Medicine. References [1] Ranson J. Etiological and prognostic factors in human acute pancreatitis: a review. Am J Gastroenterol 1982;77:633– 8. [2] Samuel I, Toriumi Y, Wilcockson DP, et al. Bile and pancreatic juice replacement ameliorates early ligation- induced acute pancreatitis in rats. Am J Surg 1995;169:391–9. [3] Samuel I, Joehl RJ. Bile-pancreatic juice replacement, not cholinergic and cholecystokinin-receptor blockade, reverses acinar cell hyperstimulation after bile-pancreatic duct ligation. Am J Surg 1996;171: 207–11. [4] Williams JA. Intracellular signaling mechanisms activated by cholecystokinin- regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Annu Rev Physiol 2001;63:77–97. [5] Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807– 69. [6] Samuel I, Zaheer A, Nelson JJ, et al. p38 MAP kinase and CCK-A receptor protein expression increase in parallel in duct ligation-induced acute pancreatitis in rats. Pancreas 2002;25:448. [7] Kaplan R, Zaheer A, Jaye M, Lim R. Molecular cloning and expression of biologically active human glia maturation factor-beta. J Neurochem 1991;57:483–90. [8] Zaheer A, Lim R. In vitro inhibition of MAP kinase (ERK1/ERK2) activity by phosphorylated glia maturation factor (GMF). Biochemistry 1996;35:6283– 8. [9] Steer ML. Workshop on experimental pancreatitis. Dig Dis Sci 1985; 30:575– 81. [10] Yang J, Denham W, Tracey KJ, et al. The physiologic consequences of macrophage pacification during severe acute pancreatitis. Shock 1998;10:169 –75. [11] Yang J, Denham W, Carter G, et al. Macrophage pacification reduces rodent pancreatitis-induced hepatocellular injury through down-regulation of hepatic tumor necrosis factor alpha and interleukin-1beta. Hepatology 1998;28:1282– 8. [12] Yang J, Murphy C, Denham W, et al. Evidence of a central role for p38 map kinase induction of tumor necrosis factor alpha in pancreatitis-associated pulmonary injury. Surgery 1999;126:216 –22. [13] Fleischer F, Dabew R, Goke B, Wagner AC. Stress kinase inhibition modulates acute experimental pancreatitis. World J Gastroenterol 2001;7:259 – 65.
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