Cell Death in Pancreatitis
2005; Elsevier BV; Volume: 281; Issue: 6 Linguagem: Inglês
10.1074/jbc.m511276200
ISSN1083-351X
AutoresOlga A. Mareninova, Kai‐Feng Sung, Peggy Hong, Aurelia Lugea, Stephen J. Pandol, Ilya Gukovsky, Anna S. Gukovskaya,
Tópico(s)Cell death mechanisms and regulation
ResumoMechanisms of cell death in pancreatitis remain unknown. Parenchymal necrosis is a major complication of pancreatitis; also, the severity of experimental pancreatitis correlates directly with necrosis and inversely with apoptosis. Thus, shifting death responses from necrosis to apoptosis may have a therapeutic value. To determine cell death pathways in pancreatitis and the possibility of necrosis/apoptosis switch, we utilized the differences between the rat model of cerulein pancreatitis, with relatively high apoptosis and low necrosis, and the mouse model, with little apoptosis and high necrosis. We found that caspases were greatly activated during cerulein pancreatitis in the rat but not mouse. Endogenous caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) underwent complete degradation in the rat but remained intact in the mouse model. Furthermore, XIAP inhibition with embelin triggered caspase activation in the mouse model, implicating XIAP in caspase blockade in pancreatitis. Caspase inhibitors decreased apoptosis and markedly stimulated necrosis in the rat model, worsening pancreatitis parameters. Conversely, caspase induction with embelin stimulated apoptosis and decreased necrosis in mouse model. Thus, caspases not only mediate apoptosis but also protect from necrosis in pancreatitis. One protective mechanism is through degradation of receptor-interacting protein (RIP), a key mediator of "programmed" necrosis. We found that RIP was cleaved (i.e. inactivated) in the rat but not the mouse model. Caspase inhibition restored RIP levels; conversely, caspase induction with embelin triggered RIP cleavage. Our results indicate key roles for caspases, XIAP, and RIP in the regulation of cell death in pancreatitis. Manipulating these signals to change the pattern of death responses presents a therapeutic strategy for treatment of pancreatitis. Mechanisms of cell death in pancreatitis remain unknown. Parenchymal necrosis is a major complication of pancreatitis; also, the severity of experimental pancreatitis correlates directly with necrosis and inversely with apoptosis. Thus, shifting death responses from necrosis to apoptosis may have a therapeutic value. To determine cell death pathways in pancreatitis and the possibility of necrosis/apoptosis switch, we utilized the differences between the rat model of cerulein pancreatitis, with relatively high apoptosis and low necrosis, and the mouse model, with little apoptosis and high necrosis. We found that caspases were greatly activated during cerulein pancreatitis in the rat but not mouse. Endogenous caspase inhibitor X-linked inhibitor of apoptosis protein (XIAP) underwent complete degradation in the rat but remained intact in the mouse model. Furthermore, XIAP inhibition with embelin triggered caspase activation in the mouse model, implicating XIAP in caspase blockade in pancreatitis. Caspase inhibitors decreased apoptosis and markedly stimulated necrosis in the rat model, worsening pancreatitis parameters. Conversely, caspase induction with embelin stimulated apoptosis and decreased necrosis in mouse model. Thus, caspases not only mediate apoptosis but also protect from necrosis in pancreatitis. One protective mechanism is through degradation of receptor-interacting protein (RIP), a key mediator of "programmed" necrosis. We found that RIP was cleaved (i.e. inactivated) in the rat but not the mouse model. Caspase inhibition restored RIP levels; conversely, caspase induction with embelin triggered RIP cleavage. Our results indicate key roles for caspases, XIAP, and RIP in the regulation of cell death in pancreatitis. Manipulating these signals to change the pattern of death responses presents a therapeutic strategy for treatment of pancreatitis. Acute pancreatitis is an inflammatory disorder of exocrine pancreas, which carries considerable morbidity and mortality, and the pathophysiology of which remains obscure (1Raraty M.G. Connor S. Criddle D.N. Sutton R. Neoptolemos J.P. Curr. Gastroenterol. Rep. 2004; 6: 99-103Crossref PubMed Scopus (104) Google Scholar). During the past decade, significant progress has been achieved in our understanding of the inflammatory response in pancreatitis (2Bhatia M. Brady M. Shokuhi S. Christmas S. Neoptolemos J.P. Slavin J. J. Pathol. 2000; 190: 117-125Crossref PubMed Scopus (456) Google Scholar, 3Gukovsky I. Gukovskaya A.S. Blinman T.A. Zaninovic V. Pandol S.J. Am. J. Physiol. 1998; 275: G1402-G1414Crossref PubMed Google Scholar, 4Norman J. Am. J. Surg. 1998; 175: 76-83Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar, 5Steer M.L. Baillieres Best. Pract. Res. Clin. Gastroenterol. 1999; 13: 213-225Crossref PubMed Scopus (68) Google Scholar). By contrast, very little is known about the mechanisms mediating another major pathologic response in pancreatitis, the parenchymal cell death. In experimental models of acute pancreatitis, acinar cells have been shown to die through both necrosis and apoptosis (6Gukovskaya A.S. Perkins P. Zaninovic V. Sandoval D. Rutherford R. Fitzsimmons T. Pandol S.J. Poucell-Hatton S. Gastroenterology. 1996; 110: 875-884Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 7Kaiser A.M. Saluja A.K. Sengupta A. Saluja M. Steer M.L. Am. J. Physiol. 1995; 269: C1295-C1304Crossref PubMed Google Scholar). The apoptosis/necrosis ratio varies in different experimental models of pancreatitis. Of note, the severity of experimental pancreatitis directly correlates with the extent of necrosis and inversely with that of apoptosis (6Gukovskaya A.S. Perkins P. Zaninovic V. Sandoval D. 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Pancreatology. 2004; 4: 567-586Crossref PubMed Scopus (136) Google Scholar). Mechanisms underlying these differences are not known. Apoptosis and necrosis are two main types of cell death (13Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (760) Google Scholar, 14Assuncao G.C. Linden R. Eur. J. Biochem. 2004; 271: 1638-1650Crossref PubMed Scopus (253) Google Scholar, 15Edinger A.L. Thompson C.B. Curr. Opin. Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1121) Google Scholar, 16Leist M. Jaattela M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 589-598Crossref PubMed Scopus (1389) Google Scholar, 17Proskuryakov S.Y. Konoplyannikov A.G. Gabai V.L. Exp. Cell Res. 2003; 283: 1-16Crossref PubMed Scopus (565) Google Scholar, 18Reed J.C. Doctor K.S. Godzik A. Sci. STKE. 2004. 2004; : re9Google Scholar). Morphologically, apoptosis is manifested by cell shrinkage and chromatin condensation, whereas necrosis is characterized by swelling of the cell and its organelles and rupture of the plasma membrane. Biochemical hallmarks of apoptosis, such as activation of specific cysteine proteases, the caspases, and internucleosomal DNA fragmentation, are usually absent in necrotic cells. Apoptosis preserves the structural integrity of the plasma membrane, whereas the necrotic cell releases its constituents, which damage neighboring cells and promote inflammatory infiltration in the organ. Therefore, necrotic death is "deadlier" to the organism than apoptotic death (13Fiers W. Beyaert R. Declercq W. Vandenabeele P. Oncogene. 1999; 18: 7719-7730Crossref PubMed Scopus (760) Google Scholar, 14Assuncao G.C. Linden R. Eur. J. Biochem. 2004; 271: 1638-1650Crossref PubMed Scopus (253) Google Scholar, 15Edinger A.L. Thompson C.B. Curr. Opin. Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1121) Google Scholar). There are two distinct pathways of apoptosis (19Lee H.C. Wei Y.H. J. Biomed. Sci. 2000; 7: 2-15Crossref PubMed Google Scholar, 20Scaffidi C. Fulda S. Srinivasan A. Friesen C. Li F. Tomaselli K.J. Debatin K.M. Krammer P.H. Peter M.E. EMBO J. 1998; 17: 1675-1687Crossref PubMed Scopus (2633) Google Scholar, 21Wolf B.B. Green D.R. J. Biol. Chem. 1999; 274: 20049-20052Abstract Full Text Full Text PDF PubMed Scopus (865) Google Scholar). The extrinsic pathway is initiated by receptor-induced activation of the initiator caspase-8 (or caspase-10) followed by activation of effector caspases such as caspase-3. This pathway is typically triggered by "death receptors", e.g. tumor necrosis factor receptor or Fas. In the intrinsic pathway, a critical event is permeabilization of the mitochondrial outer membrane, resulting in the release of pro-apoptotic factors such as cytochrome c. Once released, cytochrome c forms a complex with Apaf-1 and procaspase-9, resulting in caspase-9 activation. Caspase-9 further cleaves and activates the effectors caspases, (e.g. caspase-3) leading to subsequent degradation of cellular constituents. Inhibitor of apoptosis proteins (IAPs) 2The abbreviations used are: IAP, inhibitor of apoptosis protein; FLIP, FLICE-inhibitory protein; RIP, receptor-interacting protein; Q-VD-OPH, Q-Val-Asp(non-O-methylated)-Oph; XIAP, X-linked inhibitor of apoptosis protein; Z-D-DCB, Z-Asp-2,6-dichlorobenzoyloxymethylketone; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; CCK-8, cholecystokinin-8; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; AMC, 7-amino-4-methylcoumarin; ERK, extracellular signal-regulated kinase; E3, ubiquitin-protein isopeptide ligase. are an important class of endogenous proteins that negatively regulate caspase activation (22Bratton S.B. Walker G. Srinivasula S.M. Sun X.M. Butterworth M. Alnemri E.S. Cohen G.M. EMBO J. 2001; 20: 998-1009Crossref PubMed Scopus (342) Google Scholar, 23Salvesen G.S. Duckett C.S. Nat. Rev. Mol. 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The mechanisms of RIP action, as well as its targets, are poorly understood (15Edinger A.L. Thompson C.B. Curr. Opin. Cell Biol. 2004; 16: 663-669Crossref PubMed Scopus (1121) Google Scholar, 16Leist M. Jaattela M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 589-598Crossref PubMed Scopus (1389) Google Scholar, 17Proskuryakov S.Y. Konoplyannikov A.G. Gabai V.L. Exp. Cell Res. 2003; 283: 1-16Crossref PubMed Scopus (565) Google Scholar, 30Kuida K. Zheng T.S. Na S. Kuan C. Yang D. Karasuyama H. Rakic P. Flavell R.A. Nature. 1996; 384: 368-372Crossref PubMed Scopus (1713) Google Scholar). Acinar cell necrosis, and in particular, recurrent necrosis, is one of the most serious complications of acute pancreatitis (1Raraty M.G. Connor S. Criddle D.N. Sutton R. Neoptolemos J.P. Curr. Gastroenterol. Rep. 2004; 6: 99-103Crossref PubMed Scopus (104) Google Scholar, 35Bassi C. Butturini G. Falconi M. Salvia R. Frigerio I. Pederzoli P. 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Gastroenterology. 1996; 111: 1081-1091Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 10Bhatia M. Wallig M.A. Hofbauer B. Lee H.S. Frossard J.L. Steer M.L. Saluja A.K. Biochem. Biophys. Res. Commun. 1998; 246: 476-483Crossref PubMed Scopus (119) Google Scholar, 11Bhatia M. Am. J. Physiol. 2004; 286: G189-G196Crossref PubMed Scopus (186) Google Scholar, 12Gukovskaya A.S. Pandol S.J. Pancreatology. 2004; 4: 567-586Crossref PubMed Scopus (136) Google Scholar) that switching from the necrotic pattern of cell death to apoptosis could be beneficial in treatment of acute pancreatitis. In the present study we investigated the mechanisms of apoptosis and necrosis in pancreatitis and explored the possibility of necrosis/apoptosis switch through manipulating these signaling mechanisms. For this purpose, we utilized the differences between two related rodent models of acute pancreatitis. Pancreatitis induced in rats by supramaximally stimulating doses of the CCK-8 analog, cerulein, is a mild form of the disease characterized by relatively high extent of apoptosis and low necrosis (7Kaiser A.M. Saluja A.K. Sengupta A. Saluja M. Steer M.L. Am. J. Physiol. 1995; 269: C1295-C1304Crossref PubMed Google Scholar, 9Sandoval D. Gukovskaya A. Reavey P. Gukovsky S. Sisk A. Braquet P. Pandol S.J. Poucell-Hatton S. Gastroenterology. 1996; 111: 1081-1091Abstract Full Text PDF PubMed Scopus (226) Google Scholar). By contrast, in mice the same cerulein treatment results in a more severe disease with significant necrosis and very little apoptosis (7Kaiser A.M. Saluja A.K. Sengupta A. Saluja M. Steer M.L. Am. J. Physiol. 1995; 269: C1295-C1304Crossref PubMed Google Scholar). Both are the most commonly used and well characterized in vivo models of acute pancreatitis (37Lerch M.M. Adler G. Int. J. Pancreatol. 1994; 15: 159-170PubMed Google Scholar, 38Gorelick F.S. Adler G. Kern H.F. Go V.W. DiMagno E.P. Gardner J.D. Lebenthal E. Reber H.A. Scheele G.A. The Pancreas: Biology, Pathology, and Disease. Raven, New York1993: 64-69Google Scholar). We found drastic differences in death-signaling mechanisms, namely, caspase activation, and XIAP and RIP degradation, between the rat and mouse models of cerulein pancreatitis. By manipulating these mechanisms using pharmacologic inhibitors, we were able to shift the necrosis/apoptosis ratio, making the two models more like each other. The results identify several critical mediators of acinar cell death that may represent targets for therapeutic interventions to attenuate cell-death responses of acute pancreatitis. Experimental Pancreatitis—Cerulein pancreatitis was induced in male (200–250 g) Sprague-Dawley rats and male (25–30 g) Swiss Webster CD-1 mice by up to seven hourly intraperitoneal injections of 50 μg/kg cerulein. Control animals received similar injections of physiologic saline. Caspase inhibitors Q-VD-OPH (25 mg/kg) and Z-D-DCB (10 mg/kg), or vehicle (Me2SO), were applied in rats as a single intravenous injection 30 min before the start of cerulein treatment. XIAP inhibitor embelin (20 mg/kg), or vehicle (Me2SO), was applied in mice as one daily subcutaneous injection for 5 consecutive days; treatment with cerulein started 30 min after the last embelin injection. In the cerulein models, animals were sacrificed at 30 min, 2, 4, and 7 h after the first injection. Arginine pancreatitis was induced in rats by two hourly intraperitoneal injections of 2.5 g/kg l-arginine; controls received similar injections of saline. In this model, rats were sacrificed 24 h after the first injection. Animals were euthanized by CO2-induced asphyxiation, and the blood and pancreas were harvested for measurements. Serum Amylase and Lipase Measurements—Serum amylase and lipase levels were measured in a Hitachi 707 analyzer (Antech Diagnostics, Irvine, CA). Quantification of Apoptosis—Apoptosis was quantified on pancreatic tissue sections stained with Hoechst 33258 to visualize nuclear chromatin morphology or with TUNEL assay to measure DNA breaks, as we described previously (3Gukovsky I. Gukovskaya A.S. Blinman T.A. Zaninovic V. Pandol S.J. Am. J. Physiol. 1998; 275: G1402-G1414Crossref PubMed Google Scholar, 6Gukovskaya A.S. Perkins P. Zaninovic V. Sandoval D. Rutherford R. Fitzsimmons T. Pandol S.J. Poucell-Hatton S. Gastroenterology. 1996; 110: 875-884Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar, 9Sandoval D. Gukovskaya A. Reavey P. Gukovsky S. Sisk A. Braquet P. Pandol S.J. Poucell-Hatton S. Gastroenterology. 1996; 111: 1081-1091Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 39Gukovskaya A.S. Gukovsky I. Zaninovic V. Song M. Sandoval D. Gukovsky S. Pandol S.J. J. Clin. Invest. 1997; 100: 1853-1862Crossref PubMed Scopus (344) Google Scholar). Tissue was fixed in 4% buffered formaldehyde and embedded in paraffin, and 6 μm-thick sections were adhered to glass slides. Sections were deparaffinized by washing in Hemo-De and hydrated by transferring through graded ethanol. The sections were stained with 8 μg/ml Hoechst 33258 and examined by fluorescence microscopy. Nuclei with condensed or fragmented chromatin were considered apoptotic (12Gukovskaya A.S. Pandol S.J. Pancreatology. 2004; 4: 567-586Crossref PubMed Scopus (136) Google Scholar, 39Gukovskaya A.S. Gukovsky I. Zaninovic V. Song M. Sandoval D. Gukovsky S. Pandol S.J. J. Clin. Invest. 1997; 100: 1853-1862Crossref PubMed Scopus (344) Google Scholar). In TUNEL assay (6Gukovskaya A.S. Perkins P. Zaninovic V. Sandoval D. Rutherford R. Fitzsimmons T. Pandol S.J. Poucell-Hatton S. Gastroenterology. 1996; 110: 875-884Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar), tissue sections were stained for breaks in DNA using terminal deoxynucleotidyl transferase and fluorescein isothiocyanate-labeled dUTP according to the manufacturer's protocol (Promega, Madison, WI). For these and other quantifications of histologic measurements, a total of at least 1000 acinar cells was counted on pancreatic tissue sections from each animal. Quantification of Necrosis—Quantification of necrosis was performed on pancreatic tissue sections stained with H&E. Cells with swollen cytoplasm, loss of plasma membrane integrity, and leakage of organelles into interstitium were considered necrotic. Quantification of Inflammatory Infiltration—Quantification of inflammatory infiltration was performed on pancreatic tissue sections stained with H&E. Isolation of Pancreatic Acini—Isolation of pancreatic acini from rats or mice was performed using a collagenase digestion procedure as we described previously (3Gukovsky I. Gukovskaya A.S. Blinman T.A. Zaninovic V. Pandol S.J. Am. J. Physiol. 1998; 275: G1402-G1414Crossref PubMed Google Scholar, 29Gukovskaya A.S. Gukovsky I. Jung Y. Mouria M. Pandol S.J. J. Biol. Chem. 2002; 277: 22595-22604Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 39Gukovskaya A.S. Gukovsky I. Zaninovic V. Song M. Sandoval D. Gukovsky S. Pandol S.J. J. Clin. Invest. 1997; 100: 1853-1862Crossref PubMed Scopus (344) Google Scholar). Dispersed pancreatic acini were then incubated at 37 °C in 199 medium (Invitrogen) in the presence or absence of 100 nm CCK-8. Caspase Activities—Caspase activities were measured using a fluorogenic assay as we described previously (29Gukovskaya A.S. Gukovsky I. Jung Y. Mouria M. Pandol S.J. J. Biol. Chem. 2002; 277: 22595-22604Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 40Vaquero E.C. Edderkaoui M. Nam K.J. Gukovsky I. Pandol S.J. Gukovskaya A.S. Gastroenterology. 2003; 125: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Pancreatic tissue or acinar cell samples were homogenized in a lysis buffer containing 150 mm NaCl, 50 mm Tris-HCl (pH 7.5), 0.5% Igepal CA-630, and 0.5 mm EDTA, centrifuged for 15 min at 16,000 × g, and the supernatants were collected. Proteolytic reactions were carried out at 37 °C in a buffer containing 25 mm HEPES (pH 7.5), 10% sucrose, 0.1% CHAPS, and 10 mm dithiothreitol, using substrates specific for caspase-3 (Ac-DEVD-AMC), caspase-8 (Ac-IETD-AMC), or caspase-9 (Ac-LEHD-AMC). Cleavage of these substrates relieves 7-amino-4-methylcoumarin (AMC), which emits fluorescent signal with excitation at 380 nm and emission at 440 nm. Fluorescence was calibrated using a standard curve for AMC. The data are expressed as moles of AMC/mg of protein/min. Preparation of Tissue and Cell Lysates for Western Blot Analysis—Portions of frozen tissue were homogenized on ice in radioimmune precipitation assay buffer supplemented with 1 mm phenylmethylsulfonyl fluoride and the protease inhibitor mixture containing pepstatin, leupeptin, chymostatin, antipain, and aprotinin (5 μg/ml of each), rotated for 20 min at 4 °C, and centrifuged at 4 °C for 15 min at 16,000 × g. The supernatants were collected and stored at –80 °C. Dispersed pancreatic acini were washed twice with ice-cold phosphate-buffered saline, resuspended in radioimmune precipitation assay buffer, and processed as described above for tissue samples. Protein concentrations were determined by the Bio-Rad protein assay (Bio-Rad Laboratories). Preparation of Membrane and Cytosolic Fractions—Pancreatic tissue or acinar cell samples were homogenized in a buffer containing 250 mm sucrose, 20 mm HEPES-KOH (pH 7.0), 10 mm KCl, 1 mm EGTA, 2 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and the protease inhibitor mixture in a glass Dounce homogenizer (80 strokes). Nuclei were removed by centrifugation at 1,000 × g for 10 min at 4 °C. The supernatant was centrifuged for 1 h at 100,000 × g, and both the pellet (mitochondria-enriched membrane fraction) and supernatant (cytosolic fraction) were collected separately and used for Western blotting. Western Blot Analysis—was performed on total tissue and cell lysates, or on the membrane and cytosolic fractions, as we described previously (3Gukovsky I. Gukovskaya A.S. Blinman T.A. Zaninovic V. Pandol S.J. Am. J. Physiol. 1998; 275: G1402-G1414Crossref PubMed Google Scholar, 29Gukovskaya A.S. Gukovsky I. Jung Y. Mouria M. Pandol S.J. J. Biol. Chem. 2002; 277: 22595-22604Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 39Gukovskaya A.S. Gukovsky I. Zaninovic V. Song M. Sandoval D. Gukovsky S. Pandol S.J. J. Clin. Invest. 1997; 100: 1853-1862Crossref PubMed Scopus (344) Google Scholar, 40Vaquero E.C. Edderkaoui M. Nam K.J. Gukovsky I. Pandol S.J. Gukovskaya A.S. Gastroenterology. 2003; 125: 1188-1202Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Proteins were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose membranes. Nonspecific binding was blocked by 1-h incubation of the membranes in 5% (w/v) nonfat dry milk in Tris-buffered saline (pH 7.5). The blots were then incubated for 2 h or overnight with primary antibodies in the antibody buffer containing 1% (w/v) nonfat dry milk in TTBS (0.05% (v/v) Tween 20 in Tris-buffered saline), washed three times with TTBS, and finally incubated for 1 h with a peroxidase-labeled secondary antibody in the antibody buffer. The blots were developed for visualization using enhanced chemiluminescence (ECL) detection kit (Pierce). Band intensities in the immunoblots were quantified by densitometry. Reduction/Alkylation—This was done according to a previous study (41Soares R.V. Liu B. Oppenheim F.G. Offner G.D. Troxler R.F. Arch. Oral Biol. 2002; 47: 591-597Crossref PubMed Scopus (7) Google Scholar), with minor modifications. Briefly, proteins in tissue lysate were reduced in 0.1 m Tris-HCl (pH 9.0) containing 8 m urea and 0.1 m dithiothreitol, and incubated at 37 °C for 1 h. Alkylation of reduced proteins was achieved by incubation for 30 min with 0.3 m iodoacetamide at room temperature in the dark. The samples were then separated by SDS-PAGE, transferred to nitrocellulose membranes, and subjected to Western blot analysis with antibody against cytochrome c. Antibodies and Reagents—Antibodies against XIAP and p44/42 mitogen-activate protein kinase (Erk1/2) were from Cell Signaling (Beverly, MA); caspase-3, caspase-8, and FLIPS/L, from Santa Cruz Biotechnology (Santa Cruz, CA); cytochrome c, RIP, and Pyk2, from BD Biosciences (San Diego, CA); COX IV, from Molecular Probes (Eugene, OR); and caspase-9, from Stressgen (San Diego, CA). CCK-8 was from American Peptide (Sunnyvale, CA); cerulein was from Peninsula Laboratories (Belmont, CA). Caspase fluorogenic substrates Ac-IETD-AMC, Ac-DEVD-AMC, and Ac-LEHD-AMC, and the XIAP inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) were from Biomol (Plymouth Meeting, PA). Caspase inhibitors Z-Asp-2,6-dichlorobenzoyloxymethylketone (Z-D-DCB) and Q-Val -Asp(non-O-methylated)-OPh (Q-VD-OPH) were from ALEXIS Biochemicals (San Diego, CA) and Enzyme Systems Products (Livermore, CA), respectively. Other reagents were from Sigma. In rat and mouse cerulein pancreatitis, we measured time-dependent changes in the extent of apoptosis and necrosis in pancreas (Fig. 1). In the rat model, the extent of apoptosis was unchanged at 30 min after the start of cerulein treatment and increased ∼33-fold at 4 h and ∼16-fold at 7 h. The decrease in apoptosis at 7 h could be due to an increased clearance of apoptotic cells by inflammatory cells, the number of which in pancreas increases with time. Compared with the rat model, there was much less apoptosis in mouse cerulein pancreatitis, and the increase in apoptosis was minimal. Necrosis time dependently increased in both models of cerulein pancreatitis. In the rat model, the increase in necrosis was only detected at 4 a
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