Transgenic expression of pancreatic secretory trypsin inhibitor-I ameliorates secretagogue-induced pancreatitis in mice

Background & aims Endogenous trypsin inhibitors are believed to inhibit protease activity if trypsin becomes inadvertently activated within the acinar cell. However, this action remains unproven, and the role of endogenous pancreatic trypsin inhibitors in acute pancreatitis is unknown. In this study, we tested whether increased levels of pancreatic secretory trypsin inhibitor-I (PSTI-I) in mice could prevent secretagogue-induced pancreatitis. Methods Rat PSTI-I expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the rat elastase I enhancer/promoter. Secretagogue-induced pancreatitis was achieved by 12 hourly intraperitoneal injections of caerulein. The severity of pancreatitis was assessed by measurements of serum amylase, histologic grading, and pancreas wet weight-to-body weight ratio. Trypsinogen activation and trypsin activity were measured in pancreatic extracts. Results Targeted expression of PSTI-I to the pancreas increased endogenous trypsin inhibitor capacity by 190% (P < .01) in transgenic vs. nontransgenic mice. Caerulein administration to nontransgenic mice produced histologic evidence of acute pancreatitis, and significantly elevated serum amylase and pancreas weight ratio. In caerulein-treated transgenic mice, the histologic severity of pancreatitis was significantly reduced. There was no difference in trypsinogen activation peptide levels between caerulein-treated transgenic and nontransgenic mice. However, trypsin activity was significantly lower in transgenic mice receiving caerulein compared with nontransgenic mice. Conclusions These data demonstrate that the severity of secretagogue-induced pancreatitis is significantly ameliorated in mice with higher pancreatic levels of trypsin inhibitor. We propose that PSTI-I prevents pancreatitis by inhibiting the activity of trypsin, rather than by reducing trypsinogen activation. Background & aims Endogenous trypsin inhibitors are believed to inhibit protease activity if trypsin becomes inadvertently activated within the acinar cell. However, this action remains unproven, and the role of endogenous pancreatic trypsin inhibitors in acute pancreatitis is unknown. In this study, we tested whether increased levels of pancreatic secretory trypsin inhibitor-I (PSTI-I) in mice could prevent secretagogue-induced pancreatitis. Methods Rat PSTI-I expression was targeted to pancreatic acinar cells in transgenic mice by creating a minigene driven by the rat elastase I enhancer/promoter. Secretagogue-induced pancreatitis was achieved by 12 hourly intraperitoneal injections of caerulein. The severity of pancreatitis was assessed by measurements of serum amylase, histologic grading, and pancreas wet weight-to-body weight ratio. Trypsinogen activation and trypsin activity were measured in pancreatic extracts. Results Targeted expression of PSTI-I to the pancreas increased endogenous trypsin inhibitor capacity by 190% (P < .01) in transgenic vs. nontransgenic mice. Caerulein administration to nontransgenic mice produced histologic evidence of acute pancreatitis, and significantly elevated serum amylase and pancreas weight ratio. In caerulein-treated transgenic mice, the histologic severity of pancreatitis was significantly reduced. There was no difference in trypsinogen activation peptide levels between caerulein-treated transgenic and nontransgenic mice. However, trypsin activity was significantly lower in transgenic mice receiving caerulein compared with nontransgenic mice. Conclusions These data demonstrate that the severity of secretagogue-induced pancreatitis is significantly ameliorated in mice with higher pancreatic levels of trypsin inhibitor. We propose that PSTI-I prevents pancreatitis by inhibiting the activity of trypsin, rather than by reducing trypsinogen activation. The majority of pancreatic digestive enzymes are synthesized and secreted by pancreatic acinar cells as inactive zymogens that are activated in the duodenum. In the intestine, the brush-border enzyme enteropeptidase activates trypsinogen by cleaving the amino terminal, 6 amino acid trypsinogen activation peptide (TAP) yielding active trypsin, which then catalyzes the activation of other digestive zymogens. It is generally accepted that the pathophysiology of acute pancreatitis involves autodigestion by digestive enzymes that are prematurely activated within the pancreas. Intrapancreatic activation of inactive digestive zymogens has been demonstrated to occur in rodent models of pancreatitis,1Bialek R. Willemer S. Arnold R. Adler G. Evidence of intracellular activation of serine proteases in acute cerulein-induced pancreatitis in rats.Scand J Gastroenterol. 1991; 26: 190-196Crossref PubMed Scopus (110) Google Scholar, 2Mithofer K. Fernandez-del Castillo C. Frick T.W. Lewandrowski K.B. Rattner D.W. Warshaw A.L. Acute hypercalcemia causes acute pancreatitis and ectopic trypsinogen activation in the rat.Gastroenterology. 1995; 109: 239-246Abstract Full Text PDF PubMed Scopus (111) Google Scholar as well as in patients with acute pancreatitis.3Geokas M.C. Rinderknecht H. Free proteolytic enzymes in pancreatic juice of patients with acute pancreatitis.Am J Dig Dis. 1974; 19: 591-598Crossref PubMed Scopus (79) Google Scholar A derangement of intracellular enzyme sorting in the early stages of pancreatitis has been identified by the colocalization of digestive zymogens with lysosomal enzymes in crinophagic cytoplasmic vacuoles. This defect promotes intrapancreatic activation of pancreatic enzymes4Saluja A. Saluja M. Villa A. Leli U. Rutledge P. Meldolesi J. Steer M. Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.J Clin Invest. 1989; 84: 1260-1266Crossref PubMed Scopus (141) Google Scholar, 5Watanabe O. Baccino F.M. Steer M.L. Meldolesi J. Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell early morphological changes during development of experimental pancreatitis.Am J Physiol. 1984; 246: G457-G467PubMed Google Scholar, 6Willemer S. Bialek R. Adler G. Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis.Histochemistry. 1990; 94: 161-170Crossref PubMed Scopus (66) Google Scholar and is accompanied by a block in the secretion of zymogens from acinar cells.7Saito I. Hashimoto S. Saluja A. Steer M.L. Meldolesi J. Intracellular transport of pancreatic zymogens during caerulein supramaximal stimulation.Am J Physiol. 1987; 253: G517-G526PubMed Google Scholar, 8Saluja A. Hashimoto S. Saluja M. Powers R.E. Meldolesi J. Steer M.L. Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis.Am J Physiol. 1987; 253: G508-G516PubMed Google Scholar, 9Saluja A. Saito I. Saluja M. Houlihan M.J. Powers R.E. Meldolesi J. Steer M. In vivo rat pancreatic acinar cell function during supramaximal stimulation with caerulein.Am J Physiol. 1985; 249: G702-G710PubMed Google Scholar Recent studies have helped elucidate the mechanism of intrapancreatic activation of trypsinogen and the premature activation of digestive zymogens in acute pancreatitis. Intrapancreatic activation of trypsinogen has been demonstrated within 10 minutes of beginning supramaximal stimulation of rats with the cholecystokinin analogue caerulein.10Grady T. Saluja A. Kaiser A. Steer M. Edema and intrapancreatic trypsinogen activation precede glutathione depletion during caerulein pancreatitis.Am J Physiol. 1996; 271: G20-G26PubMed Google Scholar An important step in caerulein-induced activation of trypsinogen in pancreatic acini occurs via the lysosomal hydrolase cathepsin B.11Saluja A.K. Donovan E.A. Yamanaka K. Yamaguchi Y. Hofbauer B. Steer M.L. Cerulein-induced in vitro activation of trypsinogen in rat pancreatic acini is mediated by cathepsin B.Gastroenterology. 1997; 113: 304-310Abstract Full Text PDF PubMed Scopus (173) Google Scholar Utilizing subcellular fractionation techniques and electron microscopic immunolocalization, it was shown that trypsinogen activation in caerulein-induced pancreatitis occurs in cytoplasmic vacuoles containing digestive zymogens and lysosomal hydrolases.12Hofbauer B. Saluja A.K. Lerch M.M. Bhagat L. Bhatia M. Lee H.S. Frossard J.L. Adler G. Steer M.L. Intra-acinar cell activation of trypsinogen during caerulein-induced pancreatitis in rats.Am J Physiol. 1998; 275: G352-G362PubMed Google Scholar Thus, it is believed that lysosomal hydrolases colocalized with trypsinogen during early pancreatitis cause its activation to trypsin, triggering the intrapancreatic activation of digestive enzymes and resultant pancreatic autodigestion. Because intrapancreatic activation of small quantities of trypsinogen may occur under physiologic conditions and would lead to the release of a cascade of activated digestive enzymes, barriers exist to protect the pancreas from inappropriate activation of zymogens. One such barrier is the presence in the mammalian pancreas and pancreatic juice of a trypsin inhibitor, pancreatic secretory trypsin inhibitor (PSTI).13Pubols M.H. Bartelt D.C. Greene L.J. Trypsin inhibitor from human pancreas and pancreatic juice.J Biol Chem. 1974; 249: 2235-2242Abstract Full Text PDF PubMed Google Scholar, 14Bartelt D.C. Shapanka R. Greene L.J. The primary structure of the human pancreatic secretory trypsin inhibitor Amino acid sequence of the reduced S-aminoethylated protein.Arch Biochem Biophys. 1977; 179: 189-199Crossref PubMed Scopus (88) Google Scholar PSTI is a Kazal-type trypsin inhibitor that acts by rapidly binding covalently with trypsin to form an inactive, stable complex. Studies have suggested that up to 20% of the potentially available trypsin activity in the pancreas may be inhibited by PSTI.15Rinderknecht H. Pancreatic secretory enzymes.in: Go V.L.W. DiMagno J.D. Gardner J.D. Lebenthal E. Reber H.A. Scheele G. The pancreas biology, pathophysiology, and disease. Raven Press, New York1993: 219-251Google Scholar The therapeutic uses of exogenous trypsin inhibitors in acute pancreatitis have been investigated in both animal models and human trials. In general, beneficial effects have been observed only when high amounts of trypsin inhibitor have been used and only when given as prophylaxis before the pancreatic injury. Urinastatin, a trypsin inhibitor extracted from human urine, was shown to reduce the severity of caerulein-induced pancreatitis in rats,16Tani S. Otsuki M. Itoh H. Nakamura T. Fujii M. Okabayashi Y. Fujisawa T. Baba S. The protective effect of the trypsin inhibitor urinastatin on cerulein-induced acute pancreatitis in rats.Pancreas. 1988; 3: 471-476Crossref PubMed Scopus (20) Google Scholar and the synthetic trypsin inhibitors ONO-330717Sobajima H. Hayakawa T. Kondo T. Shibata T. Kitagawa M. Sakai Y. Ishiguro H. Tanikawa M. Nakae Y. Effect of a new synthetic trypsin inhibitor on taurocholate-induced acute pancreatitis in rats.Pancreas. 1993; 8: 240-247Crossref PubMed Scopus (15) Google Scholar and E312318Sobajima H. Hayakawa T. Kondo T. Shibata T. Kitagawa M. Sakai Y. Ishiguro H. Tanikawa M. Nakae Y. Monitoring serum tryptic activity and effect of trypsin inhibitor on rat acute pancreatitis.Digestion. 1994; 55: 90-96Crossref PubMed Scopus (7) Google Scholar improved the survival rate in trypsin-taurocholate-induced acute pancreatitis in rats. Intravenous infusion of a recombinant human PSTI analogue was shown to reduce the severity of caerulein-induced pancreatitis in rats.19Funakoshi A. Miyasaka K. Jimi A. Kitani K. Teraoka H. Yoshida N. Protective effect of human pancreatic secretory trypsin inhibitor on cerulein-induced acute pancreatitis in rats.Digestion. 1992; 52: 145-151Crossref PubMed Scopus (12) Google Scholar, 20Chen Y.Z. Ikei S. Yamaguchi Y. Sameshima H. Sugita H. Moriyasu M. Ogawa M. The protective effects of long-acting recombinant human pancreatic secretory trypsin inhibitor (R44S-PSTI) in a rat model of cerulein-induced pancreatitis.J Int Med Res. 1996; 24: 59-68PubMed Google Scholar In a multicenter, prospective, randomized trial, prophylactic administration of gabexate, a nonspecific protease inhibitor, reduced the frequency of acute pancreatitis following endoscopic retrograde cholangiopancreatography.21Cavallini G. Tittobello A. Frulloni L. Masci E. Mariana A. Di Francesco V. Gabexate for the prevention of pancreatic damage related to endoscopic retrograde cholangiopancreatography Gabexate in digestive endoscopy—Italian Group.N Engl J Med. 1996; 335: 919-923Crossref PubMed Scopus (361) Google Scholar These studies indicate that inhibition of active proteases can reduce the severity of pancreatitis if administered in advance of an experimental insult and support the concept that pancreatic proteases may be involved in the pathogenesis of pancreatitis. The present study was designed to determine whether endogenous trypsin inhibitors play a protective role in a murine model of acute pancreatitis. Pancreatic secretory trypsin inhibitor-I (PSTI-I), also known as monitor peptide, is a 61-amino acid peptide produced by pancreatic acinar cells, stored in zymogen granules, and released into the duodenal lumen with digestive zymogens.22Fukuoka S. Kawajiri H. Fushiki T. Takahashi K. Iwai K. Localization of pancreatic enzyme secretion-stimulating activity and trypsin inhibitory activity in zymogen granule of the rat pancreas.Biochim Biophys Acta. 1986; 884: 18-24Crossref PubMed Scopus (29) Google Scholar, 23Fushiki T. Fukuoka S. Iwai K. Stimulatory effect of an endogenous peptide in rat pancreatic juice on pancreatic enzyme secretion in the presence of atropine evidence for different mode of action of stimulation from exogenous trypsin inhibitors.Biochem Biophys Res Commun. 1984; 118: 532-537Crossref PubMed Scopus (53) Google Scholar PSTI-I possesses trypsin inhibitor activity and bears structural homology to human PSTI. In the current study, we have created a PSTI-I transgenic mouse model by utilizing the mouse elastase promoter element to target expression of rat PSTI-I to the pancreas. This model provides a novel approach for determining the significance of trypsin inhibition in the pathophysiology of acute pancreatitis. We tested the hypotheses that pancreatic expression of rat PSTI-I in mice prevents secretagogue-induced pancreatitis and that a protective effect of PSTI-I expression is mediated by inhibition of activated trypsin. We demonstrate pancreas-specific expression of rat PSTI-I in C57BL/6J transgenic mice, which confers an increase in trypsin inhibitor capacity. The severity of caerulein-induced pancreatitis is significantly ameliorated in mice expressing PSTI-I, and pancreatic trypsin activity is significantly reduced. These findings indicate that PSTI-I ameliorates pancreatitis by inhibiting the activity of trypsin. B6SJLF1/J and C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME), housed in climate-controlled rooms with a 12:12-hour light-dark cycle and permitted water and chow ad libitum. A transgenic mouse line with pancreas-specific expression of rat PSTI-I was created, and all experiments were performed using littermates from at least the F5 generation. Transgenic and nontransgenic mice were randomly assigned to receive either vehicle or the cholecystokinin analog caerulein (Bachem California Inc., Torrance, CA). All animal experiments were performed with approval of the Duke University Institutional Animal Care and Use Committee. A chimeric construct of the rat elastase I enhancer/promoter and a rat PSTI minigene were used to target expression to the pancreas. The transgene contained the 510-base pair rat elastase I enhancer/promoter24Hammer R.E. Swift G.H. Ornitz D.M. Quaife C.J. Palmiter R.D. Brinster R.L. MacDonald R.J. The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.Mol Cell Biol. 1987; 7: 2956-2967Crossref PubMed Scopus (72) Google Scholar upstream of a 4.6-kb minigene of rat PSTI. The minigene contained all 4 exons and 2.0-kb of 3′ flanking DNA from the rat gene; for ease of handling, the first intron was shortened from 2.3 to 0.8 kb, the second from 2 to 0.6 kb, and the third from 7.6 to 0.8 kb by deleting sequences from the center of the introns. Linear DNA devoid of cloning vector sequences was injected into fertilized eggs of the B6SJLF1/J mouse strain and reimplanted into the uterus of a female B6SJLF1/J mouse. Four founder mice bearing the transgene were obtained. One founder was lost because of unanticipated death, 1 failed to produce offspring, and 2 founder lines were maintained and expanded. Integration of the minigene into the genome was confirmed by Southern blot analysis and polymerase chain reaction (data not shown). The 2 founders were backcrossed with C57BL/6J mice, and experiments were performed using littermates from at least the F5 generation. Similar levels of PSTI-I expression were found in each founder line. For experiments, transgenic animals were compared with their nontransgenic littermates, with genotypes documented by DNA analysis. Expression of PSTI-I was assessed in 10 organs by Western blot analysis using anti-rat PSTI-I (rPSTI) antiserum raised in rabbit. Extracts of brain, colon, heart, kidney, liver, lung, pancreas, small intestine, spleen, and stomach from transgenic and nontransgenic mice were separated by Tricine SDS-PAGE (Invitrogen Inc., Carlsbad, CA), transferred to nitrocellulose, and analyzed by Western blot using anti-rPSTI antiserum and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody (Amersham Biosciences Corp., Piscataway, NJ). Peroxidase-labeled antibodies were detected using an ECL+ (enhanced chemiluminescence) kit (Amersham Biosciences Corp). The anti-rat PSTI antiserum was prepared by hyperimmunization of rabbits with recombinant GST-fused PSTI, followed by purification by affinity chromatography, as previously described.25McVey D.C. Romac J.M. Clay W.C. Kost T.A. Liddle R.A. Vigna S.R. Monitor peptide binding sites are expressed in the rat liver and small intestine.Peptides. 1999; 20: 457-464Crossref PubMed Scopus (11) Google Scholar Recombinant rat PSTI protein was used as a positive control. Following incubation with anti-rPSTI antiserum, membranes were washed and reprobed with a mouse monoclonal anti-α-tubulin antibody (Amersham Biosciences Corp.) as a control for protein loading. Cholecystokinin-stimulated secretion of PSTI-I and amylase from isolated pancreatic acini were measured. Briefly summarized, for each experiment, isolated pancreatic acini were prepared from 2 transgenic mice by enzymatic digestion of pancreas with collagenase in Krebs-Henseleit bicarbonate buffer.26Williams J.A. Korc M. Dormer R.L. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.Am J Physiol. 1978; 235: 517-524PubMed Google Scholar Acini were then incubated in 40 mmol/L Tris Ringer with various dilutions of CCK-8 for 30 minutes at 37°C. PSTI-I was isolated by immunoprecipitation of the secreted proteins using the rabbit anti-rPSTI-I and protein A Sepharose (Amersham Biosciences Corp.) according to the manufacturer's recommendations for buffer and incubation time. The assay was performed in the presence of protease inhibitor cocktail (Sigma-Aldrich Corp., St. Louis, MO) and 4 mmol/L pefabloc (Roche Diagnostics, Indianapolis, IN). The immunoprecipitate was resuspended in Tricine loading buffer (Invitrogen Inc.) and loaded onto a Tricine-PAGE gel. Proteins from the gel were transferred onto a PVDF membrane (Amersham Biosciences Corp.), and PSTI-I was measured by Western blot analysis using the FEMTO kit (Pierce Biotechnology Inc., Rockford, IL) and scanning densitometry (Molecular Dynamics-30S). Amylase release was measured as previously described,26Williams J.A. Korc M. Dormer R.L. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini.Am J Physiol. 1978; 235: 517-524PubMed Google Scholar, 27Liddle R.A. Goldfine I.D. Williams J.A. Bioassay of plasma cholecystokinin in rats effects of food, trypsin inhibitor, and alcohol.Gastroenterology. 1984; 87: 542-549Abstract Full Text PDF PubMed Scopus (414) Google Scholar using procion yellow-coupled starch as substrate.28Jung D.H. Preparation and application of Procion Yellow starch for amylase assay.Clin Chim Acta. 1980; 100: 7-11Crossref PubMed Scopus (140) Google Scholar Pancreas tissue from nontransgenic and transgenic mice was removed following death by isofluorane anesthesia and placed in 3.7% buffered formalin for 12–24 hours prior to embedding in paraffin. Immunohistochemistry was performed in the Immunohistochemistry Facility of the Duke Comprehensive Cancer Center as previously described for formalin-fixed tissue and rabbit polyvalent antiserum with the following antigen retrieval step modification.29Wikstrand C.J. Hale L.P. Batra S.K. Hill M.L. Humphrey P.A. Kurpad S.N. McLendon R.E. Moscatello D. Pegram C.N. Reist C.J. et al.Monoclonal antibodies against EGFRvIII are tumor specific and react with breast and lung carcinomas and malignant gliomas.Cancer Res. 1995; 55: 3140-3148PubMed Google Scholar Following rehydration and endogenous peroxidase blocking, sections were retrieved with pepsin (Digest-All 3; Zymed Laboratories, Inc., San Francisco, CA) for 10 minutes at 37°C, followed by several washes in PBS and blocking in 10% normal goat serum; the remainder of the assay was as previously described. Primary reagents included assay buffer (1% BSA-PBS), anti-PSTI-I polyvalent rabbit antiserum, and purified normal rabbit IgG, each at a concentration of 10 μg/mL. Caerulein was dissolved in 0.1 mol/L NaHCO3, followed by dilution in isotonic saline and was administered as 12 hourly intraperitoneal injections at a supramaximal stimulating dose of 50 μg/kg per injection.30Nathan J.D. Patel A.A. McVey D.C. Thomas J.E. Prpic V. Vigna S.R. Liddle R.A. Capsaicin vanilloid receptor-1 mediates substance P release in experimental pancreatitis.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1322-G1328PubMed Google Scholar Control mice received 12 hourly intraperitoneal injections of isotonic saline. In one group, 1 hour after the last caerulein or vehicle injection, animals were killed, and mixed arteriovenous blood was collected by decapitation for measurement of serum amylase concentration. The pancreas was then quickly removed, rinsed in saline, blotted, and weighed for determination of pancreas wet weight-to-body weight ratio as a measure of pancreatic edema. The pancreas was divided for histologic grading and for determination of TAP content. In a second group of animals, 1 hour after the last caerulein or vehicle injection, mice were killed, and pancreata were harvested for the measurement of trypsin activity. Mixed arteriovenous blood was centrifuged at room temperature for 10 minutes at 1500g. The serum amylase concentration was measured using the procion yellow starch assay previously described.28Jung D.H. Preparation and application of Procion Yellow starch for amylase assay.Clin Chim Acta. 1980; 100: 7-11Crossref PubMed Scopus (140) Google Scholar A standard curve was prepared using crude Type VI-B α-amylase (Sigma-Aldrich Corp.), and serum amylase levels were expressed as mg/mL. Portions of the pancreata were fixed overnight at room temperature in a pH-neutral, phosphate-buffered, 10% formalin solution. The fixed tissue was embedded in paraffin, sectioned at 5 μm, stained with H&E, and coded for examination by a pathologist blinded to the experimental design. The pathologist graded the severity of pancreatitis using modified scoring criteria previously described.31Spormann H. Sokolowski A. Letko G. Effect of temporary ischemia upon development and histological patterns of acute pancreatitis in the rat.Path Res Pract. 1989; 184: 507-513Crossref PubMed Scopus (151) Google Scholar The results were expressed as a score of 0 to 3 for the histologic parameters of edema and neutrophil infiltration and as a score of 0 to 7 for tissue necrosis. Total histologic score was the combined scores of edema, neutrophil infiltration, and necrosis. Freshly collected pancreas tissue was fixed in 4% glutaraldehyde in 0.1 mol/L cacodylate buffer containing 7.5% sucrose overnight, washed 3 times in 1 mol/L cacodylate buffer containing 3.4% sucrose (pH 7.4) for 20 minutes each change, and postfixed in 1% osmium tetroxide in 1 mol/L cacodylate buffer for 1 hour. The tissue was washed once in cacodylate/sucrose, followed by 2 changes of 0.11 mol/L veronyl acetate buffer for 20 minutes each change. It was then fixed for 1 hour in 1% uranyl acetate in veronal acetate buffer. After a brief rinse in distilled water, it was dehydrated in a graded series of ethanol and embedded in Embed 812 resin (Electron Microscopy Sciences, Fort Washington, PA) overnight at 60°C. Ultrathin sections were cut on a Reichert Ultracut S ultramicrotome (Leica, Deerfield, IL), picked up on 200-mesh copper grids (Electron Microscopy Sciences), poststained in lead citrate and uranyl, and viewed in a Philips EM 410 or CM 12 electron microscope (FEI Co., Hillsboro, OR).32Bozzola J.J. Russell L.D. Electron microscopy. Jones and Bartlett Publishers, 1992Google Scholar Pancreas specimens were boiled at 100°C for 15 minutes in 0.2 mol/L tris(hydroxymethyl)aminomethane (Tris)-HCl buffer (pH 7.3) containing 20 mmol/L EDTA to denature proteases and stop further TAP generation. Samples were homogenized on ice for 30 seconds and then centrifuged at 1500g for 10 minutes at 4°C. TAP was quantified in an aliquot of each supernatant using an enzyme immunoassay kit from Biotrin International Ltd. (Dublin, Ireland). Each sample was assayed in duplicate, and a standard curve was generated from free TAP protein. The total protein concentration of each homogenate was determined as previously described,33Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (225757) Google Scholar and pancreatic tissue TAP levels were expressed as ng/mg total protein. Trypsin activity was measured by fluorimetric assay using the substrate Boc-Gln-Ala-Arg-MCA (Peptides International Inc., Louisville, KY) as described.34Kawabata S. Miura T. Morita T. Kato H. Fujikawa K. Iwanaga S. Takada K. Kimura T. Sakakibara S. Highly sensitive peptide-4-methylcoumaryl-7-amide substrates for blood-clotting proteases and trypsin.Eur J Biochem. 1988; 172: 17-25Crossref PubMed Scopus (245) Google Scholar Briefly, pancreas specimens were homogenized in 5 mmol/L MOPS buffer (pH 6.5) on ice and then centrifuged at 1500g for 10 minutes at 4°C. The reaction to quantify trypsin activity was initiated by adding 100 μL of supernatant to a cuvette containing the peptide substrate dissolved in assay buffer (50 mmol/L Tris [pH 8.0], 150 mmol/L NaCl, 1 mmol/L CaCl2, 0.1 mg/mL bovine serum albumin). Fluorescence emitted at 440 nm after excitation at 380 nm was monitored using a Shimadzu RF-5000 spectrophotometer (Norcross, GA). A standard curve was generated using purified trypsin (Pierce Biotechnology Inc.), and data are expressed as the percentage of the maximal value obtained for the experiment. Pancreata from either nontransgenic or PSTI-I transgenic mice were homogenized in MOPS buffer (pH 6.5), followed by centrifugation to remove debris. Trypsin inhibition was measured as previously described,35Kasher R. Oren D.A. Barda Y. Gilon C. Miniaturized proteins the backbone cyclic proteinomimetic approach.J Mol Biol. 1999; 292: 421-429Crossref PubMed Scopus (47) Google Scholar using Nα-benzoyl-L-arginine-p-nitroanilide (L-BAPNA; Sigma-Aldrich Corp.). Briefly, 50 μL of purified TPCK trypsin (Pierce Biotechnology Inc.) solution (5 μmol/L in 1 mmol/L HCl) was diluted in 700 μL buffer solution (0.2 mol/L triethanolamine-HCl, 20 mmol/L CaCl2 [pH 7.8]). Five hundred μL (2 mg) pancreatic extract as determined by the Bradford method31Spormann H. Sokolowski A. Letko G. Effect of temporary ischemia upon development and histological patterns of acute pancreatitis in the rat.Path Res Pract. 1989; 184: 507-513Crossref PubMed Scopus (151) Google Scholar or MOPS buffer was added and incubated for 20 minutes at room temperature. Two hundred μL of BAPNA (4.5 mmol/L) was then added, and the optical density of the reaction was measured by spectrophotometry at 405 nm. The optical density was recorded every minute for 10 minutes, and data were expressed as percentage trypsin inhibition. The percentage of inhibition of the reaction by pancreatic extracts from either nontransgenic or PSTI-I transgenic mice was defined as the ratio of the rate of hydrolysis in the absence of pancreatic extract (ΔT) minus the rate of hydrolysis in the presence of pancreatic extract (ΔN) divided by the rate of hydrolysis in the absence of extract (ΔT) [% inhibition = 100 × (ΔT-ΔN)/ΔT]. Results are expressed as mean ± standard error of mean (SEM). Statistical methods consisted of 1-way analysis of variance with the Tukey posttest for pancreatitis severity parameters, TAP levels, and trypsin activity and the Student t test for the trypsin inhibition assay (GraphPad Prism version 3.03, GraphPad Software Inc., San Diego, CA). Statistical significance was set at P < .05. The rat elastase I enhancer/promoter was used to target expression of rat PSTI-I to the pancreas. Western blot analysis using antiserum against rat PSTI-I was performed with extracts of brain, colon, heart, kidney, liver, lung, pancreas, small intestine, spleen, and stomach from transgenic and nontransgenic mice (Figure 1). For transgenic mice, a band, 6 kilodaltons in size, corresponding to rat PSTI-I was detected only in the pancreas, reflecting pancreas-specific expression. Western blot ana