Original Paper Digestion 1992;52:145-151
National Kyushu Cancer Center. Fukuoka, Japan; Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; First Department of Pathology, Kurume University, Kurume, Japan; Shionogi Research Laboratories, Shionogi, Osaka, Japan
Key Words Ccrulein Acute pancreatitis Pancreatic secretory trypsin inhibitor
Protective Effect of Human Pancreatic Secretory Trypsin Inhibitor on Cerulein-induced Acute Pancreatitis in Rats
Abstract We examined the protective effect of human pancreatic secre tory trypsin inhibitor (PSTI), a specific trypsin inhibitor secreted from pancreatic acinar cells into the pancreatic duct, on cerulein-induced acute pancreatitis in conscious rats. The protective effect of human PST1-RS, an analogue of PSTI with Arg-44 to Ser substitution which has a longer half-life in vitro, was also examined. Intraperitoneal administration of a phar macological dose of cerulein to conscious rats induced acute pancreatitis, characterized by light microscopy as cellular dis organization of the acini and interstitial edema. Intravenous infusion of human PSTI (10, 50 or 250 pg/rat/h) into rats with cerulein-induced acute pancreatitis decreased their pancreatic wet weight and plasma amylase concentration. It also caused a dose-dependent decrease in vacuoles in acinar cells and inter stitial edema. Human PSTI-RS, which has a longer half-life in vivo, was more effective than native PSTI at the same dose rate (10 pg/rat/h) in reducing pancreatitis. These results sug gest that human PSTI may have a beneficial effect on acute pancreatitis.
Pancreatic secretory trypsin inhibitor (PSTI) is primarily an exocrine product of pancreatic acinar cells and is found in normal plasma and urine at very low levels [ 1]. Kazal et al. [2] have assumed that the physiological role of PSTI is to prevent preactivation of
This study was supported in part by grants from the Ministry of Fiducation. Science and Culture, the Yamanouchi Foundation for Research on Metabolic Disorders and the Uchara Memorial Foundation.
Received: December 9,1991 Received in revised form: April 6, 1992
trypsinogen in the pancreatic duct and in the pancreatic tissue. There are recent reports that the plasma PSTI concentration is in creased in patients with acute pancreatitis [3, 4] and after surgery or injury [5, 6], and human PSTI has been suggested to be an
Akihiro Funakoshi, MD Department of Gastroenterology National Kyushu Cancer Center Notamc 3-1-1. Minami-ku Fukuoka 815 (Japan)
©1992 S. Karger AG. Basel 0012-2823/92/ 0524-014552.75/0
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Akihiro Funakoshia Kyoko Miyasakab Atsuo Jim ic Kenichi Kitanib Hiroshi Teraokaá Nobuo Yoshidad
Fig. 1. Primary structures of human PST1 and PSTI-RS (Arg-44 —» Ser). Shadowed amino acids at position 18-2! indicate the trypsin binding site and at position 42-44 the trypsin-sensitive site. Symbols for individual amino acids are ac cording to the letter code of the International Union of Biochemis try.
Materials and Methods .Materials Human PSTI and PSTI-RS were prepared using a recombinant DNA technique [II, 12], Syntheticcerulein was a generous gift from Kyowa Hakko. Tokyo. Japan.
146
All cannulae used in this study were of Silastic medical grade tubing (Dow-Coming, Midland. Mich.. USA; 0.6 mm inner diameter X 0.9 mm outer diame ter). Animal Preparations Male Wistar rats (314-330 g) were obtained from Shizuoka Jikkcn Dobutsu (Shizuoka. Japan). The ani mals were anesthetized with enflurane (Abbott. North Chicago. III., USA) delivered with a vaporizer through a plastic face mask, and a duodenal cannula and extra jugular vein cannula were inserted. After the opera tion, the rats were placed in modified Bollman-type restraint cages and given free access to commercial rat chow (CRF-I. Oriental. Tokyo. Japan) and water, in a room at 24 °C with filtered air and light from 05.00 through 17.00 It. Experiments were conducted 14-16 h after the operation without starvation. Experimental Design Experiment A: Cerulein-Induced Pancreatitis. Acute experimental pancreatitis was induced as re ported by Yamaguchi et al. [10]. Tw'o subsequent intraperitoncal injections of 40 pg/kg body weight of cerulein were given at hourly intervals. In all experi ments. rats were sacrificed under light ether anesthe sia. 6 h after the second cerulein injection. Blood was obtained from the abdominal aorta in a heparinized syringe and was centrifuged at 3.000 rpm for 15 min at 4°C. The pancreas was rapidly removed, freed from fat and lymph nodes, weighed and fixed in 20% for malin for light microscopy. Experiment IJ: Effects o f Intravenous Infusion o f Human PSTI on Cerulein-Induced Pancreatitis. Ani mals were treated either by continuous infusion of 50 or 250 gg/rat/h of human PSTI at a rate of 0.5 ml/h or by bolus injection of 100 gg of human PSTI or PSTI-
Funakoshi/M ivasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Cerulein-Induced Pancreatitis by Human PSTI
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acute phase reactant of inflammation like Creactive protein, because increase in its plasma level is correlated with those of acute phase reactants. Acute interstitial pancreatitis can be in duced by administration of a supramaximal dose of cholecystokinin or its analogue (cerulein) [7, 8], This method of induction of pan creatitis has the advantages that it is techni cally easy, induces highly reproducible histo logical changes and results in very low mortal ity. Preactivation of zymogen proteases in aci nar cells is known to play an important role in the development of acute pancreatitis [9, 10]. In the present study, we examined the effect of intravenous administration of hu man PSTI in preventing induction of acute experimental pancreatitis. We also examined the effect of its analogue PSTI-RS (Arg-44 to Ser substitution; fig. 1), which has a longer half-life in vitro and in vivo, in the protection against pancreatitis [11].
Fig. 2. Pancreatic histology in rats 6 h after a second injection of cerulein. Numerous cytoplasmic vacuoles in acinar cells and inter stitial edema arc observed. Left pa nel: X 40. Right panel: X 200.
Plasma concentrations of human PSTI and amy lase were determined with a human PSTI radioimmu noassay kit (Shionogi Pharmaceuticals. Osaka. Japan) [1] and with blue starch polymer as substrate (Phadcbas amylase test. Sweden) [13], respectively. Cross reactivity of PSTI-RS with human PSTI antiserum was 60% at an equal weight ratio. The antiserum for human PSTI did not react with rat PSTI-61 o r -56. Statistics Values are expressed as means ± SE. Results were analyzed by analysis of variance (Anova) followed by the Newman-Keul multiple comparison test [14]. A p level ofless than 0.05 was considered significant.
Results Effects o f Intraperitoneal Injection o f Cerulein (Experiment A) In sham-operaled rats without cerulein in jection, the plasma amylase concentration was 8,191 ± 111 IU/ml, and the pancreaticwet weight was 0.68 ± 0.13 g (mean ± SE. n = 7). Cerulein injection induced a signifi cant increase in the plasma amylase concen tration ( 19.100 ± 2.800 IU/ml. n = 5) and the pancreatic wet weight (1.10 ± 0.09 g, n = 5). Histologically, vacuolization in acinar cells and interlobular edema were observed (fig. 2). Effect o f Intravenous Infusion o f Human PSTI or PSTI-RS on Acute Experimental Pancreatitis (Experiment B) The changes of pancreatic wet weight pro duced by administration of human PSTI or PSTI-RS were significant when analyzed by Anova (fig. 3a). Intravenous infusions of hu man PSTI at doses of 250 gg/rat/h and PSTI-
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RS immediately after the first cerulein injection and then continuous infusion of human PSTI or PSTI-RS at a rate of 10 pg/rat/h (0.5 ml/h) throughout the exper iment. Experiment C: Plasma PSTI Disappearance Rate. Rats were given 100 pg of human PSTI or PSTI-RS by bolus injection into the right extrajugular vein, and blood samples were taken 0.5, 5. 10, 30. 60 and 180 min later through an indwelling catheter in the left extrajugular vein.
lOwg/h SObs/Ii 250y«/h
Fig. 3. Effects of intravenous infusion of human PSTI on acute experimental pancreatitis induced by cerulein. Control: no treatment, cerulein: administra tion of cerulein only, + PSTI: human PSTI was infused intravenously immediately after the first cerulein in jection. Numbers in parentheses indicate numbers of animals, a Changes in pancreatic wet weight. There was a significant difference among the effect of these treatments by Anova [F (4.25) = 9.23], * p < 0.01 vs. the cerulein group, b Changes in plasma amylase con centration. Significant differences in effects of treat ments were determined by Anova [F (4.25) = 9.38]. * p < 0.01 vs. the three other treatments.
Table 1. Histological findings
Cerulein + Human PSTI ( 10 pg/h) + PSTI-RS (10 pg/h) + Human PSTI (50 pg/h) + Human PSTI (250 pg/h)
Edema
Cytoplasmic Inflammatory vacuoles infiltrate
++ ± ~ +
+++ + ± ± ~ + ~ +
± ± '—
++ + ± +
The histological gradings of edema, vacuolization and inflammation in cerulcin-induced acute pancreatitis are based on the approximate percen tages of cells involved: 0 = absent. ± = < 5 %, + = 5-25 %, -1- + = 25-25 %, + + + = > 50%.
148
Funakoshi/Miyasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Cerulein-lnduced Pancreatitis by Human PSTI
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10b«/h
RS decreased the pancreatic wet weight. These values were significantly different from that for the ccrulein-treated group by Newman-Keuls multiple comparison test. The plasma amylase concentration was decreased by administration of human PSTI or PSTIRS, the decrease being significant by Anova (fig. 3b). Intravenous infusions of human PSTI at doses of 50 and 250 pg/rat/h de creased the plasma amylase concentration sig nificantly as assessed by Newman-Keuls mul tiple comparison test relative to the values for the three other treated groups. Histological examination showed that intravenous infu sion of a higher dose of human PSTI de creased the number of vacuoles in acinar cells and interstitial edema (fig. 4. table 1). Intrave nous infusion of a lower dose of human PSTI also improved histological changes in acute pancreatitis (table 1). At a dose of 10 pg/rat/h. PSTI-RS was more effective than PSTI in reducing pancreatitis (table I). On infusions of 250. 50 and lOpg of human PSTI and lOpg of PSTI-RS, the plasma human PSTI concentrations (mean ± SE) were 599 ± 80.3, 219 ± 25.2. 13 ± 3.7 and 45 ± 3.6 ng/ ml, respectively.
Fig. 4. Pancreatic histology in rats 6 h after the second injection of cerulein with continuous intra venous infusion of 50 pg/h of hu man PST1. Vacuolization of acinar cells and interstitial edema are de creased. Left panel: X 40. Right pa nel: X 200.
Plasma PSTI Disappearance Rate (Experiment C) After bolus injection, the plasma human PSTI concentration decreased rapidly in 10 min and then gradually decreased for 180 min (fig. 5). The mean half-lives of hu man PSTI and PSTI-RS were calculated as 24 and 37 min. respectively, the plasma PSTI and PSTI-RS concentrations being 4 ± 2.3 (n = 3) and 17 ± 5.6 ng/ml (n = 3), respective ly, after 180 min. Time, min
Discussion Fig. 5. Plasma PSTI disappearance rates of human PSTI and PSTI-RS. Results are expressed as mean val ues for the experimental groups.
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The present study indicated that intrave nous infusion of human PSTI had a protec tive effect against cerulein-induced acute pan creatitis. PSTI is secreted from acinar cells into the pancreatic duct and is known to pre vent preactivation of trypsin in the pancreatic duct and the pancreas [2], Activation of zy mogen proteases in pancreatic acinar cells is thought to play an important role in the devel-
150
Therefore, the present results indicate that PSTI released endogenously into the blood stream of patients with acute pancreatitis may by some unknown mechanism prevent dam age of tissue due to inflammation in acute pancreatitis. In a previous report [ 11 ], we suggested that PSTI-RS (Arg-44 to Ser), prepared by recom binant DNA technology, inhibited human trypsin to the same extent as natural PSTI. indicating that replacement of Arg-44 in hu man PSTI does not affect its binding to hu man trypsin (fig. 1). In an in vitro study, the rate of inactivation of PSTI-RS was less than that of natural PSTI [11], suggesting that cleavage of the Arg-44-Gln-45 bond causes inactivation of human PSTI (fig. 1). The present study showed that PSTI-RS had a 1.5 times longer half-life than native human PSTI in vivo and was more effective than the latter on pancreatitis at the same dose ratio, indicat ing its clinical usefulness in the treatment of pancreatitis. The present results suggest that human PSTI may have a beneficial effect on acute pancreatitis.
Funakoshi/Mivasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Ccrulein-Induccd Pancreatitis by Human PSTI
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opment of acute pancreatitis, and intracellu lar activation of trypsinogen has been demon strated in acute pancreatitis induced by cerulein [ 10], It has been shown that application of protease inhibitors has beneficial effects on cerulein-induced pancreatitis [15-17], The protective effect of human PSTI offers strong support for the theory that trypsin plays a key role in the development of pancreatitis be cause PSTI could bind trypsin and inhibit its activity, but not other proteases. Ohlsson ct al. [ 18] also demonstrated a protective effect of human PSTI in acute hemorrhagic pancre atitis induced by intraductal injection of so dium taurocholate in rats. However, they used a large dose of PSTI given intraductally. The detection of active forms of pancreatic enzyme in the circulation of patients with severe acute pancreatitis [19] suggests that trypsin may play a role in the human disease, similar to that proposed above in the rat. We showed that intravenously administered hu man PSTI may prevent the induction of acute pancreatitis by inhibiting trypsin activation. The plasma level of human PSTI during its infusion at 250 pg/rat/h was about 600 ng/ml. Rats have two kinds of PSTI. PSTI-61 and -56 [20], Their plasma levels of endogenous PSTIs must be elevated during induction of acute experimental pancreatitis. Therefore, the total plasma level of PSTI in the rats must be the sum of the plasma levels of human PSTI and rat PSTIs, endogenously released into the blood stream by the induction of acute pancreatitis, although we did not mea sure the plasma levels of rat PSTIs. The plasma level of human PSTI increases to more than several hundred nanograms per milliliter in some patients with acute pancre atitis [4], The doses of human PSTI used in this study may be sufficient for the protection against pancreatitis. Recently, PSTI was re ported to be a growth factor like EGF in addi tion to being a trypsin inhibitor [21-23].
References 8 Watanabc O. Baccino FM. Steer ML: et al: Supramaximal caerulcin stimulation and ultrastructurc of rat pancreatic acinar cell: Early mor phological changes during develop ment of experimental pancreatitis. Am J Physiol 1984;246:G457-467. 9 Steer ML: Etiology and pathophysi ology of acute pancreatitis; in Go VLW. Brooks RP. DiMagno EP. et al (eds): The Exocrine Pancreas: Bi ology. Pathobiology. and Diseases. New York. Raven Press. 1986. pp 55-67. 10 Yamaguchi H. Kimura T. Mimura K, el al: Activation of proteases in cerulein-induced pancreatitis. Pan creas 1989:4:565-571. 11 Kikuchi N. Nagata K. Shin M. et al: Site-directed mutagenesis of human pancreatic secretory trypsin inhibi tor. J Biocheni 1989; 106:1059106.3. 12 Kikuchi N. Nagata K. Horii T. et al: Production of recombinant human pancreatic secretory trypsin inhibi tor by Escherichia coli. J Biochcm 1987;102:607-612. 13 Ceska M. Birath K. Brown B: A new and rapid method for the clinical determination of a-amylase activi ties in human serum and urine. Op timal conditions. Clin Chim Acta 1969;26:437-444. 14 Snedecor GW, Cochran WG: Analy sis of variance: in Snedecor GW. Cochran WG (eds): Statistical Meth ods. ed 7. Ames. Iowa State Univer sity Press, 1980. pp 2 15-237. 15 Wisner JR. Renner IG, Grendell JH. el al: Gabcxale mesilate (FOY) protects against ceruletide-induced acute pancreatitis in the rat. Pan creas 1987:2:181-186. 16 Lankisch PG. Pohl U. Goke B. et al: Effect of FOY-305 (camostat) on se vere acute pancreatitis in two exper imental animal models. Gastroen terology 1989:96:193-199.
17 Ohshio G. Saluja AK. Lilli U. et al: Esterase inhibitors prevent lyso somal enzyme redistribution in two noninvasive models of experimental pancreatitis. Gastroenterology 1989: 96:853-859. 18 Ohlsson K. Olsson R. Bjork P. et al: Local administration of human pan creatic secretory trypsin inhibitor prevents the development of experi mental acute pancreatitis in rats and dogs. Scand J Gastroenterol 1989; 24:693-705. 19 Funakoshi A, Yamada Y. Ito T. et al: Clinical usefulness of serum phospholipase A; determination in patients with pancreatic diseases. Pancreas 1991:6:588-594. 20 Uda K. Ogawa M. Shibata T. ct al: Purification, characterization and amino-acid sequencing of two pan creatic secretory trypsin inhibitors in rat pancreatic juice. Biol Chem Hoppe Sevier 1988:369:55—6 1. 21 Fukuoka S. Fushiki T. Kitagawa Y. et al: Growth stimulating activity on 3T3 fibroblasts of the molecular weight 6.500-peptide purified from rat pancreatic juice. Biochem Biophys Res Commun 1986:139:545— 550. 22 McKeehan WL, Sakagami Y, Uoshi IT. ct al: Two apparent human endo thelial cell growth factors from hu man hepatoma cells are tumor-asso ciated proteinase inhibitors. .1 Biol Chem 1986:261:5378-5383. 23 Ogawa M. Tsushima T. Ohba Y. el al: Stimulation of DNA synthesis in human fibroblasts by human pan creatic secretory trypsin inhibitor. Res Commun Chem Pathol Phar macol 1985:50:155-158.
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1 Nakano I. Funakoshi A. Kimura T. el al: Elevated levels of serum pan creatic secretory trypsin inhibitor (PSTI) in patients with malabsorp tion syndrome. Gastroenterol Jpn 1986:21:617-622. 2 Kazal LA. Spicer DS. Brahinski RA: Isolation of a crystalline trypsin in hibitor-anticoagulant protein from pancreas. J Am Chem Soc 1948:70: 3034-3040. 3 Kitahara T, Takatuka Y. Fujimoto K. et al: Radioimmunoassay for hu man pancreatic secretory trypsin in hibitor: Measurement of serum pan creatic secretory trypsin inhibitor in normal subjects and subjects with pancreatic diseases. Clin Chim Acta 1980:103:135-143. 4 Ogawa M. Matsuda K. Matsuda Y. et al: Evaluation of immunological assays of scrum pancreatic enzymes and pancreatic secretory trypsin in hibitor for diagnosis and manage ment of acute pancreatitis: in Sato T. Yamauchi H (eds): Pancreatitis, Its Pathophysiology and Clinical As pects. Tokyo, University of Tokyo Press, 1985. pp 107-115. 5 Matsuda K. Ogawa M. Shibata T. et al: Postoperative elevation of scrum pancreatic secretory trypsin inhibi tor. Am J Gastroenterol 1985:80: 694-698. 6 Ogawa M. Matsuda K, Shibata T, et al: Elevation of serum pancreatic se cretory trypsin inhibitor (PSTI) in patients with serious injury. Res Commun Chem Pathol Pharmacol 1985;50:259-266. 7 Lampcl M. Dem HE: Acute intersti tial pancreatitis in the rat induced by excessive doses of pancreatic secretagoguc. Virchows Arch [A] 1977:373:97-117.
National Kyushu Cancer Center. Fukuoka, Japan; Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan; First Department of Pathology, Kurume University, Kurume, Japan; Shionogi Research Laboratories, Shionogi, Osaka, Japan
Key Words Ccrulein Acute pancreatitis Pancreatic secretory trypsin inhibitor
Protective Effect of Human Pancreatic Secretory Trypsin Inhibitor on Cerulein-induced Acute Pancreatitis in Rats
Abstract We examined the protective effect of human pancreatic secre tory trypsin inhibitor (PSTI), a specific trypsin inhibitor secreted from pancreatic acinar cells into the pancreatic duct, on cerulein-induced acute pancreatitis in conscious rats. The protective effect of human PST1-RS, an analogue of PSTI with Arg-44 to Ser substitution which has a longer half-life in vitro, was also examined. Intraperitoneal administration of a phar macological dose of cerulein to conscious rats induced acute pancreatitis, characterized by light microscopy as cellular dis organization of the acini and interstitial edema. Intravenous infusion of human PSTI (10, 50 or 250 pg/rat/h) into rats with cerulein-induced acute pancreatitis decreased their pancreatic wet weight and plasma amylase concentration. It also caused a dose-dependent decrease in vacuoles in acinar cells and inter stitial edema. Human PSTI-RS, which has a longer half-life in vivo, was more effective than native PSTI at the same dose rate (10 pg/rat/h) in reducing pancreatitis. These results sug gest that human PSTI may have a beneficial effect on acute pancreatitis.
Pancreatic secretory trypsin inhibitor (PSTI) is primarily an exocrine product of pancreatic acinar cells and is found in normal plasma and urine at very low levels [ 1]. Kazal et al. [2] have assumed that the physiological role of PSTI is to prevent preactivation of
This study was supported in part by grants from the Ministry of Fiducation. Science and Culture, the Yamanouchi Foundation for Research on Metabolic Disorders and the Uchara Memorial Foundation.
Received: December 9,1991 Received in revised form: April 6, 1992
trypsinogen in the pancreatic duct and in the pancreatic tissue. There are recent reports that the plasma PSTI concentration is in creased in patients with acute pancreatitis [3, 4] and after surgery or injury [5, 6], and human PSTI has been suggested to be an
Akihiro Funakoshi, MD Department of Gastroenterology National Kyushu Cancer Center Notamc 3-1-1. Minami-ku Fukuoka 815 (Japan)
©1992 S. Karger AG. Basel 0012-2823/92/ 0524-014552.75/0
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/24/2019 5:14:37 PM
Akihiro Funakoshia Kyoko Miyasakab Atsuo Jim ic Kenichi Kitanib Hiroshi Teraokaá Nobuo Yoshidad
Fig. 1. Primary structures of human PST1 and PSTI-RS (Arg-44 —» Ser). Shadowed amino acids at position 18-2! indicate the trypsin binding site and at position 42-44 the trypsin-sensitive site. Symbols for individual amino acids are ac cording to the letter code of the International Union of Biochemis try.
Materials and Methods .Materials Human PSTI and PSTI-RS were prepared using a recombinant DNA technique [II, 12], Syntheticcerulein was a generous gift from Kyowa Hakko. Tokyo. Japan.
146
All cannulae used in this study were of Silastic medical grade tubing (Dow-Coming, Midland. Mich.. USA; 0.6 mm inner diameter X 0.9 mm outer diame ter). Animal Preparations Male Wistar rats (314-330 g) were obtained from Shizuoka Jikkcn Dobutsu (Shizuoka. Japan). The ani mals were anesthetized with enflurane (Abbott. North Chicago. III., USA) delivered with a vaporizer through a plastic face mask, and a duodenal cannula and extra jugular vein cannula were inserted. After the opera tion, the rats were placed in modified Bollman-type restraint cages and given free access to commercial rat chow (CRF-I. Oriental. Tokyo. Japan) and water, in a room at 24 °C with filtered air and light from 05.00 through 17.00 It. Experiments were conducted 14-16 h after the operation without starvation. Experimental Design Experiment A: Cerulein-Induced Pancreatitis. Acute experimental pancreatitis was induced as re ported by Yamaguchi et al. [10]. Tw'o subsequent intraperitoncal injections of 40 pg/kg body weight of cerulein were given at hourly intervals. In all experi ments. rats were sacrificed under light ether anesthe sia. 6 h after the second cerulein injection. Blood was obtained from the abdominal aorta in a heparinized syringe and was centrifuged at 3.000 rpm for 15 min at 4°C. The pancreas was rapidly removed, freed from fat and lymph nodes, weighed and fixed in 20% for malin for light microscopy. Experiment IJ: Effects o f Intravenous Infusion o f Human PSTI on Cerulein-Induced Pancreatitis. Ani mals were treated either by continuous infusion of 50 or 250 gg/rat/h of human PSTI at a rate of 0.5 ml/h or by bolus injection of 100 gg of human PSTI or PSTI-
Funakoshi/M ivasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Cerulein-Induced Pancreatitis by Human PSTI
Downloaded by: Karolinska Institutet, University Library 130.237.122.245 - 1/24/2019 5:14:37 PM
acute phase reactant of inflammation like Creactive protein, because increase in its plasma level is correlated with those of acute phase reactants. Acute interstitial pancreatitis can be in duced by administration of a supramaximal dose of cholecystokinin or its analogue (cerulein) [7, 8], This method of induction of pan creatitis has the advantages that it is techni cally easy, induces highly reproducible histo logical changes and results in very low mortal ity. Preactivation of zymogen proteases in aci nar cells is known to play an important role in the development of acute pancreatitis [9, 10]. In the present study, we examined the effect of intravenous administration of hu man PSTI in preventing induction of acute experimental pancreatitis. We also examined the effect of its analogue PSTI-RS (Arg-44 to Ser substitution; fig. 1), which has a longer half-life in vitro and in vivo, in the protection against pancreatitis [11].
Fig. 2. Pancreatic histology in rats 6 h after a second injection of cerulein. Numerous cytoplasmic vacuoles in acinar cells and inter stitial edema arc observed. Left pa nel: X 40. Right panel: X 200.
Plasma concentrations of human PSTI and amy lase were determined with a human PSTI radioimmu noassay kit (Shionogi Pharmaceuticals. Osaka. Japan) [1] and with blue starch polymer as substrate (Phadcbas amylase test. Sweden) [13], respectively. Cross reactivity of PSTI-RS with human PSTI antiserum was 60% at an equal weight ratio. The antiserum for human PSTI did not react with rat PSTI-61 o r -56. Statistics Values are expressed as means ± SE. Results were analyzed by analysis of variance (Anova) followed by the Newman-Keul multiple comparison test [14]. A p level ofless than 0.05 was considered significant.
Results Effects o f Intraperitoneal Injection o f Cerulein (Experiment A) In sham-operaled rats without cerulein in jection, the plasma amylase concentration was 8,191 ± 111 IU/ml, and the pancreaticwet weight was 0.68 ± 0.13 g (mean ± SE. n = 7). Cerulein injection induced a signifi cant increase in the plasma amylase concen tration ( 19.100 ± 2.800 IU/ml. n = 5) and the pancreatic wet weight (1.10 ± 0.09 g, n = 5). Histologically, vacuolization in acinar cells and interlobular edema were observed (fig. 2). Effect o f Intravenous Infusion o f Human PSTI or PSTI-RS on Acute Experimental Pancreatitis (Experiment B) The changes of pancreatic wet weight pro duced by administration of human PSTI or PSTI-RS were significant when analyzed by Anova (fig. 3a). Intravenous infusions of hu man PSTI at doses of 250 gg/rat/h and PSTI-
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RS immediately after the first cerulein injection and then continuous infusion of human PSTI or PSTI-RS at a rate of 10 pg/rat/h (0.5 ml/h) throughout the exper iment. Experiment C: Plasma PSTI Disappearance Rate. Rats were given 100 pg of human PSTI or PSTI-RS by bolus injection into the right extrajugular vein, and blood samples were taken 0.5, 5. 10, 30. 60 and 180 min later through an indwelling catheter in the left extrajugular vein.
lOwg/h SObs/Ii 250y«/h
Fig. 3. Effects of intravenous infusion of human PSTI on acute experimental pancreatitis induced by cerulein. Control: no treatment, cerulein: administra tion of cerulein only, + PSTI: human PSTI was infused intravenously immediately after the first cerulein in jection. Numbers in parentheses indicate numbers of animals, a Changes in pancreatic wet weight. There was a significant difference among the effect of these treatments by Anova [F (4.25) = 9.23], * p < 0.01 vs. the cerulein group, b Changes in plasma amylase con centration. Significant differences in effects of treat ments were determined by Anova [F (4.25) = 9.38]. * p < 0.01 vs. the three other treatments.
Table 1. Histological findings
Cerulein + Human PSTI ( 10 pg/h) + PSTI-RS (10 pg/h) + Human PSTI (50 pg/h) + Human PSTI (250 pg/h)
Edema
Cytoplasmic Inflammatory vacuoles infiltrate
++ ± ~ +
+++ + ± ± ~ + ~ +
± ± '—
++ + ± +
The histological gradings of edema, vacuolization and inflammation in cerulcin-induced acute pancreatitis are based on the approximate percen tages of cells involved: 0 = absent. ± = < 5 %, + = 5-25 %, -1- + = 25-25 %, + + + = > 50%.
148
Funakoshi/Miyasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Cerulein-lnduced Pancreatitis by Human PSTI
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10b«/h
RS decreased the pancreatic wet weight. These values were significantly different from that for the ccrulein-treated group by Newman-Keuls multiple comparison test. The plasma amylase concentration was decreased by administration of human PSTI or PSTIRS, the decrease being significant by Anova (fig. 3b). Intravenous infusions of human PSTI at doses of 50 and 250 pg/rat/h de creased the plasma amylase concentration sig nificantly as assessed by Newman-Keuls mul tiple comparison test relative to the values for the three other treated groups. Histological examination showed that intravenous infu sion of a higher dose of human PSTI de creased the number of vacuoles in acinar cells and interstitial edema (fig. 4. table 1). Intrave nous infusion of a lower dose of human PSTI also improved histological changes in acute pancreatitis (table 1). At a dose of 10 pg/rat/h. PSTI-RS was more effective than PSTI in reducing pancreatitis (table I). On infusions of 250. 50 and lOpg of human PSTI and lOpg of PSTI-RS, the plasma human PSTI concentrations (mean ± SE) were 599 ± 80.3, 219 ± 25.2. 13 ± 3.7 and 45 ± 3.6 ng/ ml, respectively.
Fig. 4. Pancreatic histology in rats 6 h after the second injection of cerulein with continuous intra venous infusion of 50 pg/h of hu man PST1. Vacuolization of acinar cells and interstitial edema are de creased. Left panel: X 40. Right pa nel: X 200.
Plasma PSTI Disappearance Rate (Experiment C) After bolus injection, the plasma human PSTI concentration decreased rapidly in 10 min and then gradually decreased for 180 min (fig. 5). The mean half-lives of hu man PSTI and PSTI-RS were calculated as 24 and 37 min. respectively, the plasma PSTI and PSTI-RS concentrations being 4 ± 2.3 (n = 3) and 17 ± 5.6 ng/ml (n = 3), respective ly, after 180 min. Time, min
Discussion Fig. 5. Plasma PSTI disappearance rates of human PSTI and PSTI-RS. Results are expressed as mean val ues for the experimental groups.
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The present study indicated that intrave nous infusion of human PSTI had a protec tive effect against cerulein-induced acute pan creatitis. PSTI is secreted from acinar cells into the pancreatic duct and is known to pre vent preactivation of trypsin in the pancreatic duct and the pancreas [2], Activation of zy mogen proteases in pancreatic acinar cells is thought to play an important role in the devel-
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Therefore, the present results indicate that PSTI released endogenously into the blood stream of patients with acute pancreatitis may by some unknown mechanism prevent dam age of tissue due to inflammation in acute pancreatitis. In a previous report [ 11 ], we suggested that PSTI-RS (Arg-44 to Ser), prepared by recom binant DNA technology, inhibited human trypsin to the same extent as natural PSTI. indicating that replacement of Arg-44 in hu man PSTI does not affect its binding to hu man trypsin (fig. 1). In an in vitro study, the rate of inactivation of PSTI-RS was less than that of natural PSTI [11], suggesting that cleavage of the Arg-44-Gln-45 bond causes inactivation of human PSTI (fig. 1). The present study showed that PSTI-RS had a 1.5 times longer half-life than native human PSTI in vivo and was more effective than the latter on pancreatitis at the same dose ratio, indicat ing its clinical usefulness in the treatment of pancreatitis. The present results suggest that human PSTI may have a beneficial effect on acute pancreatitis.
Funakoshi/Mivasaka/Jimi/Kitani/ Teraoka/Yoshida
Inhibition of Ccrulein-Induccd Pancreatitis by Human PSTI
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opment of acute pancreatitis, and intracellu lar activation of trypsinogen has been demon strated in acute pancreatitis induced by cerulein [ 10], It has been shown that application of protease inhibitors has beneficial effects on cerulein-induced pancreatitis [15-17], The protective effect of human PSTI offers strong support for the theory that trypsin plays a key role in the development of pancreatitis be cause PSTI could bind trypsin and inhibit its activity, but not other proteases. Ohlsson ct al. [ 18] also demonstrated a protective effect of human PSTI in acute hemorrhagic pancre atitis induced by intraductal injection of so dium taurocholate in rats. However, they used a large dose of PSTI given intraductally. The detection of active forms of pancreatic enzyme in the circulation of patients with severe acute pancreatitis [19] suggests that trypsin may play a role in the human disease, similar to that proposed above in the rat. We showed that intravenously administered hu man PSTI may prevent the induction of acute pancreatitis by inhibiting trypsin activation. The plasma level of human PSTI during its infusion at 250 pg/rat/h was about 600 ng/ml. Rats have two kinds of PSTI. PSTI-61 and -56 [20], Their plasma levels of endogenous PSTIs must be elevated during induction of acute experimental pancreatitis. Therefore, the total plasma level of PSTI in the rats must be the sum of the plasma levels of human PSTI and rat PSTIs, endogenously released into the blood stream by the induction of acute pancreatitis, although we did not mea sure the plasma levels of rat PSTIs. The plasma level of human PSTI increases to more than several hundred nanograms per milliliter in some patients with acute pancre atitis [4], The doses of human PSTI used in this study may be sufficient for the protection against pancreatitis. Recently, PSTI was re ported to be a growth factor like EGF in addi tion to being a trypsin inhibitor [21-23].
References 8 Watanabc O. Baccino FM. Steer ML: et al: Supramaximal caerulcin stimulation and ultrastructurc of rat pancreatic acinar cell: Early mor phological changes during develop ment of experimental pancreatitis. Am J Physiol 1984;246:G457-467. 9 Steer ML: Etiology and pathophysi ology of acute pancreatitis; in Go VLW. Brooks RP. DiMagno EP. et al (eds): The Exocrine Pancreas: Bi ology. Pathobiology. and Diseases. New York. Raven Press. 1986. pp 55-67. 10 Yamaguchi H. Kimura T. Mimura K, el al: Activation of proteases in cerulein-induced pancreatitis. Pan creas 1989:4:565-571. 11 Kikuchi N. Nagata K. Shin M. et al: Site-directed mutagenesis of human pancreatic secretory trypsin inhibi tor. J Biocheni 1989; 106:1059106.3. 12 Kikuchi N. Nagata K. Horii T. et al: Production of recombinant human pancreatic secretory trypsin inhibi tor by Escherichia coli. J Biochcm 1987;102:607-612. 13 Ceska M. Birath K. Brown B: A new and rapid method for the clinical determination of a-amylase activi ties in human serum and urine. Op timal conditions. Clin Chim Acta 1969;26:437-444. 14 Snedecor GW, Cochran WG: Analy sis of variance: in Snedecor GW. Cochran WG (eds): Statistical Meth ods. ed 7. Ames. Iowa State Univer sity Press, 1980. pp 2 15-237. 15 Wisner JR. Renner IG, Grendell JH. el al: Gabcxale mesilate (FOY) protects against ceruletide-induced acute pancreatitis in the rat. Pan creas 1987:2:181-186. 16 Lankisch PG. Pohl U. Goke B. et al: Effect of FOY-305 (camostat) on se vere acute pancreatitis in two exper imental animal models. Gastroen terology 1989:96:193-199.
17 Ohshio G. Saluja AK. Lilli U. et al: Esterase inhibitors prevent lyso somal enzyme redistribution in two noninvasive models of experimental pancreatitis. Gastroenterology 1989: 96:853-859. 18 Ohlsson K. Olsson R. Bjork P. et al: Local administration of human pan creatic secretory trypsin inhibitor prevents the development of experi mental acute pancreatitis in rats and dogs. Scand J Gastroenterol 1989; 24:693-705. 19 Funakoshi A, Yamada Y. Ito T. et al: Clinical usefulness of serum phospholipase A; determination in patients with pancreatic diseases. Pancreas 1991:6:588-594. 20 Uda K. Ogawa M. Shibata T. ct al: Purification, characterization and amino-acid sequencing of two pan creatic secretory trypsin inhibitors in rat pancreatic juice. Biol Chem Hoppe Sevier 1988:369:55—6 1. 21 Fukuoka S. Fushiki T. Kitagawa Y. et al: Growth stimulating activity on 3T3 fibroblasts of the molecular weight 6.500-peptide purified from rat pancreatic juice. Biochem Biophys Res Commun 1986:139:545— 550. 22 McKeehan WL, Sakagami Y, Uoshi IT. ct al: Two apparent human endo thelial cell growth factors from hu man hepatoma cells are tumor-asso ciated proteinase inhibitors. .1 Biol Chem 1986:261:5378-5383. 23 Ogawa M. Tsushima T. Ohba Y. el al: Stimulation of DNA synthesis in human fibroblasts by human pan creatic secretory trypsin inhibitor. Res Commun Chem Pathol Phar macol 1985:50:155-158.
151
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1 Nakano I. Funakoshi A. Kimura T. el al: Elevated levels of serum pan creatic secretory trypsin inhibitor (PSTI) in patients with malabsorp tion syndrome. Gastroenterol Jpn 1986:21:617-622. 2 Kazal LA. Spicer DS. Brahinski RA: Isolation of a crystalline trypsin in hibitor-anticoagulant protein from pancreas. J Am Chem Soc 1948:70: 3034-3040. 3 Kitahara T, Takatuka Y. Fujimoto K. et al: Radioimmunoassay for hu man pancreatic secretory trypsin in hibitor: Measurement of serum pan creatic secretory trypsin inhibitor in normal subjects and subjects with pancreatic diseases. Clin Chim Acta 1980:103:135-143. 4 Ogawa M. Matsuda K. Matsuda Y. et al: Evaluation of immunological assays of scrum pancreatic enzymes and pancreatic secretory trypsin in hibitor for diagnosis and manage ment of acute pancreatitis: in Sato T. Yamauchi H (eds): Pancreatitis, Its Pathophysiology and Clinical As pects. Tokyo, University of Tokyo Press, 1985. pp 107-115. 5 Matsuda K. Ogawa M. Shibata T. et al: Postoperative elevation of scrum pancreatic secretory trypsin inhibi tor. Am J Gastroenterol 1985:80: 694-698. 6 Ogawa M. Matsuda K, Shibata T, et al: Elevation of serum pancreatic se cretory trypsin inhibitor (PSTI) in patients with serious injury. Res Commun Chem Pathol Pharmacol 1985;50:259-266. 7 Lampcl M. Dem HE: Acute intersti tial pancreatitis in the rat induced by excessive doses of pancreatic secretagoguc. Virchows Arch [A] 1977:373:97-117.
