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Induction of a 60-kDa heat shock protein in rat pancreas by water-immersion stress
0020-71IX/93 $6.00+ 0.00 Copyright 0 1993Pergamon Press Ltd
Inr. J. Biochem. Vol. 25, No. 12, pp. 176991773,1993 Printed in Great Britain. All rights reserved
INDUCTION OF A 60-kDa HEAT SHOCK PROTEIN IN RAT PANCREAS BY WATER-IMMERSION STRESS MICHIRO OTAKA,” HIDEAKI ITOH,~TOSHIYUKIKUWABARA,’ AKIRA ZENIYA,’ SHUSEIFUJIMORI,’ YOHTALOUTASHIMA*and O~AMU MASAMUNE’ ‘First Department of Internal Medicine and 2Department of Biochemistry, Akita University School of Medicine, l-l-l Hondo, Akita 010, Japan [Tel. 81 188 34 1111; Fax 81 188 34 86191 (Received
Abstract-l.
7 June 1993)
In this study, expression of a 60-kDa heat shock protein in rat pancreas
before and after water-immersion
stress, which has been known
as an exacerbation
was investigated factor of caerulein-in-
duced pancreatitis in rats, by Western blot. 2. A 60-kDa heat shock protein increased after water-immersion
stress in both soluble and insoluble fractions of the pancreas. 3. Serum amylase level and pancreas weight did not increase after water-immersion. 4. No pathologic alteration was observed in the pancreas after water-immersion.
characterization and sequencing of 92 amino acid residues of seven peptide fragments digested by lysil endopeptidase.
INTRODUCTION Many studies have shown the importance of heat shock proteins (HSPs) for survival of cells under stress conditions (Itoh and Tashima, 1991). Recently it has been demonstrated that a 70-kDa heat shock protein (HSP70) is induced in pancreatic islet cells during prolonged exposure to interleukin l/I (Margulis et al., 1991). On the other hand, little is known about the expression and functions of a 60-kDa heat shock protein (HSP60) in the pancreas under stress conditions, although the HSP60 family is thought to play an important role in the pathogenesis of autoimmune diabetes in rats (Elias et al., 1990, 1991). In order to study the effects of systemic stress to the pancreas, we investigated the expression of HSP60 in rat pancreas before and after water-immersion stress, which is known as an exacerbation factor of caerulein-induced pancreatitis in rats (Yamaguchi et al.. 1990) MATERIALS AND Purification
METHODS
of HSP60
HSP60 was purified from porcine liver according to our method (Itoh et al., 1993). Briefly, frozen porcine livers were homogenized with 3 vol of buffer (10 mM Tris-Cl, IS mM /I-mercaptoethanol, 0.1 mM phenylmethyl sulfonyl fluoride, pH 7.4). After centrifugation at 18,OOOg for 10 min, the supematant was fractionated by ammonium sulfate (3&60%). After dialysis against the buffer, the protein solutions were separated using DEAE-cellulose and ATPSepharose column chromatography. The 60-kDa protein was eluted in high affinity fraction from the ATP-Sepharose column. The purified 60-kDa protein was identified as HSP60 by several methods, including physicochemical
‘To whom
correspondence
should
be addressed.
Production
of antibody
Antibody to HSP60 was produced by intramuscular injection into rabbit of 1 mg of the protein, emulsified in complete Freund’s adjuvant. Booster shots were given three times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection. Animals Fifteen to twenty-week old male Sprague-Dawley (weight: 250-300 g) were used in this experiment. Water-immersion
rats
stress
Rats were placed in a restraint cage, according to the method previously described (Takagi et al., 1963). The animals were then immersed vertically to the level of the xiphoid process in a water bath (22223°C). Rats were sacrificed before and 3, 6 and 12 hr (n = 4 in each time) following the initiation of water-immersion stress, Before killing, peripheral blood was collected from tail vein in order to assay serum amylase concentration. After killing, the pancreas was quickly removed and weighed to evaluate the degree of pancreatic edema (Yamaguchi et al., 1990). The pancreas wet wt was expressed as pancreas wet wt/body wt (mg/g). For light microscopy the pancreas was fixed in 20% formalin and embedded in paraffin wax. The tissue section was stained with hematoxylin and eosin. The remaining part of the pancreas was used immediately or frozen at -80°C until use. SDS-Polyacrylamide
gel electrophoresis
The pancreas was homogenized with 5 vol of ice-cold 25mM Tris-Cl buffer @H 7.5). The homogenates were centrifuged at 18,OOOg for 20min. The supematants (soluble fraction) were collected and the protein concentrations were adjusted to 0.2 mg/ml. The protein concentration was measured by the method of Lowry et al. (1951). The pellets were rehomogenized with the same buffer containing 1.O% 1769
1770
MICHIRO OTAKA el al. Assay of serum amylase Serum amylase was measured by the method of Ceska et al. (1969) using Phadebas amylase test (Pharmacia Laboratories, Piscataway, NJ).
RESULTS AND DISCUSSION
0
3
6
l2 (h)
Fig. 1. Alterations of pancreas weight [pancreas wet wt (mg)/body wt (g)] after water-immersion. Values shown are means & SE for four rats in each time.
Triton X-100. The homogenates were centrifuged at 18,OOOg for 20min, and the supernatants (insoluble fraction) were collected and the protein concentration of each sample was adjusted to 0.1 mg/ml. Samples were analyzed by 10% polyacrylamide gel electrophoresis according to the method of Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue (R-250) in a mixture of 25% isopropyl alcohol-10% acetic acid and destained with 10% isopropyl alcohol-IO% acetic acid. Western blot Samples were electrophoresed on SDS-polyacrylamide gels, transferred electrophoretically to PVDF (polyvinylidene difluoride) membrane (Nihon Millipore, Kogyo, Japan), and processed as described by Towbin et al. (1979). The membrane was incubated with anti-HSP60 antibody (1: 1000 dilution) and treated with horseradish peroxidase conjugated anti-rabbit IgG (1: IS00 dilution) (Bio-Rad, Richmond, CA). The peroxidase substrate was 3,3-diaminobenzidine tetrahydrochloride. The density of the immunologically stained bands was analyzed using scanning densitometer.
The pancreas wet wt, which is a marker of pancreatic edema, did not increase after water-immersion (Fig. 1). In addition, serum amylase level did not increase after immersion (Fig. 2), and no pathologic alteration was observed in the pancreas (Fig. 3). The specificity of the anti-HSP60 antibody was shown in Fig. 4. Only the 60-kDa band was stained (panel B). This protein was identified as HSP60 by partial amino acid sequence. Expression of HSP60 increased in both soluble and insoluble fractions 3 hr following the initiation of water-immersion stress, and peaked 6 to 12 hr after immersion (Fig. 5). The relative densities of stained bands in Western blot were 133.7 k 4.6%, 122.7 k 9.6% (soluble fraction, insoluble fraction; 142.2 k 10.7% (6 hr) and 173.2 & 7.9%, 3 hr), with 164.0 f 3.6%, 141.6 &-8.8 (12 hr) compared control (0 hr: 100%) by densitometrical scanning. Water-immersion stress is known as an exacerbation factor of caerulein-induced acute pancreatitis in rats, although water-immersion stress itself can not be an initiator of pancreatitis (Yamaguchi ef al., 1990). In our study, no pathologic change was observed in the pancreas, and serum amylase level and pancreas weight did not increase even 12 hr after immersion. However, HSP60 has already increased 3 hr following the initiation of water-immersion. Although the real mechanism of the induction of HSP60 is not clear, it is known that water-immersion stress decrease pancreatic blood flow (Takano et al., 1991). Our results may indicate that mild or early stages of pancreatic cell damage, without pathologic alteration, by decrease of pancreatic blood flow contribute to the induction of HSP60. Furthermore, HSP60 may play an important role for cytoprotection as a stress response in the pancreatic cells. Further precise studies are required to understand the mechanism of the induction and their functions. Our report is the first report in which the induction of HSP60 in the pancreas by water-immersion stress is investigated.
SUMMARY
6
12 (h)
Fig. 2. Serum amylase levels after water-immersion. Blood was collected from tail vein, and amylase level was measured as described in the text. Values shown are means If: SE for four rats in each time.
In order to study the effects of systemic stress to the pancreas, we investigated the expression of HSPBO in rat pancreas before and after water-immersion stress, which is known as an exacerbation factor of caerulein-induced pancreatitis in rats. Expression of HSP60 increased in both soluble and insoluble fractions 3 hr following the initiation of water-immersion stress, and peaked 612 hr after immersion. The relative densities of stained bands in Western blot were
Heat shock
protein
1771
in rat pancreas
view of the pancreas before (A) and 12 hr after water-immersion (B) (original Fig. 3. Microscopic magmfication x X0. H & E). No pathologic alteration is signihcant after immersion. A
94-kDa
I3
b
1
2
1
2
antibody. 10% SDS-polyacrylamide gels were run and stained with Fig. 4. Specificity of anti-HSP60 to PVDF membrane and stained with anti-HSP60 antibody (B). In cot rmassie Blue (A) or transferred both panels: lane I, soluble fraction of rat pancreas; lane 2, purified HSP60.
MICHIRO OTAKA et al.
1772
insoluble
soluble fraction
fraction
c 190 g -
180
1
soluble
fraction
insolubie
fraction
T **
p
120. 110.
6
12 fht
0
3
6
*2(h)
Fig. 5. Expression of HSP60 after water-immersion stress. Coomassie Blue staining of SDS-polyacrylamide gel electrophoresis (A) and Western blot (B). In A and B, lane 1. before water-immersion (0 hr): lane 2, 3 hr; lane 3, 6 hr; lane 4, 12 hr after water-immersion, Relative density (C) was calculated by next formula: Relative density (%) = density (in each time)/density (before immersion) x 100. Values shown are means f SE for four rats in each time. *P < 0.05. **P < 0.01 compared with 0 hr.
1773
Heat shock protein in rat pancreas
133.7 f 4.6%, 122.7 f 9.6% (soluble fraction, insoluble fraction; 3 hr), 173.2 + 7.9%, 142.2 + 10.7% (6 hr) and 164.0 + 3 6%, 141.6 f8.8 (12 hr) compared with control (0 hr: 100%) by densitometrical scanning. REFERENCES
Ceska M., Birath K. and Brown B. (1969) A new and rapid method for the clinical determination of a-amylase activities in human serum and urine. Clin. Chim. Acfa. 26, 437444. Elias D., Reshef T., Birk 0. S., van der ‘Zee R., Walker M. D. and Cohen I. R. (1991) Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc nafn Acad. Sci. U.S.A.
88, 3088-3091.
Elias D., Markovits D., Reshef T., van der Zee R. and Cohen I. R. (1990) Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc. natn Acad. Sci. U.S.A. 87, 15761580.
Itoh H. and Tashima Y. (1991) The stress (Heat shock) proteins. Im. J. Eiochem. 23, 1185-1191.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&685. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Margulis B. A., Sandler S., Eizirik D. L., Welsh N. and Welsh M. (1991) Lipsomal delivery of purified heat shock protein hsp70 into rat pancreatic islets as protection against interleukin 1 /?-induced impaired function. Diabetes 40, 1418-1422. Takagi K., Kasuya Y. and Watanabe K. (1963) Studies on the drugs for peptic ulcer. A reliable method for producing stress ulcer in rats. Chem. Pharmac. Bull. 12,465472. Takano S., Kimura T., Kawabuchi M., Yamaguchi H. and Nawata H. (1991) Ultrastructural study of the effects of stress on the development of acute hemorrhagic pancreatitis in rats. Gasrroenterol. Jpn. 26, 236. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn Acad. Sci. U.S.A.
76, 435&4354.
Yamaguchi H., Kimura T. and Nawata H. (1990) Does stress play a role in the development of severe pancreatitis in rats? Gastroenferology 9%, 1682-1688.
Inr. J. Biochem. Vol. 25, No. 12, pp. 176991773,1993 Printed in Great Britain. All rights reserved
INDUCTION OF A 60-kDa HEAT SHOCK PROTEIN IN RAT PANCREAS BY WATER-IMMERSION STRESS MICHIRO OTAKA,” HIDEAKI ITOH,~TOSHIYUKIKUWABARA,’ AKIRA ZENIYA,’ SHUSEIFUJIMORI,’ YOHTALOUTASHIMA*and O~AMU MASAMUNE’ ‘First Department of Internal Medicine and 2Department of Biochemistry, Akita University School of Medicine, l-l-l Hondo, Akita 010, Japan [Tel. 81 188 34 1111; Fax 81 188 34 86191 (Received
Abstract-l.
7 June 1993)
In this study, expression of a 60-kDa heat shock protein in rat pancreas
before and after water-immersion
stress, which has been known
as an exacerbation
was investigated factor of caerulein-in-
duced pancreatitis in rats, by Western blot. 2. A 60-kDa heat shock protein increased after water-immersion
stress in both soluble and insoluble fractions of the pancreas. 3. Serum amylase level and pancreas weight did not increase after water-immersion. 4. No pathologic alteration was observed in the pancreas after water-immersion.
characterization and sequencing of 92 amino acid residues of seven peptide fragments digested by lysil endopeptidase.
INTRODUCTION Many studies have shown the importance of heat shock proteins (HSPs) for survival of cells under stress conditions (Itoh and Tashima, 1991). Recently it has been demonstrated that a 70-kDa heat shock protein (HSP70) is induced in pancreatic islet cells during prolonged exposure to interleukin l/I (Margulis et al., 1991). On the other hand, little is known about the expression and functions of a 60-kDa heat shock protein (HSP60) in the pancreas under stress conditions, although the HSP60 family is thought to play an important role in the pathogenesis of autoimmune diabetes in rats (Elias et al., 1990, 1991). In order to study the effects of systemic stress to the pancreas, we investigated the expression of HSP60 in rat pancreas before and after water-immersion stress, which is known as an exacerbation factor of caerulein-induced pancreatitis in rats (Yamaguchi et al.. 1990) MATERIALS AND Purification
METHODS
of HSP60
HSP60 was purified from porcine liver according to our method (Itoh et al., 1993). Briefly, frozen porcine livers were homogenized with 3 vol of buffer (10 mM Tris-Cl, IS mM /I-mercaptoethanol, 0.1 mM phenylmethyl sulfonyl fluoride, pH 7.4). After centrifugation at 18,OOOg for 10 min, the supematant was fractionated by ammonium sulfate (3&60%). After dialysis against the buffer, the protein solutions were separated using DEAE-cellulose and ATPSepharose column chromatography. The 60-kDa protein was eluted in high affinity fraction from the ATP-Sepharose column. The purified 60-kDa protein was identified as HSP60 by several methods, including physicochemical
‘To whom
correspondence
should
be addressed.
Production
of antibody
Antibody to HSP60 was produced by intramuscular injection into rabbit of 1 mg of the protein, emulsified in complete Freund’s adjuvant. Booster shots were given three times in the same manner as the original injection at 2-week intervals. The rabbit was bled 10 days after the last injection. Animals Fifteen to twenty-week old male Sprague-Dawley (weight: 250-300 g) were used in this experiment. Water-immersion
rats
stress
Rats were placed in a restraint cage, according to the method previously described (Takagi et al., 1963). The animals were then immersed vertically to the level of the xiphoid process in a water bath (22223°C). Rats were sacrificed before and 3, 6 and 12 hr (n = 4 in each time) following the initiation of water-immersion stress, Before killing, peripheral blood was collected from tail vein in order to assay serum amylase concentration. After killing, the pancreas was quickly removed and weighed to evaluate the degree of pancreatic edema (Yamaguchi et al., 1990). The pancreas wet wt was expressed as pancreas wet wt/body wt (mg/g). For light microscopy the pancreas was fixed in 20% formalin and embedded in paraffin wax. The tissue section was stained with hematoxylin and eosin. The remaining part of the pancreas was used immediately or frozen at -80°C until use. SDS-Polyacrylamide
gel electrophoresis
The pancreas was homogenized with 5 vol of ice-cold 25mM Tris-Cl buffer @H 7.5). The homogenates were centrifuged at 18,OOOg for 20min. The supematants (soluble fraction) were collected and the protein concentrations were adjusted to 0.2 mg/ml. The protein concentration was measured by the method of Lowry et al. (1951). The pellets were rehomogenized with the same buffer containing 1.O% 1769
1770
MICHIRO OTAKA el al. Assay of serum amylase Serum amylase was measured by the method of Ceska et al. (1969) using Phadebas amylase test (Pharmacia Laboratories, Piscataway, NJ).
RESULTS AND DISCUSSION
0
3
6
l2 (h)
Fig. 1. Alterations of pancreas weight [pancreas wet wt (mg)/body wt (g)] after water-immersion. Values shown are means & SE for four rats in each time.
Triton X-100. The homogenates were centrifuged at 18,OOOg for 20min, and the supernatants (insoluble fraction) were collected and the protein concentration of each sample was adjusted to 0.1 mg/ml. Samples were analyzed by 10% polyacrylamide gel electrophoresis according to the method of Laemmli (1970). Gels were stained with 0.1% Coomassie Brilliant Blue (R-250) in a mixture of 25% isopropyl alcohol-10% acetic acid and destained with 10% isopropyl alcohol-IO% acetic acid. Western blot Samples were electrophoresed on SDS-polyacrylamide gels, transferred electrophoretically to PVDF (polyvinylidene difluoride) membrane (Nihon Millipore, Kogyo, Japan), and processed as described by Towbin et al. (1979). The membrane was incubated with anti-HSP60 antibody (1: 1000 dilution) and treated with horseradish peroxidase conjugated anti-rabbit IgG (1: IS00 dilution) (Bio-Rad, Richmond, CA). The peroxidase substrate was 3,3-diaminobenzidine tetrahydrochloride. The density of the immunologically stained bands was analyzed using scanning densitometer.
The pancreas wet wt, which is a marker of pancreatic edema, did not increase after water-immersion (Fig. 1). In addition, serum amylase level did not increase after immersion (Fig. 2), and no pathologic alteration was observed in the pancreas (Fig. 3). The specificity of the anti-HSP60 antibody was shown in Fig. 4. Only the 60-kDa band was stained (panel B). This protein was identified as HSP60 by partial amino acid sequence. Expression of HSP60 increased in both soluble and insoluble fractions 3 hr following the initiation of water-immersion stress, and peaked 6 to 12 hr after immersion (Fig. 5). The relative densities of stained bands in Western blot were 133.7 k 4.6%, 122.7 k 9.6% (soluble fraction, insoluble fraction; 142.2 k 10.7% (6 hr) and 173.2 & 7.9%, 3 hr), with 164.0 f 3.6%, 141.6 &-8.8 (12 hr) compared control (0 hr: 100%) by densitometrical scanning. Water-immersion stress is known as an exacerbation factor of caerulein-induced acute pancreatitis in rats, although water-immersion stress itself can not be an initiator of pancreatitis (Yamaguchi ef al., 1990). In our study, no pathologic change was observed in the pancreas, and serum amylase level and pancreas weight did not increase even 12 hr after immersion. However, HSP60 has already increased 3 hr following the initiation of water-immersion. Although the real mechanism of the induction of HSP60 is not clear, it is known that water-immersion stress decrease pancreatic blood flow (Takano et al., 1991). Our results may indicate that mild or early stages of pancreatic cell damage, without pathologic alteration, by decrease of pancreatic blood flow contribute to the induction of HSP60. Furthermore, HSP60 may play an important role for cytoprotection as a stress response in the pancreatic cells. Further precise studies are required to understand the mechanism of the induction and their functions. Our report is the first report in which the induction of HSP60 in the pancreas by water-immersion stress is investigated.
SUMMARY
6
12 (h)
Fig. 2. Serum amylase levels after water-immersion. Blood was collected from tail vein, and amylase level was measured as described in the text. Values shown are means If: SE for four rats in each time.
In order to study the effects of systemic stress to the pancreas, we investigated the expression of HSPBO in rat pancreas before and after water-immersion stress, which is known as an exacerbation factor of caerulein-induced pancreatitis in rats. Expression of HSP60 increased in both soluble and insoluble fractions 3 hr following the initiation of water-immersion stress, and peaked 612 hr after immersion. The relative densities of stained bands in Western blot were
Heat shock
protein
1771
in rat pancreas
view of the pancreas before (A) and 12 hr after water-immersion (B) (original Fig. 3. Microscopic magmfication x X0. H & E). No pathologic alteration is signihcant after immersion. A
94-kDa
I3
b
1
2
1
2
antibody. 10% SDS-polyacrylamide gels were run and stained with Fig. 4. Specificity of anti-HSP60 to PVDF membrane and stained with anti-HSP60 antibody (B). In cot rmassie Blue (A) or transferred both panels: lane I, soluble fraction of rat pancreas; lane 2, purified HSP60.
MICHIRO OTAKA et al.
1772
insoluble
soluble fraction
fraction
c 190 g -
180
1
soluble
fraction
insolubie
fraction
T **
p
120. 110.
6
12 fht
0
3
6
*2(h)
Fig. 5. Expression of HSP60 after water-immersion stress. Coomassie Blue staining of SDS-polyacrylamide gel electrophoresis (A) and Western blot (B). In A and B, lane 1. before water-immersion (0 hr): lane 2, 3 hr; lane 3, 6 hr; lane 4, 12 hr after water-immersion, Relative density (C) was calculated by next formula: Relative density (%) = density (in each time)/density (before immersion) x 100. Values shown are means f SE for four rats in each time. *P < 0.05. **P < 0.01 compared with 0 hr.
1773
Heat shock protein in rat pancreas
133.7 f 4.6%, 122.7 f 9.6% (soluble fraction, insoluble fraction; 3 hr), 173.2 + 7.9%, 142.2 + 10.7% (6 hr) and 164.0 + 3 6%, 141.6 f8.8 (12 hr) compared with control (0 hr: 100%) by densitometrical scanning. REFERENCES
Ceska M., Birath K. and Brown B. (1969) A new and rapid method for the clinical determination of a-amylase activities in human serum and urine. Clin. Chim. Acfa. 26, 437444. Elias D., Reshef T., Birk 0. S., van der ‘Zee R., Walker M. D. and Cohen I. R. (1991) Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc nafn Acad. Sci. U.S.A.
88, 3088-3091.
Elias D., Markovits D., Reshef T., van der Zee R. and Cohen I. R. (1990) Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc. natn Acad. Sci. U.S.A. 87, 15761580.
Itoh H. and Tashima Y. (1991) The stress (Heat shock) proteins. Im. J. Eiochem. 23, 1185-1191.
Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 68&685. Lowry 0. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurements with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Margulis B. A., Sandler S., Eizirik D. L., Welsh N. and Welsh M. (1991) Lipsomal delivery of purified heat shock protein hsp70 into rat pancreatic islets as protection against interleukin 1 /?-induced impaired function. Diabetes 40, 1418-1422. Takagi K., Kasuya Y. and Watanabe K. (1963) Studies on the drugs for peptic ulcer. A reliable method for producing stress ulcer in rats. Chem. Pharmac. Bull. 12,465472. Takano S., Kimura T., Kawabuchi M., Yamaguchi H. and Nawata H. (1991) Ultrastructural study of the effects of stress on the development of acute hemorrhagic pancreatitis in rats. Gasrroenterol. Jpn. 26, 236. Towbin H., Staehelin T. and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. natn Acad. Sci. U.S.A.
76, 435&4354.
Yamaguchi H., Kimura T. and Nawata H. (1990) Does stress play a role in the development of severe pancreatitis in rats? Gastroenferology 9%, 1682-1688.