Int. J. Pancreatol. 9 Copyright 1991 by The Humana Press Inc. All rights of any nature whatsoever reserved. 0169-4197/91/8(2): 187-201/$3.00

The Role of Phospholipase A2 in Pancreatic Acinar Cell Injury r

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A n t t i J. Hietaranta, t Z o l t h n G. L a s z l k , H e i k k i J. A h o , 1 Pirjo I". Kortesuo, t a n d T i m o J. N e v a l a i n e n *,~ IDepartment of Pathology, University of Turku, Turku, Finland," and 2Department of Pathology, Albert Szent-Gy'orgyi B4edical University, Szeged, Hungary Received May 3, 1990; Accepted June 26, 1990

Summary The integrity of rat pancreatic acinar cells under the influence of human phospholipase A2 (PLA2) was studied. Isolated pancreatic acini showed no increased discharge of aspartylaminotransferase (ASAT) when incubated either in solutions containing human pancreatic PLA2 or the bile salt sodium deoxycholate (DEC), the latter in concentrations that augment PLA2 activity but have no destructive detergent effect. When human pancreatic PLA2 was injected into the rat pancreatic duct, uneven distribution was observed at 15 rain and 3 h in immunohistochemical sections. Edema and a mild inflammatory reaction were the main changes in the pancreas. The necrotic areas seen by light and electron microscopy were quite small and located mostly at the periphery of lobules corresponding the spread of the injected material. Necrosis was of the coagulation type and showed equal extent after the injection of PLA2 with or without DEC. Internalized human pancreatic PLA2 was present already 15 rain after the injection in the cytoplasm of some intact acinar cells, indicating a functioning protective mechanism. It was concluded that pancreatic acinar cells are quite resistant to PLA2-catalyzed hydrolysis of membrane phospholipids in vitro, but additional trauma, e.g., pressure caused by intraductal injection, and tissue related factors, such as the mediators of the inflammatory reaction, make acinar cells susceptible to the effect of PLA2. Key Words: Acinar cell; acute pancreatitis; necrosis; phospholipase A2. INTRODUCTION Acute pancreatitis is regarded as an autodigestive disease in which pancreatic tissue is destroyed by activated digestive enzymes and the resulting inf l a m m a t o r y reaction within the gland. Studies on various experimental *Author to whom all correspondence and reprint requests should be addressed.

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models have revealed a complicated mechanism of tissue destruction in acute pancreatitis. Pancreatic digestive and lysosomal proteases (1,2), lipolytic enzymes (3), free radicals (4,5), prostaglandins (6, 7), various inflammatory modulators (8), bacterial toxins (9,10), stimulaton of pancreatic secretion (11,12), and the obstruction of the pancreatic duct (13) participate in the development of tissue damage. The trigger mechanisms of these pathways of cell injury are largely unknown. However, two different but mutually compatible theories emphasize either the primary role of direct bile-induced damage in pancreatic duct and acinar cells (14,15), or altered synthesis and secretion of digestive enzymes in the acinar cells (16), caused, e.g., by the consumption of ethanol (17). The purpose of the present study was to investigate the cause and development of necrosis in rat pancreatic tissue by evaluating the toxicity of human pancreatic phospholipase A2 (PLA2) and the bile salt sodium deoxycholate (DEC) on acinar cells both in vitro on isolated pancreatic acini and in vivo after an intraductal injection of PLA2. The role of PLAz in the pathogenesis of acute pancreatitis has been emphasized by numerous authors (for reviews, see 18-20). PLA2 acts both as a digestive enzyme (19) and a key enzyme in the production of inflammatory mediators, especially those of the prostaglandin and leukotriene pathways (21,22). Both the catalytic activity of PLA2 and concentration of the immunoreactive pancreatic PLA2 are increased in sera of patients suffering from acute pancreatitis (23). Experimental studies on acute pancreatitis have shown signs of hydrolysis of phospholipids by PLAz in the destroyed pancreas (24,25). The digestive and the inflammation associated PLAzs differ from each other as to their cellular source and molecular structure (20). The toxicity of PLA2 on pancreatic tissue has been studied since the 1960s (19,26). However, we considered a reevaluation of this aspect of acute pancreatitis worthwhile, because recent reports have presented evidence that phospholipase Az might be quite a harmless substance to cell membranes per se (27,28), a suggestion that is in contrast to the results of a number of earlier studies (18,24,29,30). In addition, highly purified human pancreatic PLA2, as used in the present study, has not been available earlier. Previous investigations were based on the use of either porcine or bovine pancreatic PLA2s or enzymes contained in snake venoms. MATERIALS AND METHODS

Experiments with Isolated Pancreatic Acini In Vitro Preparation o f Isolated Pancreatic A cini Pancreatic acini were prepared from male Spraque Dawley rats (200-260 g) by digestion of the pancreatic tissue with collagenase type V (Sigma, St. Louis, USA) at 37 ~ in a shaking water bath according to the methods of Williams et al. (31) and Duan and Erlanson-Albertsson (32), with some modifications. After digestion, the acini were dissociated by passing them

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through a series of polypropylene pipettes with decreasing tip diameters, filtered through a 150 ~m mesh nylon cloth, and further purified by centrifugation at 50g through a 4~ albumin gradient. The dissociation and incubation solutions contained 103 m m o l / L NaC1, 4.78 m m o l / L KC1, 1.16 m m o l / L MgSO4, 1.16 m m o l / L KHzPO4, 11.1 m m o l / L D-glucose, 25 m m o l / L HEPES, 2~ (v/v) minimal Eagles medium amino acid supplement (Flow Laboratories, Irvine, Scotland), and 1 ~ (v/v) L-glutamine (Merck, Darmstadt, FRG). The pH of this basic medium was 7.4. The dissociation solution contained 180 u / m L collagenase, 2 m m o l / L CaC12, and 0.1 m g / m L soy bean trypsin inhibitor type II-S (Sigma) in the basic medium. The incubation solution contained 1.3 m m o l / L CaC12, 0.5070 BSA, and 0.1 m g / m L soy bean trypsin inhibitor in the basic medium. All solutions were equilibrated with 100~ O2.

Release of Aspartylaminotransferase (ASAT) from Isolated Pancreatic Acini The release of the intracellular enzyme ASAT into the incubation medium was measured in order to detect cell damage caused by the added PLA2 or DEC (deoxycholic acid, sodium salt, Sigma) in the pancreatic acini in vitro. The ASAT activity was assayed using an enzymatic colorimetric test, according to the manufacturer's instructions (ASAT Test Kit, Medix Biochemica, Kauniainen, Finland). In each experiment, acini obtained from one rat pancreas were suspended in 20 mL incubation medium and preincubated at 37 ~ in a shaking water bath incubator (60 cpm) for 60 rain. After preincubation, 1 mL incubation solutions containing the agents to be tested were mixed with aliquots (0.5 mL) of acini and incubated for 30 rain. At the end of the incubation period, 0.5 mL aliquots were removed and centrifuged at 10,000 rpm for 30 s in a microcenrifuge and the supernatant was assayed for ASAT. Aliquots (0.5 mL) of acini collected before incubation were added to 1 m L incubation solution and centrifuged immediately. The activities of ASAT in these aliquots were taken as the basal value. At the same time, aliquots (0.5 mL) of acini were added to 4.5 mL of incubation solution, followed by sonication for 20 s. The ASAT activities of these aliquots were taken as the total value. The difference between the total and the basal value was taken as 100%. The ASAT values determined after incubations were expressed in percentages of 100~ Values reported are the mean for three experiments. Groups were compared statistically by the Kruskal-Wallis one-way analysis of variance test, with the acceptance criteria of p < 0 . 0 5 .

Experimental Protocol The following substances were added to isolated pancreatic acini incubated in vitro: 0.5, 1.0, 1.5, 3.0, 4.5, and 6.0 m m o l / L DEC; 0.0278, 0.278, 2.78, and 27.8 ~g/mL PLA2; and 1.0 m m o l / L DEC and either 0.0278, 2.78 or 27.8 t~g/mL PLA2o H u m a n pancreatic PLA2 was purified as described earlier (33). The catalytic activity of PLA2 (IU = 1 txmol free palmitate released in

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one minute) was measured by the method of Schgdlich et al. (34), and the immunoreactive PLA2 by the method of Eskola et al~ (35). Protein was measured by the method of Lowry et alo (36). The SA of the PLA2-preparation used was 37 U/mg.

Morphology After the incubation, two aliquots of acini from each group were fixed by immersion in 3% glutaraldehyde in 0.1 m o l / L phosphate buffer at p H 7.4, postfixed in 2% osmium tetroxide, dehydrated, and embedded in Epon. Semithin sections were stained with alkaline toluidine blue for light microscopy. Thin sections were stained with uranyl acetate and lead citrate, and studied in an electron microscope.

Experiments with Intraductally Administered Solutions In Vivo Fifty two male Spraque-Dawley rats weighing 350-500 g were fasted overnight. The animals were anesthetized with ip pentobarbital (Mebunat, Orion, Finland) and a blunt needle was introduced into the pancreatic duct, as described previously (15). All injections were given by an injection p u m p (Injectomat, Fresenius, Bad Homburg, FRG) at vol of 0.1 mL/100 g of body wt, and at a rate of 0.1 m L / m i n .

Experimental Protocol The animals were divided into the following groups: Group 1. Ten animals received physiological saline intraductally. Five animals were killed 15 min and 3 h, respectively, after the injection. The pancreas was fixed by perfusion with 4~ paraformaldehyde in phosphate buffer at pH 7.4 in two animals both at 15 min and at 3 h after the injection. The pancreases of the remaining six animals were removed and fixed by immersion in 10% buffered formalin. Group 2. Ten animals received 675 #g (25 U)/100 g of body wt PLA2 dissolved in saline intraductally. The pancreases were sampled as in Group 1. Group 3. Ten animals received 1.5 m m o l / L DEC in saline intraductally. The pancreases were sampled as in Group 1. Group 4. Ten animals received 675 ~g PLA2 (25 U)/100 g of body wt and 1.5 m m o l / L DEC in saline intraductally. The pancreases were sampled as in Group 1.

Morphology and Immunohistochemistry Pancreatic tissue fixed in 4~ paraformadehyde or 10~ buffered formalin was dehydrated and embedded in paraffin. Five micrometer thick sections were stained with hematoxylin and eosin, and consecutive sections were processed for the immunohistochemical detection of rat pancreatic trypsin by a polyclonal antibody raised in rabbits and of human pancreatic PLA2 by a

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monoclonal antibody produced by mouse hybridoma cells. The biotin-avidin method (37) was used for the immunolocalization by using diaminobenzidine (DAB) as the chromogen. The sections were incubated in 0.4% pepsin for the removal of overfixed antigenic sites. Endogenous peroxidase activity was blocked with 0.3~ hydrogen peroxide in methanol, and the background reaction was blocked with normal horse serum before the application of the specific antibodies. The nuclei were counterstained with Meyer's haematoxylin. Anionic trypsin from rat pancreatic juice was purified, and antibodies were produced in rabbits as described elsewhere (38). Monoclonal antibodies against purified human pancreatic PLAz were produced according to standard methods (39), as will be described elsewhere (Nevalainen et al., to be published). Small pieces of pancreatic tissue perfused with paraformaldehyde were further fixed by immersion with 3% glutaraldehyde, followed by fixation in 2% osmium tetroxide for electron microscopy. RESULTS

Incubation of Pancreatic A cini Incubation of acini in solutions containing 0.0278, 0.278, 2.78, and 27.8 ~g/mL of PLA2 did not increase the release of ASAT significantly. DEC had no effect in concentrations of 0.5 or 1.0 retool/L, but significant increase of ASAT activities as compared with preincubation values were observed at concentrations of 1.5 m m o l / L or higher (Fig. 1). When 1.0 m m o l / L DEC was added to the solutions containing 0.0278, 0.278, 2.78, or 27.8 ~g/mL PLA2, no statistically significant effect on the release of ASAT was observed when compared with the corresponding PLAz-containing solutions without DEC (Fig. 2). Small peripheral cytoplasmic blebs were occasionally found by light microscopy in the acinar cells after preincubation. Similar blebs were present also after 30 min incubation in various concentrations of PLAz, or PLA2 plus 1.0 m m o l / L DEC or DEC alone. Electron microscopy showed slight mitochondrial swelling and a few peripheral blebs as the only pathologic alterations (Fig. 3).

Pancreatic Morphology after Intraductal Injection of PLA2 Group 1. Edema was observed in the pancreatic tissue 15 min after the intraductal injection of saline. Edema was partly resolved at 3 h, and some animals showed occasional cytoplasmic vacuoles in acinar cells. Polymorphonuclear leucocytes were sparse or absent. Immunohistochemical staining for rat trypsin was confined to the zymogen granules and ductal contents. Electron microscopy showed occasional necrotic acinar cells or small groups of acinar cells both at 15 rain and 3 h (Fig. 4). Group 2. Intercellular edema was observed after the intraductal injection of PLA2o Small periductal or focal necroses and areas of acinar cell necrosis

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in the peripheral parts of pancreatic lobuli were observed 15 rain and 3 h after the injection. These areas were devoid of immunostainable rat pancreatic trypsin. The immunostaining o f injected human pancreatic PLA2 was intense but quite focal at 15 min. This reaction was observed under the peritoneum in the periphery o f pancreatic lobules (Fig. 5), between acinar cells or lobules, and also in the interstitium and in the walls of blood vessels. Granular reaction was present in the cytoplasm of occasional nonnecrotic acinar cells (Fig. 6), and sometimes, also in the acinar lumina. The i m m u n o reaction had mostly disappeared at 3 h, and only occasional acinar cells showed internalized h u m a n pancreatic PLA2. Occasional acinar cells were severely damaged in electron microscopy. Mitochondria were swollen and the endoplasmic reticulum was finely vesiculated 15 min and 3 h after the injection (Fig. 7). Zymogen granules were quite resistant. The cytoplasm of m a n y acinar cells contained vacuoles of varying size (Fig. 8). Group 3. In addition to focal edema, narrow areas o f peripheral lobular necrosis was seen in some animals 15 min after the intraductal DEC injection. Such necrosis was present also at 3 h when polymorphonuclear leucocytes and occasional necroses in the ductal epithelium were observed. The immunoreaction for rat pancreatic trypsin was absent (Fig. 9) or restricted to some preserved zymogen granules in necrotic acinar cells (Fig. 10). In the electron microscope, necrotic acinar cells contained structureless endoplasmic reticulum. Sometimes, only nuclear remnants were discernible. Some necro-

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Fig. 3. Electron micrograph of isolated rat pancreatic acini after 30 min incubation with 27.8 ~g/mL h-PLA2 plus 1 mmol/1 DEC. The normal cellular structure is well preserved in spite of slight mitochondrial swelling.

Fig. 4. Electron micrograph of a normal and a necrotic acinar cell (N) 3 h after an intraductal injection of saline. The endoplasmic reticulum and mitochondria (arrows) are swollen and many zymogen granules (Z) are disintegrating. tic areas were surrounded by cell fragments that contained preserved cell orgenelles. G r o u p 4. The changes were similar after the combined injection o f PLA2 and D E C to those seen in Groups 2 and 3 (Fig. 11). Ductal lesions similar to those observed after D E C injections in Group 3 were also present. Immunostainings for rat pancreatic trypsin and the injected human pancreatic PLA2 gave similar results to those seen in Groups 2 and 3.

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Fig. 5. Immunostaining for injected human pancreatic phospholipase A2 is seen at the periphery of acini and under the peritoneal surface (arrows) 15 min after PLA2 injection. Hematoxylin counterstained. ABC.

Fig. 6. Granular internalized human pancreatic PLA2 (arrows) in rat pancreatic acinar cells 15 rnin after an intraductal injection of PLA2. Hematoxylin, ABC.

Fig. 7. Necrotic acinar ceils (N) 3 h after the intraductal injection of human pancreatic PLA2. Mitochondria (arrows) show dilatation in the cell near a necrotic acinarcell.

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Fig. 8. Acinar cells contain vacuoles (V) 3 h after the intraductal injection of human pancreatic PLA2. Some concentric cytoplasmic fragments are seen in the interstitium (arrows). Since necrotic areas in the above groups were quite small, and as a rule, did not exceed 1-2% of the total acinar parenchyma as estimated by light and electron microscopy, no morphometric data was obtained. It was very difficult to find damaged cells in Group 1 (saline injection). DISCUSSION Incubation of isolated rat pancreatic acini with human pancreatic PLAz or PLA2 plus DEC did not result in acinar cell necrosis or other measurable cell injury~ However, when PLA2 and/or DEC were injected into the pancreatic duct system, small areas of acinar parenchyma became necrotic. The concentration of the PLA2 solutions were chosen so that the highest doses injected did not exceed the potential maximal amount of PLA2 activity contained in rat pancreas (28). The present result largely support those obtained by Nagai et al. (28) in analogous in vitro experiments with isolated pancreatic acini. They found that the release of labeled pancreatic proteins in the incubation medium was not significantly altered even when porcine pancreatic PLA2 was used at a concentration 50 t higher than that measured in the pancreatic homogenate. Acinar cell damage could be induced only when the action of triglyceride lipase was combined with added fatty acids, that in several earlier experiments have been shown to cause pancreatic acinar cell injury (40,41). Isolated pancreatic acinar cells are very resistant to trypsin, too. Lysis of cell membranes was induced only when anoxia was combined with tryptic activity (42).

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Fig. 9. Necrotic acinar cells at the periphery of two pancreatic lobules are devoid of immunohistochemical reaction of rat trypsin 3 h after an intraductal injection of DEC. Only the nuclei are stained with hematoxylin in the necrotic areas. The acinar cell cytoplasm is intensely labeled in nonnecrotic areas. Hematoxylin, ABC.

Fig. 10. I m m u n o h i s t o c h e m i c a l staining for rat pancreatic trypsin is seen in the zymogen granules of necrotic acinar cells (arrows) 3 h after an intraductal injection of DEC. The cytoplasm of necrotic cells is devoid of stainable structures in the hematoxylin counterstain. Hematoxylin, ABC. The a m o u n t o f PLA2 for the intraductal injection in the present study was chosen according to calculations based on our determinations of rat pancreatic PLA2 concentration (unpublished) and data reported by others (43,44). The a m o u n t of PLA2 injected, 675/~g/100 g of body wt, corresponds to that which can be expected to be activated in an intact rat pancreas. Necrosis, although quite limited in extent, was present in the acinar cells after the intraductal injection of human pancreatic PLA2. Necrotizing pancreatitis has been produced in animal pancreas by intraductal instillation of PLAE (29,45). Also other tissues, e.g., testis and lung, seem to be vulnerable to PLA2 directly, or indirectly via histamine liberation or changes in prostaglandin metabolism (30,46). However, conflicting results have also been published, e.g., Schiller et al. (47) did not describe acinar cell necrosis after intraductal injection o f snake venom PLAE. Snake venom PLAz did not induce pancreatic damage when administered intraarterially to an isolated cat pancreas (27).

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Fig. 11. Necrotic acini at the periphery of a pancreatic lobulus 15 min after an intraductal injection of human pancreatic PLA2 and DEC. Hematoxylin and eosin. PLA2 catalyzes the hydrolysis of the ester bound in position 2 of glycerophospholipids to form lysophospholipids, e.g., lecithin is converted into lysolecithin. Bile salts and calcium ions are needed for the catalytic action of the enzyme (33, 48). Our measurements (unpublished) showed maximal augmentation of human pancreatic PLA2 activity at DEC concentration of 1.5 mmol/L, which was used in the present study in solutions injected into the pancreatic duct. This low concentration seemed to cause only minimal detergent damage (49) to pancreatic acinar cells in vitro (Fig. 1). Bile acids in low concentrations are known to stimulate enzyme secretion from isolated acinar cells (50). It is well known that very high concentrations (e.g., 20-100 mmol/L of sodium taurocholate) cause necrotizing pancreatitis if injected into the pancreatic duct (51). Lysolecithin is also lytic to pancreatic acinar cells in a similar way as high concentrations of bile salt (29,52). The results of the present study indicate that concentrations of PLA2 and DEC that were nontoxic to isolated pancreatic acini in vitro caused acinar cell injury when injected into the pancreatic duct system. The pressure created by the infusion may damage occasional acinar cells. Such damage is seen after saline injection, which may compromise the blood circulation in pancreas (47). Considerably more severe injury and inflammatory response was seen when high pressures, in the range of 140 mm Hg, were employed (47). We have earlier described rupture of the rat pancreatic duct system when the infusion pressure exceeded 82 mm Hg, a level constantly reached in our in vivo experimental model (53). Coagulation necrosis was seen in the most severely injured acinar cells in the present study. This finding is consistent with earlier reports on necrosis in acute pancreatitis in human (54) and rat (15,55) pancreas. Decreased immunoreactivity of rat pancreatic trypsin in necrotic acinar cells corresponds to earlier reports on immunohistochemistry of human pancreas in acute pancreatitis (55-57). However, interstitial or diffuse cytoplasmic staining was not found in the present study.

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The monoclonal antibody raised against human pancreatic PLA2 did not crossreact with rat pancreatic PLA2. Thus, the immunostaining seen in rat pancreas after the intraductal injection of human pancreatic PLA2 reflected the distribution of the injected foreign PLA2. The infused PLA2 rapidly and unevenly spread into the pancreatic interstitium, and most probably was capable to hydrolyse the cell membranes with which it came into contact and which were injured, e.g., by the pressure effect. Pancreatic acinar cells are capable of reinternalizing exocytosed digestive enzymes. Radioactivity labeled amylase was internalized at the apical cell surface, and within 2-5 min transferred to the Golgi cisternae and condensing vacuoles (58). Extracellular proteins can be internalized into isolated pancreatic acinar cells from the apical surface via small vesicles (59). Lateral and basal plasma membranes engulfed extracellutar ferritin that reached a juxtanuclear position in 30 min (59). We propose that the granular intracytoplasmic immunoreactive PLA2 seen in the present study represents the internalized injected human pancreatic PLA2 that seems to be harmless to the integrity and function of the pancreatic acinar cells of the experimental animal. The amount of pancreatic necrosis has a poor correlation with the severity of acute pancreatitis in humans (60)~ The number of positive Ranson signs, or the cause of the disease did not influence on the tissue PLAe content and activity (61). However, determination of serum PLA2 activity but not the values of serum immunoreactive PLAz, revealed a highly significant differentiation between patients with mild edematous and severe necrotizing acute pancreatitis, the latter associated with pulmonary damage (23). Elevated PLA2 catalytic activity could also be measured in sera of patients with other severe life-threatening diseases, such as, septic shock (62) and multiple injuries (63). The PLA2 activity that reflects the severity of disease state most probably is derived from inflammatory cells, but not from the pancreas. Therefore, we conclude that pancreatic PLA2 has no deleterious effect on intact pancreatic acinar cells, and that the necrosis induced in the rat pancreas after intraductal injection most probably requires augmentation by other factors, e.g., pressure, impaired circulation, or inflammatory mediators. The role of pancreatic PLA2 in the development of multiorgan failure in acute pancreatitic remains to be determined in further studies.

ACKNOWLEDGMENTS The authors thank Anne Mattila and Pirjo Koskivaara for technical assistance, and Kaarina Jokinen for typing the manuscript. Harry Kujari is gratefully acknowledged for his help in the statistical analysis.

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4 KoiwaiI, Oguchi H, Kawa S, Yanagisawa Y, Kobayashi T, Homma T. The role of oxygen free radicals in experimental acute pancreatitis in the rat. Int. J. Pancreatol. 1989; 5: t35-143. 5 Nonaka A, Manabe T, Asano N, Kyogoku T, Imanishi K, Tamura K, Tobe T, Sugiura Y, Makino K. Direct ESR measurement of free radicals in mouse pancreatic lesions. Int. J. Pancreatol. 1989; 5: 203-211. 6 Dtugosz J, Gabryelewicz A, Andrejewska A, Triebling A, Brozozowski J, Wereszczynska U. Prostacyclin (PGI2) stabilizes hepatic lysosomes during acute experimental pancreatitis in dogs. Z. Exp. Chit. 1982; 15: 210-218. 7 van Ooijen B, Kort W J, Tinga C J, Wilson JHP, Westbroek DL. Significance of prostaglandin E2 in acute necrotizing pancreatitis in rats. Gut 1989; 30: 671-674. 8 Horn JK, Ranson JHC, Ong R, Poulis D, Perez HD, Golstein IM. Complement catabolism and chemotaxis in acute pancreatitis. J. Surg. Res. 1982; 32: 569-575. 9 Williams LF, Byrne JJ. The role of bacteria in hemorrhagic pancreatitis. Surgery 1968; 64: 967-972. 10 Rattner DW, Compton CC, Gu Z-Y, Wilkinson R, Warshaw AL. Bacterial infection is not necessary for lethal necrotizing pancreatitis in mice. Int. J. Pancreatot. 1989; 5: 99-105. 11 Evander A, Ihse I. Cimetidine treatment in acute experimental pancreatitis. Euro Surg. Res. 1980; 12: 301-309. 12 Gomez G, Townsend CM, Green D, Rajaraman S, Uchida T, Thompson JC. Involvement of cholecystokinin receptors in the adverse effect of glucocorticoids on diet-induced necrotizing pancreatitis. Surgery 1989; 105: 230-238. 13 Saluja A, Saluja M, Villa A, Leli U, Rutledge P, Meldolesi J, Steer M. Pancreatic duct obstruction in rabbit causes digestive zymogen and lysosomal enzyme colocatization. J. Clin. invest. 1989; 84: 1260-1266. 14 Opie EL, Meakins JC. Data concerning the etiology and pathology of hemorrhagic necrosis of the pancreas (acute hemorrhagic pancreatitis). J. Exp. Med. 1909; 11: 561-578. t5 Aho H J, Koskensalo SM-L, Nevalainen TJ. Experimental pancreatitis in the rat. Sodium taurocholate-induced acute hemorrhagic pancreatitis. Scand. J. Gasterenterol. 1980; 15: 411-416. 16 Steer ML, Meldolesi JM. The cell biology of experimental pancreatitis. N. Engl. J. Med. 1987; 346: 144-150. 17 Wilson JS, Korsten MA. Pirola RC. Alcohol-induced pancreatic injury (part 2). Evolution of pathogenetic theories. Int. J. Pancreatol. 1989; 4: 233-250. 18 Creutzfetdt W, Schmidt H. Aetiology and pathogenesis of pancreatitis. Current concepts. Scand. J. Gastroenterol. 1970; 5 Suppl. 6: 750-764. 19 Nevalainen TJ. The role of phospholipase A in acute pancreatitis. Scand. J. Gastroenterol. 1980; 15: 641-650. 20 Nevalainen TJ. The role of phospholipase A2 in human acute pancreatitis. Klin. Wochensehr. 1989; 67: 180-182. 21 Flesch I, Schonhardt T, Ferber E. Phospholipase and acyltransferases in macrophages. Klin. Wochenschr. 1989; 67:119-122. 22 Sipka S, Dinya Z, Gergely P, Farkas T, Szegedi G. Simultaneous presence of platelet activating factor, leukotriene B4, prostaglandin Fla and F2c~in the supernatant of human neutrophils treated with phosphotipase A2 of human monocytes. Klin. Wochenschr. 1989; 67: 123-125. 23 Biichler M, Malfertheiner P, Sch~idlich H, Nevalainen T J, Friess H, Beger H. Role of phospholipase A2 in human acute pancreatitis. Gasterenterology 1989; 97: 1521-1526. 24 Aho H J, Nevalainen T J, Lindberg RLP, Aho AJ. Experimental pancreatitis in the rat. The role of phospholipase A in sodium taurocholate-induced acute hemorrhagic pancreatitis. Scand. J. Gastroenterol. 1980; 15: 1027-1031. 25 Kahle M, K6nig H, Filler RD. Lysolecithin-concentration in pancreatic tissue during therapy with phospholipase A2-inhibitors in acute necrotizing pancreatitis. Klin. Wochenschr. 1989; 67: t77-179. 26 Schmidt H, Creutzfeldt W. The possible role of phospholipase A in the pathogenesis of acute pancreatitis. Scand. J. Gastroenterol. 1969; 4: 39-48~

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Hietaranta et al. Hong SS, Case RM, Kim KH. Analysis in the isolated perfused cat pancreas of factors implicated in the pathogenesis of pancreatitis. Pancreas 1988; 3: 450-458. Nagai H, Henrich H, Wiinsch P-H, Fischbach W, M/Sssner J. Role of pancreatic enzymes and their substrates in autodigestion of the pancreas. Gastroenterology 1989; 96: 838-847. Aho H J, Nevalainen, TJ. Experimental pancreatitis in the rat. Light and electron microscopical observations on early pancreatic lesions induced by intraductal injection of trypsin, phospholipase A2, lysolecithin and non-ionic detergent. Virchows Arch. Cell Pathol. 1982; 40: 347-356. St~immer P. Investigations into various pancreatic enzymes. Exp. Pathol. 1982; 22: 203-209. Williams A J, Korc M, Dormer RL. Action of secretagogues on a new preparation of functionally intact, isolated pancreatic acini. Am. J. Physiol. 1978; 235: E517-E524. Duan RD, Erlanson-Albertsson C. Effect of bile salt on amylase release from rat pancreatic acini. Scand. J. Gastroenterol. 1985; 20: 1239-1245. Eskola JU, Nevalainen T J, Aho HJ. Purification and characterization of human pancreatic PLA2. Clin. Chem. 1983; 29: 1772-1776. Sch~idlich H, Bitchier M, Beget HG. Improved method for the determination of phospholipase A2 catalytic activity concentration in human serum and ascites. J o Clin. Chem. Clin. Biochem. 1987; 25: 505-509. Eskola JU, Nevalainen T J, L6vgren TN-E. Time-resolved fluoroimmunoassay of human pancreatic phospholipase A2. Clin. Chem. 1983; 29: 1777-1780. Lowry OH, Rosenbrough N J, Farr AL, Randall RJ. Protein measurement with the folin phenol reagant. J. Biol. Chem. 1951; 193: 265-275. Hsu S-M, Raine L, Fanger H. Use of avidin-biotin complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J. Histochem. Cytochem. 1981; 29: 577-580. Gr~inroos JM, Aho H J, Hietaranta A J, Nevalainen TJ. Early acinar cell changes in caerulein induced interstitial acute pancreatitis in the rat. Exp. Pathol. in press. Galfre G, Milstein C. Preparation of monoclonal antibodies: Strategies and procedures. Methods in Enzymology 1981; 73: 3-46. Nagy Z, Papp M, B~ilint A. Vascular injury associated with acute pancreatitis induced by oil or Na-deoxycholate. Acta Morph. Acad: Sci. Hung. 1971; 19: 175-185. Saharia P, Margolis S, Zuidema GD, Cameron JL. Acute pancreatitis with hyperlipemia. Studies with an isolated perfused canine pancreas. Surgery t977; 82; 60-67. Letko G, Falkenberg B, Matthias R. Isolated acinar ceils from rat pancreas in pathogenic studies on acute pancreatitis. Z. Exp. Chir. Transplant Kiinstl. Organe 1989; 22: 197-203. Arnesj6 B, Filipek-Wender H. Intracellular distribution of lipolytic enzymes in the rat pancreas. Acta Physiol. Scand. 1968; 74: 616-628. Arnesj6 B. Intracellular distribution of phospholipases in the rat pancreas. Acta Physiol. Scand. 1970; 81: 170-175. Wanke M, Nagel W, Willig F. Formen der experimentellen Pankreatitis patho-anatomisch gesehen. Frankfurt Z. Pathol. 1966; 75: 207-227. St/Smmer P, Steinmann U. Phospholipase A2 induced diffuse alveolar damage--Effect of indomethacin and dexamethasone upon morphology and plasma-histamine level. Klin. Wochenschr. 1989; 67: 171-176. Schiller WR, Suriyapa C, Anderson MC. A comparison of pancreatitis induced by high or low pressure injections of enzymes into the canine pancreatic duct. Int. J. Pancreatol. 1989; 4: 281-290. Arnesj6 B, Grubb A. The activation, purification and properties ot rat pancreatic jmce phospholipase A2. Acta Chem. Scand. 1971; 25: 577-589. Helenius A, Simons K. Solubilization of membranes by detergents. Biochim. Biophys. Acta (Amst.) 1975; 415: 29-79. Duan RD, Erlanson-Albertsson C. Effects of extracellular calcium and magnesium on bile-salt-stimulated amylase release from rat pancreatic acini. Scand. J. Gastroenterol. 1986; 21: 1211-1216.

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51 Aho HJ, Nevalainen TJ. Experimental pancreatitis in the rat. UItrastructure of sodium taurocholate-induced pancreatic lesions. Scand. J. Gastroenterol. 1980; t5: 41%424. 52 Terry TR, Hermon-Taylor J, Grant DAW. The generation of lysolecithin by enterokinase in trypsinogen prophospholipase A2 lecithin mixtures, and its relevance to the pathogenesis of acute necrotizing pancreatitis. Clin. Chim. Acta 1985; 150: 151-163. 53 Aho H J, Suonpg~i K, Ahola RA, Nevalainen TJ. Experimental pancreatitis in the rat. Ductal factors in sodium taurocholate-induced acute pancreatitis. Exp. Pathol. 1984; 25: 73-79. 54 Aho H J, Nevalainen TJ, Havia VT, Heinonen RJ, Aho AJ. Human acute pancreatitis. A light and electron microscopic study. Acta Pathol. Microbiol. Immunol. Scand. Sect. A 1982; 90: 367-373. 55 Aho H J, Putzke H-P, Nevalainen T J, L6bel D, Pelliniemi L J, Dummler W, Suonp~i~i AK, Tessenow W. Immunohistochemical localization of trypsinogen and trypsin in acute and chronic pancreatitis. Digestion 1983; 27: 21-28. 56 Kl6ppel G, Dreyer T, Willemer S, Kern HF, Adler G. Human acute pancreatitis: its pathogenesis in the light of immunocytochemical and ultrastructural findings in acinar cells. Virchows Arch. (Pathol. Anat.) 1986; 409: 791-803. 57 Willemer S, K16ppel G, Kern HF, Adler G. Immunocytochemical and morphometric analysis of acinar zymogen granules in human acute pancreatitis. Virchows Arch. (Pathol. Anat.) 1989; 415:1 t5-123. 58 Romagnoli P, Herzog V, Reinternalization of secretory proteins during membrane recycling in rat pancreatic acinar cells. Eur. J. Cell Biol. 1987; 44: 16%t75. 59 Livne E, Oliver C. Internalization of cationized ferritin by isolated pancreatic acinar cells. J. Histochem. Cytochem. 1986; 34: 167-176. 60 Nordback I, Pessi T, Auvinen O, Autio V. Determination of necrosis in necrotizing pancreatitis. Br.J. Surg. 1985; 72: 225-227. 61 Nordback I, Teerenhovi O, Auvinen O, Koivula T, Thuren T, Kinnunen P, Eskola J, N~int6 V. Human pancreatic phospholipase A2 in acute necrotizing pancreatitis. Digestion 1989; 42: 128-134. 62 Vadas P. Plasma phospholipase A2 levels corrleate with the hemodynamic and pulmonary changes in Gram-negative septic shock in man. J. Lab. Clin. Med. 1984; 104: 873-881. 63 Biichter M, Deller A, Malfertheiner P, Kleine HO, Wiedeck H, Uhl W, Samtner M, Friess H, Nevalainen T, Beger HG. Serum phospholipase A2 in intensive care patients with peritonitis, multiple injury, and necrotizing pancreatitis. Klin. Wochenschr. 1989; 67: 217-221.

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Volume 8, 1991