Esterase Inhibitors Prevent Lysosomal Enzyme Redistribution in Two Noninvasive Models of Experimental Pancreatitis

Transcription

1 GASTROENTEROLOGY 1989;96:853-9 Esterase nhibitors Prevent Lysosomal Enzyme Redistribution in Two Noninvasive Models of Experimental Pancreatitis G. OHSHO, A. K. SALUA, U. LEL, A. SENGUPTA, and M.1. STEER Department of Surgery and Division of Gastroenterology, Harvard Digestive Diseases Center and Charles A. Dana Research Laboratories, Beth srael Hospital and Harvard Medical School, Boston, Massachusetts Earlier studies have indicated that lysosomal enzymes such as cathepsin B become redistributed within pancreatic acinar cells during the early stages of both diet- and secretagogue-induced acute pancreatitis. As a result, cathepsin B and digestive zymogens became colocalized within large cytoplasmic vacuoles. As cathepsin B can activate trypsinogen, this co localization could result in intracellular digestive enzyme activation. The present study investigates the protective effects of gabexate mesilate (FOY) and camostate (FOY 35) on both ofthese noninvasive models of experimental pancreatitis. These esterase inhibitors prevented the hyperamylasemia, pancreatic edema, and acinar cell vacuolization that characterize secretagogue-induced pancreatitis and the hyperamylasemia and mortality that characterize diet-induced pancreatitis. n addition, FOY and FOY 35 were found to significantly decrease the subcellular redistribution of cathepsin B that occurs in both models. These findings indicate that enzyme activity sensitive to inhibition by FOY and FOY 35 may be critical to the redistribution phenomenon that characterizes both of these models of pancreatitis. t is generally believed that intra pancreatic digestive enzyme activation, as well as injury to the gland by activated proteolytic and lipolytic enzymes, is an important early event in the pathogenesis of acute pancreatitis. Until recently, it was assumed that digestive enzyme activation occurred within the pancreatic ductal space or in the duodenum. Recent studies, however, have suggested that digestive enzyme activation might occur within acinar cells. This conclusion was based on the results of studies using the secretagogue- and diet-induced models of experimental acute pancreatitis that indicated that, early in the evolution of those forms of the disease, digestive enzymes and lysosomal hydrolases become colocalized within cytoplasmic vacuoles (1-4). As the lysosomal enzyme cathepsin B is known to be capable of activating trypsinogen (5) and trypsin can activate the remaining digestive enzyme zymogens, the observed colocalization could result in intravacuolar digestive enzyme activation. n the present communication we report the results of studies that evaluated the effects of the potent new low molecular weight enzyme inhibitors gabexate mesilate (FOY) and camostate (FOY 35) on (a) edematous pancreatitis induced by infusing rats with a dose of the cholecystokinin-pancreozymin analogue caerulein in excess of that which causes maximal rates of digestive enzyme secretion from the pancreas and (b) necrotizing pancreatitis induced by feeding young female mice a cholinedeficient ethionine-supplemented (CDE) diet. n accord with reports by others (6), we have found that FOY protects against the interstitial pancreatic edema and hyperamylasemia that characterize the secretagogue-induced model of pancreatitis. We have also noted that FOY 35 reduces the mortality rate and degree of hyperamylasemia that follow ingestion of the CDE diet. These observations are consistent with the previous report that FOY has a similar effect on this model (7). We have also found thatredistribution of the lysosomal enzyme cathepsin B from the lysosome- and mitochondria-enriched Abbreviation used in this paper: ede. choline-deficient ethionine supplemented by the American Gastroenterological Association /89/$3.5

2 854 OHSHO ET AL. GASTROENTEROLOGY Vol. 96, No.3 subcellular fraction to the zymogen granule-enriched subcellular fraction is also reduced by these enzyme inhibitors. This latter observation suggests that activated enzymes may play an important role in the redistribution phenomenon, which is the earliest event that has been noted to characterize these models of acute pancreatitis. Materials and Methods Male Wi star rats (9-12 g) from Charles River Breeding Laboratories, Wilmington, Mass. were used for the secretagogue-induced pancreatitis experiments. A PE-5 cannula was placed in the jugular vein and its patency was maintained by continuous infusion of a solution containing NaCl (15 mm) and heparin (3 Ulml) at a rate of.2 mllh as described earlier (8). The animals were housed in shoe-box cages and allowed h to recover from the effects of anesthesia. Animals were subsequently divided into the following three groups: (a) control-infused only with the heparin-saline solution at a rate of.58 mllh for 3.5 h; (b) caerulein-infused with the heparin-saline solution as above (.58 mllh, 3.5 h) but with caerulein added to the infusate such that each animal received 5 JLg/kg h; (c) FaY plus caerulein-identical to caerulein but FaY infused for 2 h before and throughout the 3.5 h of caerulein infusion. The dose of FaY administered was 5 mg/kg. h. Female CD-l mice (1-14 g) from Charles River Breeding Laboratories were used for the diet-induced pancreatitis experiments. After an initial 24-h period of fasting, they were fed g/mouse of either regular laboratory diet (control group) or of the CDE diet containing.5% ethionine (U.S. Biochemical Corp., Cleveland, Ohio). When tested, FaY 35 was added to the diets and administered over 24 h such that a total dose of 6 mg/kg was consumed. After completion of the 24-h period during which animals were fed one of the diets with or without FaY 35, all mice were again fasted for 24 h. They were then either killed for use in biochemical experiments or given regular laboratory diet ad libitum for the following 3 days to allow for determination of mortality rates. Gabexate mesilate (FaY) and camostate (FaY 35) were the kind gifts of Ono Pharmaceuticals, Osaka, Japan. Other reagents were obtained from previously reported sources. n Vitro Amylase Secretion Dispersed rat and mouse pancreatic acllll were prepared by collagenase (Cooper Diagnostics, Freehold, N.J.) digestion and gentle shearing as previously described (9). Acini were suspended in HE PES-Ringer buffer (ph 7.4) containing (mm) NaC (115), KC (5), MgS4 (), Na 2 HP 4 (1), HEPES (1), CaCl 2 (1.26), and glucose (15). n addition, the buffer contained Eagle's basal amino acids, bovine serum albumin (.1%), and soybean trypsin inhibitor (.1 %, Cooper Diagnostics) and was saturated by bubbling with O 2, The acini were pre incubated in HEPES Ringer buffer (37 C) with or without FaY (1-4 M) for 3 min and then suspended in the same buffer containing varying concentrations of secretagogues. Amylase secretion was measured over the ensuing 3 min and expressed as the percentage of total amylase content. Net stimulated amylase secretion was calculated by subtracting the percentage of amylase secreted in the absence from that in the presence of the secretagogue. Subcellular Fractionation At selected times, rats and mice were killed by cervical dislocation, portions of the pancreas were quickly removed and trimmed of fat, and the pancreas was homogenized with a glass-teflon homogenizer in buffer (ph 6.5) containing 5 mm MOPS, 1 mm MgS4, and 25 mm sucrose. Unbroken cells and debris were removed by low-speed centrifugation (15 g. 15 min, 4 C) and the resulting supernatant was used for subcellular fractionation as described by Tartakoff and Jamieson (1) with minor modifications (8). The low-speed supernatant was considered to be the entire sample for later calculations. That supernatant was centrifuged (13 g. 15 min. 4 C) and the resulting zymogen granule-rich pellet was harvested. The remaining supernatant was centrifuged (12, g, 12 min. 4 C) to obtain a pellet that was rich in lysosomes and mitochondria. The remaining supernatant was centrifuged (15. g. 6 min, 4 C) to obtain a microsome-rich pellet and a supernatant whose contents were considered "soluble." The purity of each of these subcellular fractions was evaluated using marker enzymes with findings identical to those previously reported from this laboratory (8). Other portions of the pancreas were homogenized in phosphate-buffered saline containing.5% Triton X- using a Brinkman polytron (Brinkman nstruments, nc.. Westbury, N.Y.) and used to quantitate total enzyme content of the pancreas. Assays Amylase activity in serum and in subcellular fractions from the pancreas was measured using soluble starch as the substrate according to the method of Bernfield (11). Cathepsin B activity in pancreas fractions was measured fluorimetrically using the substrate CBZ-arginyl-arginine,B-naphthylamide (Bacham Bioscience nc.; Philadelphia, Pa.) as described by McDonald and Ellis with minor modifications (3.12). Deoxyribonucleic acid was measured according to the method of Labarca and Paigen (13) using calf thymus deoxyribonucleic acid (DNA) as the standard. The development of pancreatic edema was quantitated by comparing the pancreas weight obtained immediately after killing the animals ("wet weight") to that of the same sample after incubation at 15 C for 48 h ("dry weight"). Microscopy Samples of rat pancreatic tissue were fixed by immersion for 12 h in a mixture containing 95% ethanolsaturated picric acid/formalin/acetic acid (8: 15: 5. voll vollvol). After paraffin embedding, sectioning. and stain-

3 March 1989 ESTERASES N PANCREATTS 855 ing with hematoxylin-eosin, the sections were examined by a blinded observer and changes were graded on a -4 + scale. Mouse pancreatic fragments were fixed by immersion for 12 h in Zenker's fluid followed by overnight washing in running water. They were immersed in 15% sucrose-enriched phosphate-buffered saline and cryosectioned at 6 p.m. The sections were treated with Lugol's iodine-thiosulfate, stained with hematoxylin-eosin, and examined, as were rat samples, by a blinded observer. A 25 3 E 2 E "- " D D 2 15 " " " 1 E E D 5 D B Analysis of Data The results reported in this communication represent the mean ± SEM for n determinations. Differences between groups were evaluated using Student's t-test, with significant differences defined as those associated with a probability value of <.5. Mortality data were analyzed using the technique of Kaplan-Meier and evaluated using the. method with Yates' correction (14). Results Effects of FOY and FOY 35 on Serum Amylase and Morphologic Changes n agreement with previous reports from this (3,8) and other laboratories (15), infusion of a supramaximally stimulating dose of caerulein (5 JLg/kg. h) for 3.5 h was found to cause an increase in serum amylase levels (Figure la) and the appearance of pancreatic edema. The latter could be objectively quantitated by measuring the pancreatic dry to wet weight ratio, which was found to be 28.3% ± 1.6% in control samples and 13.1% ±.6% in samples taken from supramaximally stimulated rats. Other histologic changes that were noted included acinar cell vacuolization and the appearance, within the pancreatic parenchyma, of an inflammatory cell infiltrate (Table 1). When FOY (5 mg/kg. h) was infused before as well as with caerulein, the caerulein-induced increase in serum amylase was markedly attenuated (Figure 1). n addition, the dry to wet weight ratio of the pancreas was increased to 24.7% ±.2%, indicating that pancreatic edema had been prevented by FOY. These effects of FOY were all highly significant (p <.1). n addition, neither an inflammatory cell infiltrate nor evidence of acinar cell vacuolization was noted in animals given FOY (Table 1). Administration of the CDE diet was also found to cause hyperamylasemia (Figure B) and acinar cell vacuolization. n addition, ingestion of the CDE diet was associated with a 6% mortality rate (Figure 2). Similar observations have been reported previously from this laboratory (16). Addition of FOY 35 to the CDE diet resulted in a reduction in the degree of hyperamylasemia (Figure Control CER FOY Control CDE FOY35 +CER +CDE Figure 1. Effects of FOY and FOY 35 on hyperamylasemia of pancreatitis. A. Rats were infused with heparin-saline ("control"), heparin-saline plus 5 ltg/kg. h caerulein ("CER"), or heparin-saline plus caerulein plus 5 mg/ kg h FOY ("FOY + CER"), and serum amylase activity was measured as described in the text. Results represent mean and vertical bars the SEM value of duplicate measurements, each made using 7-12 different animals in each group. B. Mice were fed regular laboratory diet ("control") the CDE diet ("CDE"), or the CDE diet combined with FOY 35 ("FOY 35 + CDE") and serum amylase activity was measured as described in the text. Results represent mean and vertical bars the SEM value of duplicate measurements, each made using 9-15 different animals in each group. * p <.1 when caerulein or CDE alone were compared with control. * * p <.1 when FOY + caerulein or FOY 35 + CDE were compared with caerulein or CDE alone. B) and in the mortality rate (Figure 2). Others have noted that FOY has a similar effect when tested using the CDE diet-induced model of pancreatitis (7). The degree of acinar cell vacuolization was not altered by administration of FOY 35 with the CDE diet. Effect of FOY on Caerulein Stimulation of n Vitro Amylase Secretion Caerulein caused a dose-dependent stimulation of amylase secretion from dispersed rat pancre- Table 1. Effect of Caerulein and FOY on Microscopic Changes nduced by Supramaximal Stimulation with Caerulein nflammatory Acinar cell nfusion group n c ell infiltrate vacuolization Control 8 Caerulein Caerulein + FOY 5 Rats were infused with heparin-saline ("control"), heparin-saline plus caerulein ("caerulein"), or heparin-saline plus FOY plus caerulein ("caerulein + FOY "), for 3.5 h as described in the text. n addition, the animals given FOY received that agent for 2 h before caerulein infusion. There were n rats in each group. Histologic changes were blindly graded on a scale from (no change) to 4+ (maximum change).

4 856 OHSHO ET AL. GASTROENTEROLOGY Vol. 96, No.3 l _._ i 6 Jl O t r - - r J Days Figure 2. Effects of FOY 35 on mortality rate of CDE dietinduced pancreatitis. Mice were fed the CDE diet alone (-"-, n = 32) or the CDE diet plus FOY 35 (e- --e, n = 32) for 24 h as described in the text. The statistical significance of changes was evaluated by comparing the mortality rates. * p <.1. and ** p <.1. atic acini with maximal rates of net amylase secretion (1.2% ± 1.4% per 3 min) observed in the presence of 1-1 M caerulein. Higher doses of the secretagogue resulted in lower rates of amylase secretion (17). Addition of FOY (1-4 M) to a suspension of dispersed acini did not alter the response of the acini to caerulein, i.e., in the presence of FOY, maximal caerulein-stimulated net amylase secretion (1.9% ±.5% per 3 min) was still observed when the caerulein concentration was 1-1 M and lower rates of secretion were noted when higher concentrations of the secretagogue were present. n vitro caerulein-stimulated amylase secretion in mouse pancreatic acini was 7.3% ±.2% of total content per 3 min. n acini taken from mice fed the CDE diet, in vitro caerulein-stimulated amylase secretion was reduced to only 1.6% ±.4% of total content per 3 min. This phenomenon has been reported previously (9). A similar reduction in caerulein-stimulated amylase secretion was observed when acini taken from mice fed the CDE diet with FOY 35 were evaluated (net stimulated amylase secretion = 1.2% ±.3% of total content per 3 min). Effects of FOY and FOY 35 on Subcellular Distribution of Amylase The distribution of amylase in the various subcellular fractions of pancreas obtained from control animals was similar to that previously reported (8) (Figures 3A and 3B). Although most of the amylase activity was recovered from particulate fractions (zymogen granules, endoplasmic reticulum) a significant amount was present in the soluble fraction. This soluble amylase activity presumably results from organelle disruption during tissue processing. Alternatively, some or all of it may represent enzymes already discharged into the ductal space at the time the animals were killed. Supramaximal 6 A -:J' 5 ll ' 5 ll KP 12KP 15KP 15KS B 1.3KP 12KP 15KS 15KP Figure 3. Effect of FOY and FOY 35 on subcellular distribution of amylase during pancreatitis. A. Results from rats infused with or without caerulein with or without FOY. B. Results from mice fed the CDE diet with or without FOY 35. Open bars depict control animals, solid bars represent those given only caerulein or the CDE diet, and cross-hatched bars indicate the animals given either caerulein with FOY or the CDE diet with,foy 35. The subcellular fractions are the 13 g x 15 min pellet (1.3 KP), the 12, g x 12 min pellet (12 KP), the 15, g x 6 min pellet (15 KP), and the 15, g x 6 min supernatant (15 KS). Results represent mean and vertical bars the SEM values for 5-12 different animals and separate subcellular fractionations in each group. * p <.1 when caerulein or CDE alone was compared with control group. ** p <.1 when FOY + caerulein was compared with caerulein alone. Unless indicated, such comparisons revealed p values >.5. stimulation with caerulein resulted in an increase in the amount of amylase recovered from the soluble fraction, a change that had previously been reported and interpreted to indicate that supramaximal stimulation causes amylase to localize in fragile organelles that are subject to disruption during tissue processing (8). A corresponding decrease in the amylase activity present in the particulate fractions was also noted. Administration of FOY at the time of caerulein infusion prevented the alterations in amylase distribution induced by caerulein infusion (Figure 3A). Thus, in samples obtained from animals infused with caerulein plus FOY, the amylase con-

5 March 1989 ESTERASES N PANCREATTS 857 tent of the soluble fraction was similar to that noted in control animals not given a supramaximal dose of caerulein and significantly lower than that observed after caerulein infusion without FOY (p <.1). Administration of the CDE diet to mice also resulted in a significant increase in the amylase activity of the soluble fraction (Figure 3B). FOY 35, however, did not alter this redistribution of amylase activity. The CDE diet caused the digestive enzyme content of the pancreas to rise from a control value of 1.33 ±.35 D/JLg DNA to a level of 3.56 ±.3 U/JLg DNA (p <.1)' as enzyme synthesis continues while enzyme secretion is reduced (18,19). Administration of FOY 35 did not reduce the elevation in pancreatic amylase content that occurred after the ingestion of the CDE diet. After ingestion of the CDE diet containing FOY 35, the amylase content of the pancreas was found to be 3.27 ±.29 U/JLg DNA. Effects of FOY on Subcellular Distribution of Cathepsin B The cathepsin B content of the pancreas, expressed as units per microgram of DNA, was not altered by either supramaximal stimulation with caerulein on administration of the CDE diet, and was unaffected by either FOY or FOY 35 (not shown). Subcellular fractionation of samples obtained from control animals revealed that cathepsin B activity was recovered, primarily, in the 12, g pellet (Figures 4A and 4B) identified earlier (3) as being enriched in mitochondria and lysosomes. A significant portion of the cathepsin B activity was also present in the 13 g pellet. This observation has been interpreted to indicate the presence of heavy lysosomes. We have previously reported that supramaximal stimulation with caerulein causes a redistribution of cathepsin B. n subcellular fractionation experiments such as this, the redistribution of cathepsin B causes an increased amount to be recovered in the 13 g pellet, whereas that in the 12, g pellet declines (3). The redistribution of cathepsin B caused by caerulein infusion was found to be markedly reduced by infusion of FOY (p <.1) (Figure 4A). The CDE diet also caused the cathepsin B activity to increase in the 13 g pellet and to decrease in the 12, g pellet (Figure 4B). Administration of FOY 35 with the CDE diet partially, but significantly (p <.1), reduced this redistribution of cathepsin B (Figure 4B). Discussion FOY and FOY 35 are synthetic guanidino acid esters that inhibit a number of esterases including trypsin, phospholipase A z, kallikrein, plasmin, 5 A,..." -' 4 g l- LL. 3...,!1i!,.z. 2 1 nil 7 6 B 1 Lo KP 12KP 15KP 15KS 1.3KP 12KP 15KP 15KS Figure 4. Effect of FOY and FOY 35 on subcellular distribution of cathepsin B. Experimental groups and subcellular fractions are as described in Figure 3. A. Results for rats given caerulein with or without FOY. B. Results for mice fed the CDE diet with or without FOY 35. n panel A: * p <.1 when caerulein was compared with control; * * p <.1 when FOY + caerulein was compared with caerulein alone. n panel B: * p <.1 when CDE was compared with control; * * p <.1 when FOY + CDE was compared with CDE alone. Unless otherwise indicated, such comparisons revealed p values >.5. thrombin, and the C 1R and C 1 esterases (2-22). FOY 35, in contrast to FOY, is absorbed from the gastrointestinal tract, and circulating levels of the active esterase inhibitor can be detected after oral administration. The relatively low molecular weight of FOY and FOY 35 (417 and 495 daltons, respectively) has suggested that they might gain entry into the pancreas from the vascular space and has encouraged studies evaluating their use in the therapy of acute pancreatitis. n this communication, we have confirmed several recent reports that have indicated that these agents can exert a protective effect against secretagogue- (6,23) as well as diet- (7) induced pancreatitis. FOY was found to prevent the hyperamylasemia, pancreatic edema, inflammatory cell infiltrate, and acinar cell vacuolization that follow supramaximal stimulation with caerulein (Figure la, Table 1). Similarly, FOY 35 was noted to reduce the hyperamylasemia and mortality rate that occur after administration of the CDE diet (Fig-

6 858 OHSHO ET AL. GASTROENTEROLOGY Vol. 96, No.3 ures 1B and 2). These observations indicate that enzyme activity subject to FOY inhibition plays an important role in the evolution of both of these models of pancreatitis but, by themselves, these findings do not further define the events that are modulated by the esterase inhibitors. The ability of FOY to reduce the severity of secretagogue-induced pancreatitis might be explained by interference by FOY with either the binding or subsequent acinar cell stimulation by caeruleini.e., that FOY might act by preventing secretagogueinduced supramaximal stimulation. This possibility was tested by measuring in vitro caerulein-stimulated amylase secretion from acini in the presence and absence of 1-4 M FOY. That concentration of FOY was chosen after preliminary calculations indicated that infusion of 5 mg/kg. h of FOY in 1-g rats for 3 h would achieve a FOY level in blood :51-4 M. The observation that 1-4 M FOY does not interfere with the stimulation of amylase release induced by low concentrations of caerulein and does not prevent the inhibition of secretion that follows exposure to high concentrations of the secretagogue indicates that FOY does not interfere with binding or the subsequent stimulus-secretion coupling events that follow the binding of caerulein to surface membrane receptors. We have previously shown that the CDE diet interferes with stimulus-secretion coupling at a step subsequent to hormone-receptor binding and before membrane phosphoinositide hydrolysis by phospholipase C (9). As a result, acini taken from mice fed the CDE diet are unresponsive to in vitro stimulation by secretagogues such as caerulein. We considered the possibility that FOY 35 might protect against CDE diet-induced pancreatitis by preventing the development of this defect in stimulus-secretion coupling. To test this possibility, acini taken from mice given the CDE diet with or without FOY 35 were prepared and in vitro caerulein stimulation of amylase secretion was evaluated. FOY 35 did not prevent the reduction in caerulein-stimulated amylase secretion that occurred after administration of the CDE diet. This observation indicates that FOY 35 does not protect against CDE diet-induced pancreatitis by preventing the defect in stimulus-secretion coupling caused by the ethionine-containing diet. Both the secretagogue and the diet-induced models of experimental pancreatitis are characterized by the formation of large cytoplasmic vacuoles that contain both digestive enzyme zymogens and lysosomal hydrolases. n the diet model, these vacuoles arise by crinophagy (i.e., fusion between zymogen granules and lysosomes), whereas in the secretagogue model, the vacuoles appear to develop as a result of both crinophagy and a defect in the normal sorting mechanisms that segregate lysosomal hydrolases from digestive zymogens during intracellular transport (1-4). n both models, colocalization of lysosomal hydrolases and digestive zymogens in large cytoplasmic vacuoles has been noted to precede the appearance of cell injury. As the lysosomal hydrolase cathepsin B can activate trypsinogen and trypsin can activate the other zymogens, this colocalization phenomenon might result in intracellular digestive enzyme activation and be an important event in the evolution of these forms of pancreatitis. n our previously reported studies, colocalization of digestive enzymes and lysosomal hydrolases within the large vacuoles was demonstrated using the morphologic techniques of immunolocalization and enzyme cytochemistry (1-4). n the secretagogue-induced model, subcellular fractionation studies were also performed (3). They indicated that a redistribution of cathepsin B from the lysosomeand mitochondria-enriched fraction to the more dense zymogen granule-enriched fraction corresponded to the morphologically demonstrated colocalization. n this communication, we report the results of similar subcellular fractionation studies, performed after administration of the CDE diet. Once again, redistribution of cathepsin B to the zymogen granule-enriched fraction was noted. The most intriguing observation reported in this communication is the finding that FOY and FOY 35 prevent the phenomenon of cathepsin B redistribution that characterizes both of these models of pancreatitis. Thus, FOY administration markedly reduces the increase in cathepsin B noted in the 13 g pellet and the decrease in the 12, g pellet after subcellular fractionation of samples taken from rats subject to supramaximal stimulation with caerulein (Figure 4A) and from mice fed the CDE diet (Figure 4B). As a result of these observations, we conclude that the redistribution phenomenon (and presumably the co localization of digestive zymogens and lysosomal hydrolases) is dependent on esterase activity, which is susceptible to inhibition by FOY and FOY 35. Further identification and characterization of that enzyme activity may shed important light on the pathophysiology of acute pancreatitis. Candidate enzymes should include the phospholipases as well as the proteases that can be inhibited by FOY. Administration of FOY was found to prevent both cytoplasmic vacuole formation (Table 1) and the increase in soluble amylase activity that is observed when the pancreas is fractionated after supramaximal stimulation with caerulein (Figure 3A). t is likely that these two effects are interrelated as the increase in soluble amylase activity probably results from rupture of the large cytoplasmic vacuoles that

7 March 1989 ESTERASES N PANCREATTS 859 have been noted to become increasingly fragile during supramaximal stimulation with caerulein (3). On the other hand, FOY 35 does not prevent vacuole formation (not shown) or alter the increase in soluble amylase activity in fractions taken from mice fed the CDE diet (Figure 3B). As both FOY and FOY 35 reduce cathepsin B redistribution, an explanation for the differing effect of FOY and FOY 35 on vacuole formation and on the distribution of amylase activity is not readily apparent from these studies. These observations, however, might suggest that vacuole formation is not the consequence of cathepsin B redistribution. Further, these findings may indicate that vacuole formation either precedes or occurs independently of cathepsin B redistribution, and that ii the secretagogue model, but not in the diet model, of pancreatitis, vacuole formation involves the action of esterases that are subject to inhibition by FOY. Further clarification of these issues must await the results of future studies. References 1. Koike H, Steer ML, Meldolesi J. Pancreatic effects of ethionine: blockade of exocytosis and appearance of crinophagy and autophagy precede cellular necrosis. Am J Physiol 1982;242:G Watanabe O. Baccino FM. Steer ML. Meldolesi J. Effects of supramaximal caerulein stimulation on the ultrastructure of rat pancreatic acinar cell: early morphological changes during the development of experimental pancreatitis. Am J Physiol 1984;246:G Saluja A. Hashimoto S. Saluja M. Powers RE. Meldolesi J. Steer ML. Subcellular redistribution of lysosomal enzymes during caerulein-induced pancreatitis. Am J Physiol 1987; 253:G Saito. Hashimoto S. Saluja A. Steer ML. Meldolesi J. ntracellular transport of pancreatic zymogens during caerulein supramaximal stimulation. Am J Physio1987;253:G Greenbaum LA. Hirshkowitz A. Endogenous cathepsin activates trypsinogen in extracts of dog pancreas. Proc Soc Exp Bio Med 1961;17: Wisner JR. Renner G. Grendell JH. Niederau C. Ferrell LD. Gabexate mesilate (FOY) protects against ceruletide-induced acute pancreatitis in the rat. Pancreas 1987;2: Niedereau C. Liddle RA. Ferrell LD. Grendell JH. Beneficial effects of cholecystokinin-receptor blockade and inhibition of proteolytic enzyme activity in experimental acute hemorrhagic pancreatitis in mice. Evidence for cholecystokinin as a major factor in development of acute pancreatitis. J Clin nvest 1986;78: Saluja A. Saito. Saluja M. et al. n-vivo rat pancreatic acinar cell function during supramaximal stimulation with caerulein. Am J Physio1985;249:G Powers RE. Saluja AK. Houlihan MJ, Steer ML. Diminished agonist-stimulated inositol trisphosphate generation block stimulus-secretion coupling in mouse pancreatic acini during diet-induced experimental pancreatitis. J Clin nvest 1986;77: Tartakoff A. Jamieson JE. Fractionation of guinea pig pancreas. Methods Enzymo1974;31: Bernfield P. Amylases X and B. n: Colowick SP. Kaplan ND. eds. Methods in enzymology. Volume 1. New York: Academic. 1955: McDonald JK. Ellis S. On the substrate specificity of cathepsin B, and B2 including a new flu orogenic substrate for cathepsin B,. Life Sci 1975;17: Labarca C. Paigen K. A simple. rapid and sensitive DNA assay procedure. Anal Biochem 198;12: Colten T. Statistics in medicine. Boston: Little. Brown. 1974: Lampel M. Kern H. Acute interstitial pancreatitis in the rat induced by excessive doses of a pancreatic secretagogue. Virchows Arch [Aj1977;373: Manabe T. Steer ML. Experimental acute pancreatitis in mice: protective effects of glucagon. Gastroenterology 1979;76: Williams JA. Korc MA. Dormer RL. Action of secretagogues on a new preparation of functionally intact isolated pancreatic acini. Am J Physio1978;235:E Lombardi B. Estes LW. Longnecker DS. Acute hemorrhagic pancreatitis (massive necrosis) with fat necrosis induced in mice by DL-ethionine fed with a choline deficient diet. Am J Patho1975;79: Gilliland L. Steer ML. Effects of ethionine on digestive enzyme synthesis and discharge by mouse pancreas. Am J Physio198;239:G Muramatsu M. Fujii S. nhibitory effects of w-guanidino esters on trypsin. plasmin. plasma kallikrein and thrombin. Biochim Biophys Acta 1972;268: Tamura Y. Hirado M. Okamura Y. Minato Y. Fujii S. Synthetic inhibitors of trypsin. plasmin. kallikrein. thrombin. C,r and C, esterase. Biochim Biophys Acta 1977;484: Freise J, Magerstedt p. Schmid K. nhibition of phospholipase A2 by gabexate mesilate. camostate and aprotinin. Enzyme 1983;3: Okegawa T. Fujita T. Yamamoto Y. et al. Effects of FOY on experimental acute pancreatitis. Gendai ryo (Current Medicine) 1974;6:1-15. Received March Accepted September Address requests for reprints to: Michael L. Steer. M.D. Department of Surgery. Beth srael Hospital. 33 Brookline Avenue. Boston. Massachusetts This study was supported by National nstitutes of Health grants AM and AM G. Ohshio was supported by funds from the Uehara Memorial Foundation.