New Perspectives on Acute Pancreatitis S . D. LEACH. F. S. GORELICK L I . M. MODLIN Dept. of Surgery, Yale University School of Medicine, New Haven, Connecticut, USA

Leach SD, Gorelick FS, Modlin IM. New perspectives on acute pancreatitis. Scand J Gastroenterol 1992;27 Suppl 192:29-38.

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The past decade has witnessed considerable changes in the clinical management of acute pancreatitis. Simultaneously. significant advances have bcen made in understanding the cellular and biochemical events involved in the initiation of this disease. This review summarizes recent clinical and scientific progress regarding acute pancreatitis and suggests areas for future investigation. Key words: Benzamidine: cerulein pancreatitis: chloroquine; choline-deficient. ethione-supplemented diet-induced pancreatitis; cysteine protease inhibitor; pancreatitis; zymogen activation Iroin M. Modlin, M . D . , Ph. D.. Depr. of Surgery, Yale University School of Medicine, 333 Cedar 9 , New Huueti, CT 06510, USA

Acute pancreatitis continues to represent a vexing clinical entity. The pathobiology of the disease process is ill understood, few effective therapies exist, and several difficulties plague investigation of this condition. First, the clinical course of acute pancreatitis is extremely variable, and few patients with fulminant disease may be encountered in any single institution. Second, human pancreatic tissue remains essentially inaccessible for clinical investigation, and, finally, there exists no ideal experimental model of the clinical disease process. Nevertheless, in the past decade considerable progress has been made, in both the clinical and scientific arenas, regarding several aspects of acute pancreatitis. In this review, we summarize newer concepts in the clinical management of acute pancreatitis and present recent information relevant to the cellular biology and biochemistry of pancreatic inflammation. CLINICAL PROGRESS Within the past decade several specific concepts related to the treatment of acute pancreatitis have undergone significant evolution. The disease remains clinically problematic; no specific medical or surgical therapy is capable of directly limiting pancreatic autodigestion and inflammation (1). Care of the patient with acute pancreatitis thus remains largely custodial, with attention directed towards maintaining an adequate circulatory volume, maximizing renal perfusion. supporting respiration, correcting electrolyte abnormalities, and providing adequate nutrition. More direct therapies are aimed largely at addressing the numerous complications of pancreatitis. The therapeutic efficacy of 'putting the pancreas at rest' by inhibiting acinar cell secretion has largely been discounted. Indeed, randomized prospective clinical trials involving nasogastric suction (2), cimetidine (3). atropine (4), glucagon ( 5 ) , calcitonin (6), and somatostatin (7) have met with

almost uniformly disappointing results (Table I). These trials have often been plagued by problems of statistical 'power' (8) and by difficulties in initiating therapy during the early stages of pancreatitis, when treatment is likely to be most effective. Nevertheless, the repetitive failure of these diverse anti-secretory therapies calls into question the role of acinar cell secretory activity in contributing to the severity of pancreatitis. In a wide number of experimental models of acute pancreatitis (9-11), acinar cell secretion already appears to be markedly diminished. Further data from our laboratory (12) and others (13) suggest that digestive zymogen activation during acute pancreatitis may actually occur within the acinar cell. Further inhibition of secretion in this setting may actually increase the amount of intracellular zymogen available for activation and potentially worsen the disease. Thus, it seems clear that attempts at diminishing the severity of pancreatitis by inhibiting export of digestive enzymes from the acinar cell may be theoretically flawed and clinically ineffective. The apparent intracellular location of digestive enzyme activation during acute pancreatitis must also be considered in contemplating the utility of protease inhibitor therapy during acute pancreatitis. Like anti-secretory therapy, studies investigating the efficacy of anti-proteolytic therapy in acute pancreatitis have been plagued by difficulties with

Table I . Failure of anti-secretory therapy in acute pancreatitis

Form of therapy

Reference

Nasogastric suction Cimetidine Atropine Glucagon Calcitonin Somatostatin

2 3 4 5 6 7

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regard to the timing of drug administration, and delays of up to 24 h after the onset of symptoms are common. Nevertheless, systemic administration of the antiprotease aprotinin (TrasylolB) has proven ineffective in clinical trials (14). Future efforts to suppress proteolytic activity during acute pancreatitis should involve combinations of different protease inhibitors to achieve maximum effect. Additional approaches may involve strategies to provide increased antiprotease activity within the acinar cell itself. Included in these strategies may be the development of clinically safe membrane-permeant protease inhibitors and the administration of protease inhibitors directly into the pancreatic duct to make them available for acinar cell endocytosis. In contrast to the largely unsuccessful outcome of pharmacologic therapies, substantial progress has been made in the application of endoscopic retrograde cholangiopancreatography (ERCP) to the treatment of gallstone pancreatitis. While the precise pathophysiology of gallstone pancreatitis remains obscure, the concept of common bile duct stone migration with resultant pancreatic ductal hypertension and/or bile reflux remains widely accepted (15). The vast majority of such stones seem to pass spontaneously (16), and a substantial fraction of patients with gallstone pancrzatitis have spontaneous resolution of the disease. A few patients, however, have a fulminant course, often related to ampullary stone impaction and accompanying cholangitis. In this setting, ERCP, papillotomy, and stone extraction have been demonstrated to be both safe and potentially effective in certain clinical subgroups (17). In a recent provocative clinical trial comparing ERCP with conservative therapy in 121 patients, there was no difference in outcome for patients with clinically mild disease, but a marked reduction in morbidity and mortality for those patients judged to have severe gallstone pancreatitis (18). Thus, while the vast majority of patients with gallstone pancreatitis need no such invasive therapy, ERCP may prove useful in patients with severe gallstone pancreatitis who fail to improve within 24 to 48 h. Additional studies from other centers are needed to confirm the utility of such an approach. The question remains open as to whether cholecystectomy is required after endoscopic papillotomy for gallstone pancreatitis. Although the practice has been to exclude the elderly, high-risk patient from cholecystectomy in this setting, this approach must now be reconsidered and prospectively studied in the light of the increasing availability of laparoscopic cholecystectomy (19). In addition to ERCP, the question of whether peritoneal lavage may provide effective therapy in acute pancreatitis has received considerable recent attention. Previous trials (20,21) produced conflicting results. While the prompt initiation of peritoneal lavage seems to lessen early cardiopulmonary morbidity, the demonstration of a decrease in late pancreatic sepsis or an improved overall mortality has proven elusive. Recently, Ransom & Berman (22) have applied an extended (7-day) period of large-volume lavage to

selected patients with severe pancreatitis. In a randomized, prospective clinical trial involving a total of 29 patients with 3 or more signs of severity, 7-day lavage was associated with a reduction in septic complications and overall mortality when compared with a 2-day lavage. While these data require confirmation from other centers, the findings once again have raised interest in the potential for peritoneal lavage to influence favorably the outcome of acute pancreatitis. Whether there is any incremental benefit to lavage delivered directly into the lesser sac via surgically placed catheters (23) remains an additional question for future investigation. While progress towards an effective primary therapy for acute pancreatitis has been limited, considerable advances have been made in the detection and treatment of the complications of pancreatitis. Notable among these recent developments are the use of computerized dynamic pancreatography in the detection of pancreatic necrosis (24), the application of fine-needle aspiration for the diagnosis of infected pancreatic necrosis (25), and the application of percutaneous drainage techniques for peripancreatic fluid collections and pancreatic pseudocysts (26,27). Pancreatic necrosis remains a significant source of morbidity and mortality in patients with acute pancreatitis. Previous investigations have demonstrated a direct relationship between clinically severe disease and necrotizing pancreatic histology (28). Pancreatic necrosis is often suspected on the basis of persistent fever or cardiopulmonary instability. However, when subjected to rigorous clinicopathologic analysis, these and other clinical criteria have proven ineffective in predicting the presence or absence of pancreatic necrosis (29). Thus, the demonstration of necrotizing pancreatitis has previously required formal laparotomy. It should be recognized, however, that even direct surgical inspection may be inaccurate in predicting the presence or absence of deep parenchymal necrosis. AS initially reported by Kivisaari et al. in 1983 (30), contrast-enhanced computerized tomography using rapidbolus contrast injection and thin tomographic sections through the pancreatic parenchyma has recently been shown accurately to predict the presence or absence of pancreatic necrosis. Bradley et al. (24) have applied the term 'dynamic pancreatography' to this technique. Specifically, the absence of contrast enhancement in all or part of the pancreatic parenchyma is indicative of pancreatic hypoperfusion and necrosis (Fig. 1). Necrosis of peripancreatic tissues may be similarly demonstrated. In several series (23,31) such findings are predictive of a fulminant clinical course. Whereas such methods now enable the diagnosis of pancreatic necrosis to be made non-operatively, the precise significance of these findings remain difficult to interpret. The natural history of computerized tomography (CT)-documented pancreatic hypoperfusion remains incompletely documented, and the need for surgical debridement in all cases of pancreatic necrosis is unsubstantiated. Furthermore, whether pancreatic parenchymal necrosis and peripancreatic

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Fig. I . Dynamic contrast-enhanced computerized tomography of pancreas demonstrating acute necrotizing pancreatitis. Areas of low attenuation represent hypoperfused areas of pancreatic parenchyma. These areas were confirmed to represent pancreatic necrosis at the time of laparotomy.

tissue necrosis contribute equally to overall morbidity remains unknown. It is clear that limited areas of necrosis in the absence of infection are not infrequently associated with a fairly benign clinical course (32) and may not necessarily mandate surgical intervention. To identify further those patients who may benefit from early surgical debridment, various investigators have combined CT with percutaneous fine-needle aspiration to determine the presence of infected pancreatic necrosis (24,25,31). These series have clearly defined CT-guided aspiration as a safe and accurate means of detecting bacterial contamination of pancreatic necrosis. Some authors have argued that the presence of bacterial contamination provides a more reliable prediction of the need for surgical intervention than does the appearance of pancreatic necrosis on CT scan (31,32). Together, these modalities provide a significant supplement to clinical judgement in determining the need for surgical intervention in acute pancreatitis. Whereas most patients with uncomplicated disease need not undergo such investigation, we advocate early contrast-enhanced CT scanning for all patients with severe pancreatitis, as evidenced by three or more Ranson criteria. If no pancreatic necrosis is evident, the patient may be observed and subjected to serial scanning if indicated. If CT scanning demonstrates a hypoperfused pancreatic parenchyma in a patient with clinical evidence of infection, percutaneous needle aspiration to determine the presence or absence of bacterial con-

tamination should be performed. Sterile collections may be observed, whereas infected areas of pancreatic or peripancreatic necrosis demand aggressive surgical debridement. In this setting, death rates as high as 15% may still be expected (31,32). A significant evolution in the management of pancreatic pseudocysts and peripancreatic fluid collections has also occurred in the past decade. A notable feature contributing to these changes has been an increased awareness of the high incidence of such collections on the basis of the frequent application of CT scanning in patients with pancreatitis (33). Unfortunately, significant inconsistency exists in the literature with regard to the use of the term ‘pseudocyst’ as applied to fluid collections observed on CT. It is clear that peripancreatic fluid collections occur frequently during bouts of acute pancreatitis (34). These collections occur in association with a recent disease attack; they are often amorphous and conform to the confines of the lesser sac; there is no distinct fibrotic wall and no communication with the pancreatic duct system. Such collections may be best referred to as lesser sac effusions (35). True pseudocysts, however, represent the sequelae of pancreatic ductal disruption. They are typically spherical or ovoid in shape, develop a distinct wall, and often continue to communicate with the pancreatic duct (34). Although the natural history of these two entities continues to be defined, it has been suggested that peripancreatic fluid collections show a particularly high rate of spontaneous

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resolution (34). True pseudocysts, however, appear to be less likely to resolve spontaneously, especially when they occur in the setting of chronic pancreatitis (35). The confusion regarding the use of these terms has made it difficult to evaluate the wisdom of new therapies for pancreatic pseudocyst. In particular, there has been much recent interest in percutaneous drainage of these collections. Experience with percutaneous drainage has generally been disappointing, with resolution typically 60% or less (26). However, Van Sonnenberg et al. (27) have recently reported a substantial experience with this technique in dealing with both infected and non-infected pseudocysts. In their series, a 90% success rate was reported after percutaneous drainage of 101 pseudocysts in 77 different patients. Unfortunately, the report was accompanied by limited clinical information with regard to whether these collections represented true pseudocysts as opposed to peripancreatic fluid collections. Thus the anticipated rate of spontaneous resolution remains unclear, and the benefit of percutaneous drainage for noninfected pseudocysts remains undocumented. In the case of infected pseudocysts, percutaneous aspiration clearly represents a safe method to detect infection (36). and percutaneous drainage may provide a reasonable means of temporizing until cyst wall maturity makes definitive enteric decompression possible. In summary, the past decade has witnessed substantial evolution in the management of patients with acute pancreatitis. New technologies including ERCP and dynamic CT scanning have augmented diagnostic accuracy and generated new approaches to patients with complicated pancreatitis. In contrast, little therapeutic progress has been made in addressing the primary pathophysiology of the disease. With this problem in mind, our laboratory has become interested in the cellular biology and biochemistry of acute pancreatitis, in the hope that understanding the basic biology of this disease might lead to the development of a rational primary therapy. SCIENTIFIC PROGRESS Acute pancreatitis: fundamental issues Under normal circumstances the pancreatic acinar cell functions as an efficient synthesizer and exporter of digestive enzymes. Under pathologic conditions, however, marked derangements of acinar cell function may play a critical role in the development of acute pancreatitis. In the 100 years following the original characterization of acute pancreatitis by Reginald Fitz (37), numerous investigations have been directed at determining the mechanism by which pancreatitis is initiated. These investigations have commonly used several different animal models of pancreatitis. Experimental work in this area has recently been reviewed (38). A fundamental assumption common to many of these investigations has been the proposal that premature activation of proteolytic and lipolytic zymogens may play a

Table 11. Mechanisms of trypsinogen activation to trypsin and their respective pH optima Mechanism

Optimum pH

Brush border enteropeptidase Trypsinogen autoactivation Lysosomal cathepsin B

8.0 5.0 3.6

central role in permitting progression of the disease. Initially articulated by Chiari in 1896 (39), this theory of ‘autodigestion’ was rapidly embraced despite a relative paucity of supporting data. Within the past 2 decades, however, intrapancreatic activation of digestive zymogens has been reported in both clinical (40) and experimental (41,42) forms of acute pancreatitis. Thus, in the absence of other tenable theories, the concept that pancreatitis results from premature activation of digestive zymogen within the exocrine pancreas remains a widely accepted creed. Recently, the demonstration of digestive zymogen activation during clinical acute pancreatitis has been facilitated by the development of antibodies against the cleaved trypsinogen activation peptide. Utilizing this probe to analyze both serum and urine, Hermon-Taylor and co-workers (43) have confirmed activation of trypsinogen during severe human pancreatitis. The amount of activation peptide present appears to correlate with disease severity. The mechanism and location of pathologic zymogen activation within the pancreas remain unknown. In addition to the well-described proteolytic cascade that follows trypsinogen activation by the brush border enzyme enteropeptidase, two other enzymatic mechanisms for the conversion of trypsinogen to trypsin have been demonstrated in in vitro biochemical investigations. These include trypsinogen-mediated autoactivation (44) and activation mediated by the lysosomal cysteine protease, cathepsin B (45). These three known mechanisms of trypsinogen activation and their respective pH optima are summarized in Table 11. The acinar cell is normally protected from inappropriate activation of its proteolytic and lipolytic content proteins by several safety mechanisms, including: 1) synthesis of enzymes as inactive zymogen precursors; 2) effective segregation and condensation of zymogens within membrane bound-secretory granules; 3) zymogen processing and storage at a pH that minimizes enzyme activity; and 4) co-synthesis and co-segregation of several potent protease inhibitors, including the pancreatic secretory trypsin inhibitor (PSTI) (46). Early in the process of acute pancreatitis it is presumed that these safety mechanisms become ineffective, and digestive zymogens become inappropriately activated. Precisely how and where this zymogen activation occurs remains unknown. Theoretically, zymogen activation may take place in one of three pancreatic tissue compartments: intraductal, interstitial, or intracellular.

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Acute Pancreatitis

The concept of intraductal zymogen activation is often invoked when considering bile reflux as an important factor in the pathogenesis of pancreatitis (47). In the presence of a normal ductal epithelium, however, it is unclear whether zymogens activated within the ductular lumen are capable of causing tissue injury. Reber and co-workers (48) have demonstrated abnormalities in ductal permeability during various forms of experimental pancreatitis which may give zymogens activated within the pancreatic duct access to the interstitial space. In addition to this ‘back-diffusion’ hypothesis, other investigators have demonstrated the direct entry of zymogens into the interstitial space by way of basolateral exocytosis during secretagogue-induced pancreatitis (49). Whether zymogens may become activated by interstitial tissue proteases remains a subject of debate. These previously developed concepts regarding the pathophysiology of pancreatitis emphasize extracellular aspects of the disease. While such events may result in the activation of digestive enzymes within the pancreatic duct or even in the periductal interstitium of the pancreas, it is unclear how such a process might lead to the extensive acinar cell necrosis frequently observed during acute pancreatitis. Indeed, acinar cells in short-term tissue culture seem to be relatively resistant to injury by external proteases and lipases (50). In contrast to these theories of extracellular zymogen activation, recent investigations have evaluated the possibility that digestive zymogens may become activated within the acinar call itself. The concept that pathologic zymogen activation might represent an intracellular event was initially proposed by Rao et al. in 1980 (5 1) and has formed the basis for a large resurgence in pancreatitis research in the past decade. A series of elegant studies undertaken by Steer and coworkers (reviewed in Ref. 5 2 ) have identified several intracellular events that may play a role in the activation of digestive zymogens within the acinar cell and the initiation of acute pancreatitis. Utilizing several experimental models of pancreatitis, it has been demonstrated that acinar cell secretion is markedly reduced during the early stages of the disease. In pancreatitis induced by the administration of a choline-deficient, ethionine-supplemented (CDE) diet, this decrease in the secretion of digestive zymogens appears to result from a specific disruption in acinar cell stimulussecretion coupling ( 5 3 ) . This inhibition of secretion appears to occur despite continued synthesis of digestive zymogens (52). Thus, early in the process of pancreatitis, increased amounts of digestive zymogens may be present within the acinar cell. In addition to an increased zymogen protein content, acinar cells harvested from animals with acute pancreatitis seem to be affected by an abnormal subcellular distribution of digestive zymogens and lysosomal hydrolases. In experimental pancreatitis induced either by hyperstimulation with the cholecystokinin analog cerulein (54) or by administration of a CDE diet (53), Steer et al. (52) have proposed that an

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abnormal co-mixture of digestive zymogens and lysosomal hydrolases exists within the acinar cell. Biochemically, this co-localization of digestive and lysosomal enzymes can be demonstrated by a shift in lysosomal hydrolase activity from a lighter lysosomal pellet to a heavier zymogen granuleenriched fraction during centrifugation of pancreatic homogenates. Similar biochemical evidence of an abnormal colocalization of digestive and lysosomal enzymes has been reported after obstruction of the pancreatic duct in the rabbit (55). Morphologically, this co-localization of secretory zymogens and lysosomal hydrolases is correlated with vacuolization of the acinar cell. In several different forms of experimental pancreatitis, large membrane-bound cytoplasmic vacuoles develop within the acinar cell (56,57). In at least two models, these vacuoles have an acidic internal pH (58). Similar vacuoles have been reported to develop within the human pancreas during acute pancreatitis (59). At least two different mechanisms for the co-mixing of zymogen and lysosomal proteins may be proposed. In CDE diet-induced pancreatitis, abnormal fusion events between zymogen granules and lysosomal derivatives are observed (57). This process is known as crinophagy and may represent a ubiquitous mechanism by which secretory cells degrade excess secretory product (60). In cerulein-induced pancreatitis, similar fusion events are also observed (56). However, co-localization of digestive zymogens and lysosomal hydrolases has been reported in early condensing vacuoles adjacent to the Golgi apparatus, suggesting that a defect in the initial sorting of lysosomal and zymogen proteins may also be present. On the basis of these observations, Steer et al. (52) have proposed that the ‘trigger mechanism’ responsible for the initiation of digestive zymogen activation within the acinar cell may be the co-localization of digestive zymogens with lysosomal hydrolases. As previously mentioned, the lysosoma1 enzyme cathepsin B is capable of converting trypsinogen to trypsin in vitro. Thus the observed mixture of zymogen and lysosomal proteins may indeed provide the basis for pathologic zymogen activation. The concepts developed from this series of investigations have proven seminal in promoting the concept that specific subcellular events may lead to the intracellular activation of digestive zymogens during the early phases of acute pancreatitis. Nevertheless, these concepts currently remain somewhat speculative. While a co-localization of digestive zymogens and lysosomal hydrolases may indeed occur during the development of experimental pancreatitis, the relationship between this event and actual zymogen activation remains unproven. Several aspects of this proposal require further clarification. In purified biochemical systems, the activation of trypsinogen by lysosomal cathepsin B is observed only over a very narrow pH range (2.5-4.0), with an optimal rate of activation occurring at pH3.6 (45). The presence of an acinar cell compartment with a pH in this

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range has yet to be demonstrated (61). In addition, these theories regarding the mechanisms of intracellular zymogen activation during acute pancreatitis were formulated at a time when such intracellular activation had yet to be demonstrated. Thus the temporal relationship between the COlocalization of lysosomal and zymogen proteins and the onset of zymogen activation during various forms of in vivo experimental pancreatitis remains unknown. Subsequent investigations by Adler and co-workers (62) have provided further comment on these issues. These investigators report a significant degree of co-localization of lysosomal hydrolases even in untreated animals. They have further confirmed an increase in lysosomal enzyme activity in the ‘zymogen granule’ fraction of homogenized pancreas using cerulein-induced pancreatitis in the rat. Utilizing immunolabeling and electron microscopy, however, these investigators suggested that such biochemical changes do not correlate morphologically with an increased co-localization of secretory zymogens and lysosomal hydrolases. Specifically, the increased activity of lysosomal enzymes within the ‘zymogen granule’ pellet after cerulein infusion was ascribed to the increased contamination of this pellet with autophagosomes that labeled exclusively for lysosomal enzymes. These authors concluded that true co-localization of lysosomal hydrolases and digestive zymogens is not enhanced during cerulein-induced pancreatitis (62). The interpretation of such data on the redistribution of lysosomal hydrolases during various forms of experimental pancreatitis remains controversial. We feel that the current weight of evidence suggests the following: 1) under normal conditions there is some co-localization of secretory zymogens and lysosomal hydrolases; 2) under certain pathologic conditions the amount of lysosomal hydrolases in the secretory compartment increases; and 3) the relationship between these findings and actual zymogen activation remains unknown. Before firm conclusions can be drawn, further rigorous examination of this concept is required. Irrespective of the true role of lysosomal hydrolases in the pathogenesis of acute pancreatitis, the possibility that digestive zymogen activation represents an intracellular event remains intriguing. In an additional study (13), Adler et al. have recently demonstrated that activated zymogens are detectable within pancreatic homogenates after the introduction of cerulein-induced pancreatitis. After subcellular fractionation these activities appear to be concentrated within a 1000g post-nuclear pellet. These findings support the hypothesis that intracellular zymogen activation may indeed occur. However, there are substantial theoretic difficulties in confirming intracellular zymogen activation by investigations utilizing intact animals and whole pancreatic homogenates. Specifically, it remains unclear whether activated digestive enzymes present in a specific subcellular fraction actually result from zymogen activation within the acinar cell. Alternatively, zymogens activated in the extracellular space

may secondarily partition in a specific subcellular fraction during tissue homogenation. Further elucidation of this question has been hindered by the lack of a suitable cellular system in which to investigate this process. Recognizing this difficulty, we sought to develop a new cellular model that might recapitulate the early events of acute pancreatitis. In such a system subcellular events might be studied in isolation from potentially confounding in vivo factors such as extrinsic neuro-humoral input, inflammation, and pancreatic blood flow.

Acute pancreatitis: a cellular model of zymogen activation On the basis of the ability of high doses of secretagogues to induce pancreatitis in the intact animal (63,64), we examined the effect of high-dose cholecystokinin (CCK) and highdose carbachol on isolated rat pancreatic acini in short-term tissue culture (12,65). In our system isolated rat pancreatic acini were exposed to incremental doses of secretagogue for various intervals. To detect zymogen activation in this cellular system, we developed an immunologic method for the detection of activated zymogens based on immunoblot analysis. After treatment acini were solubilized in detergent, and acinar cell proteins separated by sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. These proteins were then transferred to nitrocellulose and exposed to a polyclonal antisera raised against the digestive zymogen procarboxypeptidase A l . This antiserum recognizes both the inactive zymogen precursor (molecular mass, 46 kDa) and the activated enzyme, carboxypeptidase A1 (molecular mass, 35 kDa). Similar antisera were used to detect activation of procarboxypeptidase B and chymotrypsinogen 2. After localization with ’251-labeledsecondary antibody, the relative amounts of inactive and active enzyme were visualized by autoradiography and quantified by measurement of gamma emissions. Utilizing this technique, control acini incubated with medium alone were found to contain small amounts of the 35kDa activated carboxypeptidase A1 relative to the content of the 46-kDa inactive precursor, procarboxypeptidase A l . After exposure to incremental doses of either CCK or carbachol, however, activated enzyme accumulated within the acinar cell. Similar activation of both procarboxypeptidase B and chymotrypsinogen 2 were observed (Fig. 2). Zymogen activation occurred fairly rapidly (within 15 min) after the addition of secretagogue. The activated enzyme forms were detectable in acinar cell pellets and not in the incubation medium, suggesting an intracellular site of conversion. T o our knowledge, these findings represent the first direct demonstration that digestive zymogens may be activated within the acinar cell. Our recent understanding of the CCK receptor suggests that it exists in two interconvertable states, high-affinity and low-affinity (66). These two different forms of the CCK receptor exert differential effects on various aspects of acinar

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ACTIVATION OF ZYMOGENS WITHIN PANCREATIC AClNl Carboxypeptidase A1

Carboxypeptidase B

Chymotrypsin 2

ZYMOGEN

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ACTIVE ENZYME

Fig. 2. Secretagogue-induced activation of digestive zymogens in isolated pancreatic acini. After treatment with either cholecystokinin (CCK) or carbachol, acinar cell proteins are separated by sodium dodecyl sulfate-polyacrylamidegel electrophoresis, immunoblotted with anti-zymogen antisera, and visualized by autoradiography after application of '251-labeledgoat anti-rabbit IgG. The higher molecular weight species (solid brackets) correspond to inactive zymogen. The lower molecular weight species (dotted brackets) represent activated carboxypeptidase A1 , carboxypeptidase B, and chymotrypsin 2, respectively. Abbreviations: Cont = control; CARB = 100 micromolar carbachol: and CCK = 100 nanomolar CCK.

cell physiology. Occupation of the high-affinity form of the receptor appears to mediate CCK-induced zymogen secretion, whereas occupation of the low-affinity form of the receptor is associated with an inhibition of zymogen release. On the basis of dose-response experiments and additional investigations utilizing a specific high-affinity CCK receptor antagonist (65), we have demonstrated that CCK induces activation of digestive zymogens through occupancy of the low-affinity CCK receptor. With regard to carbacholinduced zymogen activation, the effect is mediated by muscarinic receptors. Identification of specific second messenger system(s) that may mediate these effects remains the subject of active investigation. Using this new cellular model, we examined the mechanism responsible for the conversion of inactive zymogens to their active forms. As previously noted, at least two mechanisms exist for the initiation of pathologic zymogen activation within the acinar cell: 1) autoactivation of trypsinogen, a member of the class of serine proteases, and 2) activation of trypsinogen by the lysosomal enzyme cathepsin B, a member of the class of cysteine proteases. Both of these enzymatic events require an acidic pH to proceed optimally. To determine which of these mechanisms might be responsible for the initiation of intracellular zymogen activation, acini were preincubated with either a serine protease inhibitor (benzamidine) or a cysteine protease inhibitor (EM) before incubation with CCK. Benzamidine pretreatment completely prevented the activation of procarboxypeptidase induced by CCK, whereas pretreatment with E64 did not block this conversion (12). Although these findings do not exclude other proteolytic pathways, they favor the hypothesis that trypsinogen autoactivation is the

mechanism responsible for initiating zymogen activation in this system. The means by which autoactivation of trypsinogen is initiated may represent a fundamental point in understanding the pathogenesis of acute pancreatitis. In purified enzyme systems, autoactivation of trypsinogen occurs optimally at a pH of 5.0 (44). Whereas the pH of pancreatic zymogen granules is usually maintained at just less than 7.0, pH values approaching 5.0 do occur within various compartments of the acinar cell, including lysosomes and immature condensing vacuoles (61). Of note, the cytoplasmic vacuoles observed during various in vivo forms of experimental pancreatitis also have an acidic internal pH (58); in a preliminary report utilizing DAMP-electron microscopy (61), we have estimated the pH of vacuoles induced by acinar cell hyperstimulation to be approximately 5.2 (67). These observations indicate that a low pH compartment may be required for zymogen activation to proceed. To test this hypothesis, we utilized two different agents capable of perturbing subcellular pH gradients. The weak base chloroquine acts to neutralize acidic subcellular compartments by directly binding to protons; monensin, on the other hand, neutralizes subcellular compartments by acting as a selective protonophore (68). We investigated the ability of both chloroquine and monensin to alter subcellular pH within the acinar cell and influence the intracellular activation of digestive zymogens induced by CCK (12). Neutralization of acidic subcellular compartments with either chloroquine or monensin was associated with a marked reduction in CCK-induced zymogen activation within the acinar cell. Although these agents may affect numerous metabolic pathways, the results indicate that an acidic sub-

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cellular compartment may be required for the initiation of intracellular zymogen activation. To extend these findings to the in vivo situation, in preliminary studies we have examined the effect of subcellular pH manipulation with chloroquine on the course of dietinduced pancreatitis in the mouse. Similar to our in vitro observations, continuous infusion of high-dose chloroquine limited the amount of active trypsin detectable in pancreatic homogenates and significantly improved survival (69). These findings should be interpreted with caution; chloroquine infusion in vivo may exert many effects unrelated t o changes in acinar cell pH. Nevertheless, these results are consistent with the hypothesis that a low p H compartment may be involved in zymogen activation during experimental pancreatitis. In summary, our investigations suggest that hyperstimulation of isolated pancreatic acini with high-dose CCK may serve as a useful cellular model of the early events in acute pancreatitis. On the basis of our findings in this system, we propose the following: 1) Occupation of the low-affinity form of the CCK receptor leads to the generation of specific second messengers within the acinar cell. 2) Activation of these second messenger pathways results in the exposure of trypsinogen to a low pH environment. 3) In this acidic environment, trypsinogen autoactivates t o trypsin and initiates activation of other zymogens within the acinar cell.

In addition t o further clarifying the mechanism by which intracellular zymogen activation occurs, future investigations may provide useful information about the means by which activated zymogens are degraded. In our system the amount of activated zymogen becomes maximal after 30 to 45 min of stimulation and subsequently decreases (12). In a preliminary report we have demonstrated that inhibition of cathepsin B by E64 actually prolongs the period during which activated zymogens are detectable in CCK-treated acini (70). It is thus possible that the morphnlogic and biochemical alterations involving lysosomal hydrolases observed during acute pancreatitis may actually represent protective, degradative mechanisms that function to limit the amount of activated zymogen present within the acinar cell.

Unanswered questions Several questions have been left unanswered by these investigations. Prominent among these is the precise mechanism by which zymogens become exposed to an acidic pH within the acinar cell. Several possibilities exist. First, zymogens may enter an acidic compartment by way of the crinophagic fusion events previously described by Steer and co-workers (57). Thus the significance of any co-localization of digestive zymogens and lysosomal hydrolases during the early stages of acute pancreatitis may actually lie not in the exposure of digestive zymogens to lysosomal cysteine proteases but in the attendant exposure of zymogens to the acidic pH of the lysosomal interior. Alternatively, exposure of zymogens t o an acidic p H may occur within a component of the classical secretory pathway. Under physiologic conditions, zymogens are briefly exposed to an acidic environment on entry into condensing vacuoles. Interestingly, some activated zymogen is detectable even in unstimulated acinar cells and may result from this transient exposure to a low pH. Under normal circumstances condensing vacuoles rapidly neutralize as they are transformed into mature zymogen granules (61). A pathologic alteration in condensing vacuole maturation may result in the prolonged exposure of digestive zymogens to an acidic pH, resulting in enhanced trypsinogen autoactivation. An examination of subcellular p H changes within the acinar cell after short-term CCK treatment might provide some information about this possibility.

REFERENCES

CONCLUSION

The previous decade has witnessed substantial progress in clarifying some of the fundamental mechanisms involved in the initiation of acute pancreatitis. Much of this progress has relied on the investigation of subcellular events within the acinar cell. The confirmation that digestive zymogen activation represents an intracellular event and the identification of the molecular mechanisms responsible for this event may provide the basis for novel new therapeutic strategies in acute pancreatitis.

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