Zymogen proteolysis within the pancreatic acinar cell is associated with cellular injury

T. Grady1, M. Mah'Moud2, T. Otani2, S. Rhee2, M. M. Lerch3, and F. S. Gorelick2

Departments of 1 Surgery and 2 Medicine, Veterans Affairs Connecticut Healthcare System, West Haven 06516; and Yale University School of Medicine, New Haven, Connecticut 06510; and 3 Department of Medicine B, Westfaelische Wilhelms-Universitaet, Münster 48129, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The pathological activation of digestive zymogens within the pancreatic acinar cell probably plays a central role in initiating many forms of pancreatitis. To examine the relationship between zymogen activation and acinar cell injury, we investigated the effects of secretagogue treatment on isolated pancreatic acini. Immunofluorescence studies using antibodies to the trypsinogen-activation peptide demonstrated that both CCK (10-7 M) hyperstimulation and bombesin (10-5 M) stimulation of isolated acini resulted in trypsinogen processing to trypsin. These treatments also induced the proteolytic processing of procarboxypeptidase A1 to carboxypeptidase A1 (CA1). After CCK hyperstimulation, most CA1 remained in the acinar cell. In contrast, the CA1 generated by bombesin was released from the acinar cell. CCK hyperstimulation of acini was associated with cellular injury, whereas bombesin treatment did not induce injury. These studies suggest that 1) proteolytic zymogen processing occurs within the pancreatic acinar cell and 2) both zymogen activation and the retention of enzymes within the acinar cell may be required to induce injury.

pancreas; caerulein; bombesin

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

ACTIVATION OF DIGESTIVE zymogens within the acinar cell is likely to be a precipitating factor in the development of acute pancreatitis (2, 15). Hyperstimulating concentrations more than 10 times the amount required to generate a maximal secretory response of CCK-8 stimulate the conversion of procarboxypeptidase A1 (PCA1) to carboxypeptidase A1 (CA1) and the processing of procarboxypeptidase B, chymotrypsinogen 2, and trypsinogen to their respective active forms in isolated pancreatic acini (10, 14, 22). It is likely that CCK hyperstimulation results in the conversions of digestive zymogens to their active forms both in isolated acini and in vivo. However, the ability of such activation to result in acinar cell injury is unknown.

Cellular injury is evident within hours after the induction of acute pancreatitis. In addition to intracellular zymogen activation, oxidant stress, decreased ATP levels, ischemia, inflammation, and cytokines may contribute to the injury (11, 15, 23; reviewed in Ref. 9). Although recovery from caerulein hyperstimulation is almost always complete, the insult is associated with acinar cell injury and cell death by necrosis and apoptosis (11). The present study uses isolated pancreatic acini to examine the potential for zymogen activation to generate cellular injury in the absence of other factors such as inflammation and hypoxemia. Antibodies that selectively detect the trypsinogen activation peptide (TAP), a marker for trypsinogen processing, were used in immunofluorescence studies to confirm that zymogen processing takes place in the pancreatic acinar cell. Although little TAP was detected in unstimulated pancreatic acini, treatment with high concentrations of CCK (10-7 M), caerulein (10-8 M), or bombesin (10-5 M) resulted in the intracellular generation of TAP. These agents also stimulated the processing of PCA1 to CA1. However, the distribution of the active enzyme form, CA1, was different for the two secretagogues. After caerulein hyperstimulation, CA1 was largely retained in the acinar cell, but after bombesin stimulation CA1 was found in the media. Finally, the effects of secretagogues on cellular injury were examined using the release of lactate dehydrogenase (LDH), the retention of trypan blue, and morphological criteria. CCK caused injury in a time- and concentration-dependent manner, whereas bombesin did not cause injury. These studies suggest that activation of digestive zymogens may induce acinar cell injury. Furthermore, both activation and retention of enzymes within the pancreatic acinar cell may be required to induce cell injury.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

Rats were obtained from the Charles River Breeding Laboratories (Wilmington, MA) and housed at the Veterans Affairs Connecticut Healthcare System, West Haven, CT. Caerulein, CCK-8, and bombesin were purchased from Research Plus (Bayonne, NJ). The o-phenylmethyl ester of CCK (OPE) was generously provided by L. J. Miller, Rochester, MN (7). An inability to obtain caerulein during the study resulted in the use of CCK-8 for some studies. However, the two agents were found to have an equivalent effect in all in vitro assays.

The anesthetics used were Rompun (xylazine hydrochloride; Mobay, Shawnee, KS), Ketaset (ketamine hydrochloride; Aveco, Fort Dodge, IA), and Metofane (methoxyflurane, Aveco). Heparin was purchased from Elkins-Sinn, Cherry Hill, NJ. Trypan blue stain was purchased from GIBCO BRL, Gaithersburg, MD. Dithiothreitol, beta -napthylamine, 3-(N-morpholino)propanesulfonic acid, HEPES, hexadecyltrimethylammonium bromide, tetramethylbenzidine (TMB), catalase, N,N-dimethylformamide, HPLC-grade water for the electrophoresis procedures, and the LDH determination assay kit LDH-500 were all purchased from the Sigma Chemical, St. Louis, MO. 125I-labeled goat anti-rabbit IgG was purchased from DuPont-New England Nuclear, Billerica, MA.

Materials used for protein electrophoresis and Western blot analysis were purchased from Bio-Rad Laboratories, Hercules, CA. Immobilon-P membranes were purchased from Millipore, Bedford, MA. Nylon meshes (400, 200, and 20 µm) were purchased from Small Parts, Miami, FL. The Phadebas amylase assay kit was purchased from Pharmacia Diagnostics, Atlanta, GA. The lipase turbidimetric assay kit was obtained from Boehringer Mannheim Diagnostics, Indianapolis, IN.

In Vivo Experimental Design

Hyperstimulation with caerulein and bombesin. Male Wistar rats (125-150 g) were fed laboratory chow and water ad libitum. Fasted rats were given a constant intravenous infusion for 3 h of 0.9% NaCl as a control, caerulein (5 µg · kg-1 · h-1), or bombesin (500 µg · kg-1 · h-1) dissolved in 0.9% NaCl. This caerulein dose has been shown to induce pancreatitis in rats (9). The dose of bombesin was chosen because our in vitro studies revealed that bombesin is ~100-fold less potent than caerulein in stimulating amylase release from acinar cells.

Markers of pancreatitis. Serum amylase and lipase activity were determined as described (4, 24). The water content of the pancreas was quantified by comparing the wet weight to the same sample after desiccation and expressed as a percentage. Pancreatic myeloperoxidase (MPO) activity was determined using a spectrophotometeric assay that detects its oxidation of TMB as described (8) and was expressed as units per gram wet tissue weight.

In Vitro Experimental Design

Preparation of isolated pancreatic acini. Isolated pancreatic acini were prepared from fasted male Wistar rats (40-80 g) by enzymatic digestion as described (19). Acini were suspended in Tris-Ringer buffer (pH 7.4) containing (in mM) 40 trisma base, 95 NaCl, 4.7 KCl, 0.6 MgCl2, 1.3 CaCl2, 0.5% bovine serum, and 0.01% soybean trypsin inhibitor and saturated with oxygen at 37°C. The dispersed cells then underwent a 3-h equilibration period unless otherwise stated. Preparations that demonstrated at least 95% viability using trypan blue exclusion were used for experimentation.

Detection of TAP. Affinity-purified antibodies to the five amino acids (Asp-Asp-Asp-Asp-Lys) adjacent to the activation site in trypsinogen were prepared as described (5). For immunofluorescence studies, the treated pancreatic acini were fixed with a buffer (pH 7.2) containing (in mM) 50 HEPES, 100 NaCl, 1 EGTA, 5 benzamidine, and 10 alpha -phenylmethylsulfonyl fluoride that contained 0.05% glutaraldehyde and 2% paraformaldehyde for 2 h and stored in 1% paraformaldehyde. For labeling the TAP antigen, fixed acini were permeabilized in 0.05% saponin in buffer without fixative and incubated for 4 h with antibody to TAP diluted 1:300 (~3 ng/µl), followed by FITC-conjugated goat anti-rabbit F(ab')2 (Biosource, Burlingame, CA). After washing, the acini were mounted in 15 µl ProLong Antifade Kit (Molecular Probes, Eugene, OR). Labeling was viewed through a Zeiss Axiophot (Thornwood, NY), and the images were recorded on Tmax film (ASA 100; Kodak, Rochester, NY) at a magnification of ×630. Fluorescence micrographs were exposed for 20 s, developed using constant conditions, and digitized using a Nikon Coolscan slide scanner. Images were processed identically in Adobe Photoshop.

Measurement of amylase release from acini. Amylase secretion (60 min) was measured as described using the assay method of Ceska et al. with the Phadebas assay and measuring amylase content in the incubation medium and acini (4, 21). Amylase secretion was expressed as a percentage of the total amylase. The net stimulated amylase secretion was determined by subtracting the percentage of basal (unstimulated) secretion from that of the stimulated acini. Basal amylase release ranged from 2 to 5%.

Detection of zymogen proteolysis (conversion) in vitro. Dispersed acini were incubated in a 24-well plate with 250 µl of cells distributed into each well. After 30 min of treatment, the acini were solubilized in sample buffer (13) with 10% beta -mercaptoethanol and boiled for 10 min and the solubilized proteins (~60 µg/lane) were subjected to electrophoresis on SDS-polyacrylamide (10%) gels at a constant voltage (150 V) and processed for quantitative immunoblot analysis as described (14). The relative level of conversion of PCA1 to CA1 was expressed as the ratio of stimulated over basal conversion (14). In a few studies, the percent of PCA1 converted to CA1 was calculated by quantifying PCA1 and CA1 and expressed as a percent conversion (CA1/CA1 + PCA1).

Detection of converted zymogens in the incubation medium. To detect PCA1 and CA1 in the incubation medium, it was separated from gravity-sedimented acini by aspiration and passed through a 10-µm Nytex mesh to remove residual acini. Proteins in the acinar pellet and the medium were solubilized in sample buffer (13), normalized to volume, and subjected to immunoblot analysis.

Assessment of acinar cell injury. Trypan blue retention in isolated acini was quantitated with the use of a spectrophotometric assay and expressed as an A583/280 ratio (12). LDH was measured in the medium and in isolated acini that had been solubilized in 0.1% Triton X-100. To prevent interference with substances added to the incubation medium, the enzyme was precipitated from the fractions with ice-cold methanol (80%) and resuspended in 25 mM HEPES, pH 7.5, and 100 mM NaCl. LDH release was expressed as the activity in the medium as a percent of the total. Preliminary studies demonstrated that longer equilibration periods reduced markers of cell injury in control and physiologically stimulated cells without changing their secretory responsiveness. Accordingly, assays of cell injury were performed on acini after a 3-h equilibration period unless otherwise noted.

Data Analysis

The data are presented as means ± SE values for multiple determinations from at least five separate animals for in vivo studies. All in vitro experiments were performed in duplicate for each individual observation. The statistical significance of observed changes was performed by analysis of variance followed by Dunnett's or by Student's t-test for unpaired observations. Significant changes were defined as those with a P value <0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In Vivo Studies

Caerulein, but not bombesin, hyperstimulation generates pancreatitis in vivo. As reported, caerulein hyperstimulation (5 µg · kg-1 · h-1) for 3 h resulted in edematous pancreatitis indicated by hyperamylasemia, hyperlipasemia, and increased tissue water content and MPO activity (Table 1). Bombesin hyperstimulation (500 µg · kg-1 · h-1) of rats resulted in none of the parameters indicative of acute pancreatitis (Table 1), with the exception of a small increase in tissue water. These studies confirm that caerulein hyperstimulation causes pancreatitis and that high doses of bombesin do not (21). Because zymogen activation within the pancreatic acinar cell has been linked to generation of pancreatitis, the effects of CCK or its analog caerulein and bombesin on zymogen processing in isolated acini were examined.

                              
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Table 1.   In vivo induction of pancreatitis by caerulein and not bombesin

In Vitro Studies

Secretagogue stimulation is associated with the generation of TAP in isolated pancreatic acini. In the absence of stimulation (Fig. 1A) or after physiological stimulation by CCK (10-10 M) (Fig. 1B), little or no TAP immunoreactivity was detected within pancreatic acinar cells. However, after hyperstimulation using CCK (Fig. 1C), caerulein (Fig. 1D), or bombesin (Fig. 1E), TAP immunoreactivity appeared in irregular vesicular structures that were located in a juxtanuclear distribution in acinar cells. These structures were similar in size, morphology, and distribution to those generated after in vivo caerulein hyperstimulation (20). Although the effects of caerulein on TAP generation were expected, the appearance of TAP after bombesin stimulation was surprising in light of the inability of bombesin to generate pancreatitis. To confirm that both caerulein and bombesin stimulated zymogen proteolysis, the processing of PCA1 to CA1 was examined in isolated acini.


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Fig. 1.   Secretagogues stimulate trypsinogen processing within pancreatic acinar cell. Isolated pancreatic acini were given saline treatment (A), physiological stimulation with CCK (10-10 M) (B), hyperstimulation with CCK (10-7 M) (C), hyperstimulation with caerulein (10-8 M) (D), and hyperstimulation with bombesin (10-5 M) for 30 min (E), then processed for trypsinogen activation peptide (TAP) labeling (see MATERIALS AND METHODS). Although there was virtually no labeling in control or after physiological stimulation (A and B), high-dose secretagogues generated TAP immunoreactivity associated with structures in the supranuclear regions of acinar cells (C, D, and E; arrows). No immunoreactivity was detected in absence of primary antibody 30 min after hyperstimulation with CCK (10-7 M) (F). Original magnification, ×630.

PCA1 processing to CA1 is stimulated by caerulein and bombesin. The dose-related effects of caerulein and bombesin on amylase secretion and proteolytic zymogen processing were examined in isolated acini. The maximal stimulatory concentration of caerulein for amylase secretion was 10-10 M; higher concentrations led to a progressive decrease in enzyme discharge (Fig. 2A). Bombesin stimulated secretion in a monophasic manner, with maximal secretion observed at 10-6 M. The processing of PCA1 to CA1 was also concentration dependent, increasing between 10-11 and 10-7 M caerulein (Fig. 2A). This result is indistinguishable from that previously found for CCK-8 (14). Similar to caerulein and CCK (14), bombesin stimulated the proteolytic conversion of PCA1 to CA1; a concentration of 10-7 M resulted in maximal processing. In unstimulated acini, 1-2% of PCA1 was processed to CA1; in caerulein-hyperstimulated acini, 4-8% of PCA1 was converted to the active form. The levels of CA1 generated by bombesin were ~60-70% of that generated by caerulein. The addition of OPE (10-10-10-6 M for 30 min), a selective agonist of the high-affinity CCK receptor that stimulates the monophasic release of amylase (7), did not stimulate the processing of PCA1 to CA1 (not shown).


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Fig. 2.   Caerulein and bombesin stimulate amylase secretion and zymogen conversion. Concentration dependence for amylase secretion (60-min stimulation; A) and conversion of procarboxypeptidase A1 (PCA1) to carboxypeptidase A1 (CA1; 30-min stimulation; B) in isolated acini is shown. , Caerulein; black-diamond , bombesin.

The release of CA1 from the cell would be predicted to follow the same pattern as amylase secretion if the processing of zymogens to active forms takes place in a secretory compartment. Accordingly, the release of CA1 into the incubation medium was examined. In unstimulated acini, little of the mature enzyme CA1 was found in the medium (Fig. 3). Although caerulein (10-7 M) hyperstimulation induced maximum levels of CA1 (Fig. 2B), virtually none of the active form was detected in the medium (Fig. 3). In contrast, CA1 appeared in the medium after bombesin stimulation (Fig. 3). Thus CA1 generated by caerulein hyperstimulation is retained within the acinar cell while much of that generated by bombesin is released into the medium. The conversion induced by caerulein (10-7 M) and bombesin (10-7 M) was blocked by 10 mM benzamidine, but not by the addition of soybean trypsin inhibitor (1 mg/ml), a high molecular-weight trypsin inhibitor that would not be expected to enter the cell. This observation demonstrates that PCA1 is likely processed to CA1 in a secretory compartment. Next, we sought evidence that caerulein might also stimulate CA1 processing in a secretory compartment.


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Fig. 3.   CA1 is released into incubation medium after bombesin stimulation. Representative autoradiograph depicting presence of PCA1- and CA1-immunoreactive bands in incubation medium is shown at top. A, unstimulated; B, caerulein (10-7 M) treatment; C, bombesin (10-7 M) treatment (n = 12). Equal volumes of incubation medium were subjected to immunoblot analysis. Only faint band CA1 is observed in unstimulated and caerulein condition, but a prominent band is observed after bombesin treatment. PCA1 band followed a similar pattern and its appearance increased in medium after bombesin treatment. Relative secretion of CA1 into medium is quantified at bottom and expressed as a ratio to basal condition from same experiment (means ± SE; n = 6).

The secretion of CA1 into the medium was measured as a function of increasing concentrations of caerulein (Fig. 4) in parallel to its effect on amylase secretion (Fig. 2A). The amount of CA1 released into the medium decreased when the concentration of caerulein increased above 10-10 M. These findings demonstrate that CA1 generated by low concentrations of stimulation is released from the acinar cell and that higher concentrations of caerulein (>10-10 M) cause both reduced amylase secretion and the retention of CA1 within the acinar cell.


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Fig. 4.   CA1 is released into incubation medium as an inverse function of caerulein concentration. Relative levels of CA1 in medium (black-triangle) detected in incubation medium after stimulation with caerulein were expressed as a ratio to unstimulated controls. Acini were stimulated with caerulein for 30 min, and medium was processed for immunoblot analysis. Relative levels of CA1 released into medium were also expressed as a ratio to total CA1 generated in acini (medium/total; ). P < 0.05 for all medium values compared with unstimulated control.

High concentrations of CCK generate cell injury. To determine whether zymogen activation and the retention of active enzymes are associated with acinar cell injury, pancreatic acini were exposed to CCK for up to 2 h. Unstimulated controls or those maximally stimulated (10-10 M) by CCK exhibited the same levels of LDH release and trypan blue retention (not shown). However, supraphysiological (10-7 M) concentrations of CCK released more LDH and retained more trypan blue, and the effects of CCK hyperstimulation on both markers of cell injury were time dependent; LDH release and trypan blue retention were elevated after 30 min of treatment and continued to increase during the next 90 min (Fig. 5). To determine whether serine proteases contributed to cell injury, the effects of the inhibitor benzamidine were examined.


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Fig. 5.   CCK hyperstimulation results in lactate dehydrogenase (LDH) release and trypan blue retention. Isolated acini were treated with medium (control) or physiological concentrations of CCK (10-10 M) for 120 min or were hyperstimulated with CCK (10-7 M) for periods up to 120 min. A: LDH content measured in acini and medium and expressed as percent total released. B: trypan blue retention in acini measured spectrophotometrically and expressed as A583/280 ratio. P < 0.001 for 120 min CCK (10-7 M) compared with unstimulated or 120 min CCK (10-10 M).

Pretreatment (15 min) with benzamidine (10 mM) has been found to block CCK-stimulated PCA1 processing to CA1 in isolated acini without altering the secretory response to CCK (14). Benzamidine reduced both LDH release and trypan blue retention (Fig. 6). The fact that neither LDH release nor the degree of trypan blue retention returned to baseline levels after benzamidine suggests that some injury may be present. The protective effects of benzamidine were time dependent; after 30-60 min of hyperstimulation, it was less effective in reducing injury. Addition of the soybean protease inhibitor (1 mg/ml), a high-molecular-weight trypsin inhibitor that would not be expected to enter the cell, to the incubation medium did not prevent cellular injury (not shown). These findings suggest that activation of intracellular serine proteases soon after the initiation of CCK hyperstimulation contributes to acinar cell injury. To test the hypothesis that enzymes might not cause cell damage if they are secreted, the effects of bombesin on acinar cell injury were examined.


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Fig. 6.   Benzamidine reduces LDH release and trypan blue retention in a time-dependent manner. Isolated acini were treated with medium (control) or hyperstimulated by CCK (10-7 M) for 120 min. After initiation of 120-min CCK treatment, benzamidine (10 mM) was added to medium. Times refer to duration of CCK treatment before addition of benzamidine (e.g., 90 = 90 min CCK, 30 min CCK + benzamidine). The inhibitor reduced effect of CCK hyperstimulation on LDH release (A) and trypan blue retention (B) in a time-dependent manner.

Acini were exposed to concentrations of bombesin that induced nearly maximum conversion of PCA1 to CA1 (Fig. 2B). In contrast to CCK, bombesin did not increase either LDH release or trypan blue retention (Fig. 7). To provide additional evidence that CCK but not bombesin induced injury, the effects of treatments on the acinar ultrastructure were examined.


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Fig. 7.   Bombesin, an agent that generates active enzyme forms that are released from the acinar cell, does not cause cell injury. Acini treated with bombesin (10-5 M; BOM) for 120 min demonstrated the same levels of LDH release (A) and trypan blue retention (B) as unstimulated controls. * P < 0.01 compared with 120 min CCK (10-7 M).

The electron microscopy appearance of control acini was similar to that of those exposed to CCK (10-10 M) or bombesin (10-5 M). However, acini hyperstimulated with CCK (10-7 M) developed protrusions of the basal membrane. The membrane "blebs" are a characteristic response of isolated acini to secretagogue hyperstimulation (1, 3) and were virtually eliminated by pretreatment with benzamidine (data not shown). Mitochondrial swelling and a loss of membrane definition are characteristic of cell injury. Mitochondria from control acini were elongated and had well-defined cristae (Fig. 8A). In contrast, mitochondria from the hyperstimulated condition were rounded and swollen and demonstrated poorly defined cristae (Fig. 8B). The mitochondria from benzamidine pretreatment (Fig. 8C) with or exposure to bombesin (Fig. 8D) were not swollen and had well-defined cristae. These morphological changes were confirmed by using the ratio of the short to long axis of the mitochondria as a marker for swelling (Table 2).


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Fig. 8.   In vitro hyperstimulation induces ultrastructural changes in acinar cells. Mitochondrial morphologies of unstimulated (A), CCK (10-7 M)-treated (B), CCK (10-7 M) with benzamidine (10 mM)-treated (C), and bombesin (10-5 M)-treated (D) cells are shown. Mitochondria appear normal in control cells (A) but are rounded after CCK hyperstimulation (B). Mitochondria from condition of benzamidine with CCK (C) or bombesin condition (D) appeared normal. Changes are quantitated in Table 2. Scale bar = 0.5 µm.

                              
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Table 2.   Mitochondrial morphology in pancreatic acini is affected by secretagogues

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The existence of a relationship between enzyme activation and acinar cell injury was previously unknown. To examine these potential associations, the effects of secretagogues on zymogen processing and cell damage were examined. First, we confirmed that in vivo caerulein hyperstimulation causes pancreatitis, but high doses of bombesin do not. To correlate zymogen processing with the generation of pancreatitis, antibodies to TAP were used as a marker for trypsinogen conversion to trypsin in isolated pancreatic acini. The TAP antigen was not detected in control conditions or after physiological stimulation by CCK or caerulein. Similar to in vivo studies (20), hyperstimulation by CCK (10-7 M) or its analog, caerulein (10-7 M), resulted in the generation of TAP within the acinar cell. Surprisingly, bombesin also stimulated the intracellular generation of TAP. The morphological appearance of the TAPpositive compartment was indistinguishable among the secretagogues or from that generated by caerulein hyperstimulation in vivo (20) and did not overlap with zymogen granules. Parallel studies in isolated acini demonstrated that caerulein and bombesin also stimulated the processing of the zymogen PCA1 to the active form, CA1. However, the trafficking of CA1 differed among secretagogues. Whereas most CA1 was retained in the acinar cell after caerulein (10-7 M) or CCK hyperstimulation (10-7 M) (14), CA1 was secreted from the cell after bombesin treatment. These findings suggest that stimulation of zymogen processing may be a generalized response of the acinar cell to stimulation and that this processing takes place within the acinar cell.

A link between zymogen processing, secretion of active enzyme forms, and cell injury was next examined. With the use of the biochemical criteria of LDH release and trypan blue retention and the morphological criteria of mitochondrial swelling and vacuole formation, caerulein hyperstimulation of acini was found to cause injury, whereas bombesin did not. The caerulein-induced injury could be reduced by benzamidine, a serine protease inhibitor that blocks the conversion of PCA1 to CA1 (14). Whether the protective effect of benzamidine is a result of blocking zymogen activation or inhibiting serine proteases that become activated during hyperstimulation is unclear. Notably, a concentration of benzamidine that has been found to block CCK-induced processing of PCA1 to CA1 did not reduce injury to background levels. This suggests that serine proteases play a role in acinar cell injury but that additional mechanisms, such as activated proteases that are not inhibited by benzamidine or other factors, may contribute to cell damage.

Although both caerulein and bombesin stimulate the generation of TAP in the acinar cell and PCA1 processing, there are differences in the response to the two agents. In the case of caerulein, maximum zymogen conversion occurs at supramaximal concentrations, whereas bombesin stimulates maximum conversion at the same concentration that causes maximum amylase secretion. An agonist of the high-affinity CCK receptor, OPE, did not significantly stimulate the processing of PCA1. Notably, the OPE peptide generates a similar monophasic pattern of amylase secretion as generated by bombesin and stimulates maximal amylase secretion at ~10-7 M (7). To summarize, maximal secretory concentrations of OPE that stimulate amylase release do not stimulate processing. Maximal secretory concentrations of bombesin stimulate both maximal amylase release and processing. Finally, much higher concentrations of CCK or caerulein than required to maximally stimulate amylase secretion are required to stimulate processing. These findings suggest that different intracellular signals may regulate secretion and zymogen processing.

The observation that much of CA1 is released from the acinar cell after bombesin stimulation or low concentrations of caerulein has two important implications. First, because acinar cells are not injured under these conditions, this finding would suggest that CA1 is being released from a secretory compartment. In a related study (20), we demonstrate that in vivo caerulein hyperstimulation is associated with the generation of TAP in a compartment that overlaps with a marker of lysosomes and recycling endosomes but not zymogen granules. Although direct studies are required, it is likely that TAP and CA1 are generated in the same compartment. These findings are consistent with the speculation that TAP and CA1 may be generated in a distinct secretory compartment, as discussed in Ref. 20. Second, if these secretagogues are stimulating processing in the same compartment, why is CA1 being retained after supramaximal concentrations of CCK or caerulein and not bombesin? Previous studies have demonstrated that the apical actin cytoskeleton of the acinar cell plays a central role in regulated secretion and that disruption of this cytoskeleton blocks secretion (16). Morphological studies have demonstrated that supramaximal concentrations of caerulein or CCK that are associated with reduced amylase secretion result in disruption of the apical acinar cell cytoskeleton (6, 17). However, supramaximal concentrations of bombesin do not cause reduced secretion or disturb the actin cytoskeleton (18). It is likely that the differences in the trafficking of CA1 among secretagogues reflect the distinct effects of the agents on the apical actin cytoskeleton (6, 16, 17).

In summary, the present study provides direct evidence that zymogen processing occurs within the pancreatic acinar cell. The fact that the processed form of PCA1, CA1, is released from the acinar cell after stimulation by bombesin or low concentrations of caerulein suggests that the processing take places within a secretory compartment. The fact that caerulein or CCK and bombesin stimulate PCA1 processing but have different effects on the retention of CA1 in the acinar cell suggests that different signaling mechanisms may regulate processing and secretion. Finally, zymogen processing is associated with the generation of acinar cell injury. However, injury is observed only when zymogens are both activated and retained in the acinar cell.

    ACKNOWLEDGEMENTS

The authors thank Dr. Lisa Matovcik for reviewing this manuscript, Dr. Robert Powers for his guidance and involvement in performing some of the initial studies, and Dr. Irvin Modlin for his critical comments.

    FOOTNOTES

T. Otani is a visiting scientist from the Department of Surgery II, University of Tokyo, Tokyo, Japan. F. S. Gorelick is the recipient of a Veterans Affairs (VA) Career Development Award, and this work was supported by a VA Merit Award. The contributions of the first two authors are equal.

Address for reprint requests: F. S. Gorelick, Research Bldg. 27, VA Connecticut Healthcare, West Haven, CT 06516.

Received 6 August 1997; accepted in final form 3 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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