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
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ABSTRACT |
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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
(107 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
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INTRODUCTION |
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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
(107 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.
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MATERIALS AND METHODS |
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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, -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 · kg1 · 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 -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% -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 |
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In Vivo Studies
Caerulein, but not bombesin, hyperstimulation generates pancreatitis in vivo. As reported, caerulein hyperstimulation (5 µg · kg
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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
(1010 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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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 1010 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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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 (1010
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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DISCUSSION |
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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 (107 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
~107 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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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