Serine proteases mediate inflammatory pain in acute pancreatitis

ArticleinAJP Gastrointestinal and Liver Physiology 300(6):G1033-42 · March 2011with8 Reads
Impact Factor: 3.80 · DOI: 10.1152/ajpgi.00305.2010 · Source: PubMed
Abstract

Acute pancreatitis is a life-threatening inflammatory disease characterized by abdominal pain of unknown etiology. Trypsin, a key mediator of pancreatitis, causes inflammation and pain by activating protease-activated receptor 2 (PAR(2)), but the isoforms of trypsin that cause pancreatitis and pancreatic pain are unknown. We hypothesized that human trypsin IV and rat P23, which activate PAR(2) and are resistant to pancreatic trypsin inhibitors, contribute to pancreatic inflammation and pain. Injections of a subinflammatory dose of exogenous trypsin increased c-Fos immunoreactivity, indicative of spinal nociceptive activation, but did not cause inflammation, as assessed by measuring serum amylase and myeloperoxidase activity and by histology. The same dose of trypsin IV and P23 increased some inflammatory end points and caused a more robust effect on nociception, which was blocked by melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant trypsin isoforms. To determine the contribution of endogenous activation of trypsin and its minor isoforms, recombinant enterokinase (ENK), which activates trypsins in the duodenum, was administered into the pancreas. Intraductal ENK caused nociception and inflammation that were diminished by polypeptide inhibitors, including soybean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by melagatran. Finally, the secretagogue cerulein induced pancreatic nociceptive activation and nocifensive behavior that were reversed by melagatran. Thus trypsin and its minor isoforms mediate pancreatic pain and inflammation. In particular, the inhibitor-resistant isoforms trypsin IV and P23 may be important in mediating prolonged pancreatic inflammatory pain in pancreatitis. Our results suggest that inhibitors of these isoforms could be novel therapies for pancreatitis pain.

Full-text

Available from: Eugene P Ceppa, Jan 09, 2016
Serine proteases mediate inflammatory pain in acute pancreatitis
Eugene P. Ceppa,
1
Victoria Lyo,
2
Eileen F. Grady,
2
Wolfgang Knecht,
4
Sarah Grahn,
2
Anders Peterson,
4
Nigel W. Bunnett,
2,3
Kimberly S. Kirkwood,
2
and Fiore Cattaruzza
2
1
Department of Surgery, Duke University Medical Center, Durham, North Carolina; Departments of
2
Surgery and
3
Physiology, University of California, San Francisco, San Francisco, California; and
4
Molecular Pharmacology and Lead
Generation, AstraZeneca Research and Development, Mölndal, Sweden
Submitted 28 June 2010; accepted in final form 8 March 2011
Ceppa EP, Lyo V, Grady EF, Knecht W, Grahn S, Peterson A,
Bunnett NW, Kirkwood KS, Cattaruzza F. Serine proteases medi-
ate inflammatory pain in acute pancreatitis. Am J Physiol Gastrointest
Liver Physiol 300: G1033–G1042, 2011. First published March 24,
2011; doi:10.1152/ajpgi.00305.2010.—Acute pancreatitis is a life-
threatening inflammatory disease characterized by abdominal pain of
unknown etiology. Trypsin, a key mediator of pancreatitis, causes
inflammation and pain by activating protease-activated receptor 2
(PAR
2
), but the isoforms of trypsin that cause pancreatitis and
pancreatic pain are unknown. We hypothesized that human trypsin IV
and rat P23, which activate PAR
2
and are resistant to pancreatic
trypsin inhibitors, contribute to pancreatic inflammation and pain.
Injections of a subinflammatory dose of exogenous trypsin increased
c-Fos immunoreactivity, indicative of spinal nociceptive activation,
but did not cause inflammation, as assessed by measuring serum
amylase and myeloperoxidase activity and by histology. The same
dose of trypsin IV and P23 increased some inflammatory end points
and caused a more robust effect on nociception, which was blocked by
melagatran, a trypsin inhibitor that also inhibits polypeptide-resistant
trypsin isoforms. To determine the contribution of endogenous acti-
vation of trypsin and its minor isoforms, recombinant enterokinase
(ENK), which activates trypsins in the duodenum, was administered
into the pancreas. Intraductal ENK caused nociception and inflamma-
tion that were diminished by polypeptide inhibitors, including soy-
bean trypsin inhibitor and a specific trypsin inhibitor (type I-P), and by
melagatran. Finally, the secretagogue cerulein induced pancreatic
nociceptive activation and nocifensive behavior that were reversed by
melagatran. Thus trypsin and its minor isoforms mediate pancreatic
pain and inflammation. In particular, the inhibitor-resistant isoforms
trypsin IV and P23 may be important in mediating prolonged pancre-
atic inflammatory pain in pancreatitis. Our results suggest that inhib-
itors of these isoforms could be novel therapies for pancreatitis pain.
trypsin; trypsin IV; P23; enterokinase; pain
ACUTE PANCREATITIS IS A POTENTIALLY fatal inflammatory disease,
which typically begins with the onset of severe abdominal
pain. Although autodigestion of the pancreas by proteases such
as trypsin is considered the main mechanism of the pathogen-
esis of acute pancreatitis, the molecular basis is still poorly
understood. The activity of pancreatic digestive enzymes is
strictly controlled by redundant mechanisms, including synthe-
sis of inactive zymogens (e.g., trypsinogens, mesotrypsino-
gen), zymogen segregation in membrane-bound compartments
that are packaged with pancreatic secretory trypsin inhibitors
(PSTIs), degradation in lysosomal compartments, and re-
stricted activation by enterokinase (ENK) located separately in
the duodenum (14), where ENK cleaves trypsin-associated
peptide from trypsinogen to release activated trypsins (36, 41).
ENK is localized to the brush border membranes of duodenal
enterocytes and is thus able to activate proteases once they
have been secreted from the pancreas into the duodenum and
are in direct proximity to nutrient substrates.
Trypsinogens are a functionally diverse gene family. In
humans, protease serine type 1 (pssr1) encodes trypsinogen I
(cationic trypsin), pssr2 encodes trypsinogen II (anionic tryp-
sin), and pssr3 encodes mesotrypsinogen. Trypsinogen IV is a
splice variant of mesotrypsinogen (47). A potential homologue
of human mesotrypsinogen in rats is P23 trypsinogen, a minor
isoform. Trypsin IV/mesotrypsin and P23 are resistant to
polypeptide inhibitors, including the PSTIs and soybean tryp-
sin inhibitor, and may thus remain active for prolonged period
of time. However, the role of trypsin IV/mesotrypsin in disease
is unknown.
The biological effects of trypsins are in part attributed to the
proteolytic activation of a family of G-protein coupled recep-
tors, the protease-activated receptors (PARs) (35). Trypsins,
and other serine proteases, cleave the extracellular NH
2
-termi-
nal domain, thereby unmasking a newly formed NH
2
-terminal
that acts as a tethered ligand that binds to and activates the
cleaved receptor. PAR
2
, which is activated by trypsins and
mast cell tryptase, is strongly expressed on the luminal surface
of pancreatic acinar and ductal cells, and by pancreatic sensory
nerves. However, the contribution of PAR
2
to pancreatitis is
controversial, with reported proinflammatory and anti-inflam-
matory effects (14, 20, 26, 29, 31, 42).
In both experimental and human acute pancreatitis, prema-
ture cleavage of trypsinogen in pancreatic acinar cells liberates
the activated serine protease trypsin, leading to cellular damage
and inflammatory cell infiltration (13, 27, 29). Serine protease
inhibitors block trypsinogen activation and reduce the severity
of pancreatitis (8, 25, 34, 39). Genetic mutations in the cationic
trypsinogen gene or in the pancreatic secretory trypsin inhibitor
gene, both resulting in persistent tryptic activity (14), have
been identified in patients with hereditary pancreatitis. Little is
known about the role of trypsins in the pathogenesis of pan-
creatic inflammatory pain. Injection of a subinflammatory dose
of trypsin into the pancreatic duct increased expression of
c-Fos by spinal nociceptive neurons and caused mechanical
hyperalgesia via PAR
2
activation (16, 17). We hypothesized
that inhibitor-resistant isoforms of trypsin might produce an
augmented response. In the present study, we injected exoge-
nous trypsin II, trypsin IV, and P23 into the pancreatic duct of
rats pretreated or not with melagatran (MGT). MGT, originally
developed as a direct thrombin inhibitor, is also a potent
trypsin inhibitor (10, 11) and, as we show here, also acts as a
high-affinity inhibitor of polypeptide-inhibitor-resistant trypsin
isoforms. We then measured both pancreatic inflammation and
Address for reprint requests and other correspondence: K. S. Kirkwood,
UCSF, Dept. of Surgery, 521 Parnassus Ave., Rm. C-341, San Francisco, CA
94143-0660 (e-mail: kim.kirkwood@ucsfmedctr.org).
Am J Physiol Gastrointest Liver Physiol 300: G1033–G1042, 2011.
First published March 24, 2011; doi:10.1152/ajpgi.00305.2010.
0193-1857/11 Copyright
©
2011 the American Physiological Societyhttp://www.ajpgi.org G1033
Page 1
nociceptive signaling (46). To determine whether activation of
endogenous trypsinogen produces pancreatic inflammation and
pain via the release of trypsin isoforms, we injected ENK into
the pancreatic duct following pretreatment with trypsin inhib-
itors with different sensitivities to the various isoforms of
trypsin. Finally, to determine the contribution of inhibitor-
resistant trypsins to inflammation and pain, we induced acute
pancreatitis with supramaximal doses of cerulein in rats pre-
treated with MGT. We found that infusion of a subinflamma-
tory dose of trypsin caused pain whereas infusion of the same
dose of trypsin IV and P23 caused more robust pancreatic pain
and inflammation. These effects were blocked by pretreatment
with MGT. Premature activation of trypsin and its isoforms
induced by intraductal injection of ENK caused pancreatic
inflammation and pain. Among the trypsin inhibitors, the most
pronounced reduction in ENK-induced pancreatitis pain was
seen following pretreatment with MGT. Moreover, we also
confirmed that MGT blocked nocifensive behavior and noci-
ception induced by cerulein. Thus trypsin, including its minor
inhibitor-resistant isoforms, contributes to pancreatic pain, and
specific inhibitors of these isoforms could be novel therapies
for pancreatitis pain.
METHODS
Animals. Sprague-Dawley rats (male, 225–275 g; Charles River
Laboratories, Hollister, CA) were kept in a temperature-controlled
environment with 12:12-h light-dark cycle with free access to food
and water. All procedures performed were approved by the University
of California, San Francisco Institutional Animal Care and Use
Committee and in compliance with the “Guide for the Care and Use
of Laboratory Animals” (Institute of Laboratory Animal Resources,
National Academy of Sciences, Bethesda, MD).
Materials. Rabbit anti-c-Fos was from Chemicon (Temecula, CA)
and biotinylated goat anti-rabbit IgG was from Vector Laboratory
(Burlingame, CA). Porcine intestinal ENK and porcine pancreatic
trypsin II-S were from Sigma (St. Louis, MO), and recombinant
human trypsin IV and recombinant rat P23 have been described (22).
Soybean trypsin inhibitor type II-S (STI) and trypsin inhibitor type I-P
(TPI) were from Sigma, and MGT (10, 11), which has activity against
both thrombin and trypsins including trypsin IV, was from Astra-
Zeneca (Möldnal, Sweden).
In vitro inhibitor characterization. The inhibition constants (K
ic
)of
MGT and STI were studied for recombinant rat P23, recombinant
human trypsin IV and pancreatic trypsin. IC
50
values were determined
by using 150 M tosyl-GPR-pNA as substrate ([S]) in 100 mM
Tris·HCl, pH 8, 1 mM CaCl
2
at 25°C. For better comparisons, the IC
50
values were converted into binding affinity K
ic
values, via the Cheng-
Prusoff equation: K
ic
IC
50
/(1 [S]/K
m
) and the respective K
m
value of tosyl-GPR-pNA as described (22).
Intraductal infusion of proteases. Proteases were infused into the
pancreatic duct as described elsewhere (17), with several modifica-
tions. Rats were anesthetized with sodium pentobarbital (50 mg/kg ip)
and the abdomen was opened in the midline. The proximal common
bile duct was temporarily occluded with a hemoclip to prevent flow of
infused agents into the liver. The duodenum was punctured on the
antimesenteric side using a 30-gauge needle near the papilla of Vater
and a catheter (PE-10 tubing) that was inserted through the papilla
into the biliopancreatic duct. A 6-0 Prolene stitch secured the catheter
in the duct. ENK (100 U/kg), trypsin isoforms (trypsin II, trypsin IV,
p23; 0.01 mg/ml) or vehicle (0.1 M PBS or 0.9% NaCl, control) (all
250 l) were infused into the pancreatic duct over 10 min. The
hemoclip and the catheter were removed before the abdomen was
closed. Two and a half hours after intraductal infusion, rats were
killed with pentobarbital sodium (200 mg/kg ip) and tissues were
collected.
Cerulein-induced acute pancreatitis. Rats received hourly subcu-
taneous injections of supramaximal doses of cerulein (200
g·kg
1
·h
1
) or vehicle (0.9% NaCl) for 6 h, after which tissues were
collected (21, 22).
Administration of trypsin inhibitors. STI (8 mg/kg ip), TPI (7
mg/kg ip), or MGT (0.188 mg/kg ip) was administered 1 h before
infusion of P23 or trypsin IV or ENK into the pancreatic duct or
cerulein treatment.
Tissue collection. Blood was collected from the left ventricle,
centrifuged (10,000 g, 10 min, 4°C), and the serum was collected for
amylase activity. Rats were transcardially perfused with 100 ml of 0.1
M PBS. The body of the pancreas was snap frozen in liquid nitrogen
for assay of myeloperoxidase (MPO) activity, and the tail of the
pancreas was placed in 10% formalin (16 h, room temperature) for
histological analysis. Rats were then perfused with 300 ml of 3.7%
formaldehyde in PBS. Segments of thoracic spinal cord (T6, T8, T9,
T10, T12) were immersion-fixed in 3.7% paraformaldehyde for1hat
room temperature, cryoprotected by incubation in 30% sucrose (16 h,
4°C), and processed for localization of c-Fos by immunohistochem-
istry.
Serum amylase activity. Serum was assayed for amylase activity by
use of Infinity Amylase Liquid Stable Reagent (Thermo Electron,
Louisville, CO). Results are expressed as units per liter amylase.
MPO activity. Pancreatic tissue was assayed for MPO activity as an
index of granulocyte infiltration as previously described (4). Tissue
was thawed, homogenized in 20 mM phosphate buffer pH 7.4, and
centrifuged (10,000 g, 10 min, 4°C). Pellets were resuspended in 50
mM PBS pH 6.0 containing 0.5% hexadecyltrimethylammonium
bromide (Sigma). The suspensions underwent three cycles of freezing
and thawing, sonication for 60 s, and centrifugation (10,000 g, 20 min,
4°C). MPO activity in the supernatant was measured using the
substrate 3,3=,5,5=-tetramethylbenzidine dihydrochloride liquid sub-
strate system (Sigma). Absorbance was assessed at 650 nm, and
human neutrophil MPO (Calbiochem, San Diego, CA) was used to
generate a standard curve. Results are expressed as units of MPO
activity per milligram of proteins, measured with BCA protein assay
kit (Pierce Biotechnology, Rockford, IL).
TAP assays. Trypsin-associated peptide (TAP) is a stable NH
2
-
terminal hexapeptide (Val-Asp-Asp-Asp-Asp-Lys) that is liberated in
equimolar amounts to trypsin after cleavage of trypsinogen. TAP is
conserved among mammals (5) and is distinct from any other se-
quence within the trypsinogen or trypsin proteins, thereby allowing
for in situ assessment of trypsinogen activation (6). Pancreatic tissue
(200 mg) was placed in 1 ml of 0.2 M Tris·HCl, pH 7.3, containing
20 mM EDTA. Samples were boiled for 15 min, homogenized for 30
s, and centrifuged (10,000 g, 10 min, room temperature). Complete
protease inhibitor (Roche Biochemicals, Indianapolis, IN) was added
to the supernatant. TAP was measured by using an ELISA (Biotrin
International, Dublin, Ireland). Results are reported in nanomoles per
liter of TAP.
Pancreas HSS. Pancreatic samples were paraffin embedded, sec-
tioned (5 m), and stained with hematoxylin and eosin. Pathological
changes were evaluated by light microscopy (20 objective) as
described (18, 28, 44) by an investigator unaware of the experimental
groups. The following seven categories were assessed on a scale of
0 –5: macrolobular edema, microlobular edema, zymogen degranula-
tion, polymorphonuclear leukocyte (PMN) infiltration, PMNs in peri-
pancreatic fat, presence of vacuoles, and necrosis. The scores were
tabulated and the mean value of each group served as the histology
severity score (HSS).
Activation of spinal nociceptive neurons. To assess activation of
nociceptive spinal neurons, c-Fos immunoreactivity (IR) was local-
ized by immunohistochemistry (3, 4). Spinal sections (40–50 m)
were cut in the transverse plane by use of a sliding microtome and
were placed in 100 mM PBS, pH 7.4. Samples were incubated in PBS
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containing 3% normal goat serum (1 h, room temperature) and
incubated with c-Fos antibody (1:20,000, 16 h, room temperature).
Sections were washed and incubated with biotinylated goat anti-rabbit
antibody (1:200, 1 h, room temperature), followed by an avidin-
biotin-peroxidase complex (Vector Laboratories). Sections were
treated with 1% hydrogen peroxide. Slides were examined by light
microscopy (20 objective) by an investigator unaware of the exper-
imental groups. The number of Fos-stained nuclei in the superficial
laminae I and II of the spinal cord were counted in six sections per rat
from each spinal cord level, and mean counts were determined for
each rat.
VFF testing. Nocifensive behavior to mechanical stimuli was
measured as described elsewhere (48). Briefly, starting 2 days before
induction of pancreatitis, rats were acclimated for at least 1 h/day in
plastic cages with a mesh floor. The day before the testing, the
abdomen of rats was shaved and the baseline score was measured by
applying in ascending order calibrated von Frey filaments (VFFs;
North Coast Medical, Morgan Hill, CA) of different sizes to the
abdominal area 10 times each for 1–2 s, with at least a 10-s interval
between applications to avoid “wind-up” effects. A response was
considered positive when the rat raised or retracted its abdomen
(withdrawal response). On the experimental day, the baseline was
measured just before rats received hourly subcutaneous injections of
supramaximal doses of cerulein or saline for 6 h. After the last
injection, mechanical visceral nocifensive behavior was measured
with VFF testing. Data are expressed as number of withdrawal
responses per each filament per rat.
Statistical analysis. Data are expressed as means SE from n
6 rats per group (unless otherwise stated). Results were compared by
Student’s t-test (2 comparisons) or one-way ANOVA followed by
Dunnett (multiple comparison to 1 control) or Tukey (multiple com-
parisons) posttest comparisons. Behavioral mechanical hyperalgesia
results were analyzed by two-way ANOVA that accounts for treat-
ment and filament size comparison, whereas HSS (nonparametric
data) were analyzed by the Mann-Whitney test.
RESULTS
Effects of intraductal trypsins on pancreatitis and pancreatic
pain. Pancreatic intraductal injections of subinflammatory
doses of exogenous trypsin (0.01–1 mg/ml) have been reported
to activate spinal nociceptive neurons in a dose-dependent
manner, as shown by the increase in c-Fos expression, without
evidence of inflammation (16). Since we anticipated aug-
mented pain responses to inhibitor-resistant trypsin isoforms,
we examined effects of the lowest dose of trypsin (0.01 mg/ml)
reported to affect c-Fos expression. Trypsin II, trypsin IV, or
P23 (0.01 mg/ml) was infused into the pancreatic duct, and 2.5
h later inflammation was assessed by measurement of serum
amylase, pancreatic MPO, and HSS. Some rats were pretreated
1 h before with MGT (0.188 mg/kg ip), a trypsin inhibitor with
activity against polypeptide inhibitor-resistant trypsin iso-
forms. Infusion of trypsin II at the selected dose did not cause
pancreatic inflammation, as shown by the lack of effect on
serum amylase, pancreatic MPO, and HSS (Fig. 1, AC). In
contrast, trypsin IV and P23 caused pancreatic inflammation,
even at this low dose. In particular, P23 robustly increased
serum amylase activity (Fig. 1A), whereas trypsin IV increased
pancreatic MPO activity (Fig. 1B), and both these effects were
reduced by MGT pretreatment. Moreover, trypsin IV and P23
significantly enhanced HSS (Fig. 1, C and D). These inhibitor-
resistant isoforms of trypsin produced a pattern of pancreatic
inflammation and injury characterized by edema, marked ne-
crosis, and infiltration of neutrophils (Fig. 1D). Pretreatment
with MGT prior to trypsin IV altered some of the individual
end points assessed. Thus there was a decrease in interlobular
edema. However, this was offset by an increase in vacuoles in
pancreatic acinar cells, such that the overall HSS was un-
Fig. 1. Effects of intraductal injection of
trypsin isoforms on pancreatic inflammation.
Trypsin (Trp II), trypsin IV (Trp IV), P23
(0.01 mg/ml in 250 l of saline), or vehicle
(Veh; 250 l of saline) was injected into the
pancreatic duct. Some rats were pretreated
with melagatran (MGT; 0.188 mg/kg ip) 1 h
before the surgery. After 2.5 h, serum amy-
lase, pancreatic myeloperoxidase (MPO) ac-
tivity (A and B) and histology severity score
(HSS; C and D) were measured. Note that at
this subinflammatory dose, trypsin II did not
affect pancreatic inflammation; in contrast,
P23 increased amylase activity and human
trypsin IV increased MPO activity and these
effects were reduced by MGT. Both P23 and
trypsin IV significantly increased HSS. Ar-
rows signify inflammatory cells in edema-
tous interstitial tissue. *P 0.05 vs. Veh;
**P 0.005 vs. Veh; ***P 0.001 vs. Veh;
^P 0.05 vs. same treatment without MGT
pretreatment; ^^P 0.01 vs. same treatment
without MGT pretreatment; n 4–6.
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changed. Similarly, pretreatment with MGT prior to P23 de-
creased interlobular edema, but increased intralobular edema,
zymogen loss, neutrophil infiltration, and necrosis were appar-
ent. Thus the HSS obtained in this group was increased
compared with that seen in animals injected with P23 alone.
To examine whether injection of inhibitor-resistant trypsin
isoforms caused activation of pancreatic nociceptive pathways,
we quantified c-Fos IR in the superficial laminae I/II of the
spinal levels T6–T12. As expected, intraductal infusion of
trypsin II increased c-Fos IR in all pancreatic spinal levels,
with a significant effect in T9, compared with vehicle (Fig.
2A). The inhibitor-resistant isoforms trypsin IV and P23 had
more robust nociceptive effects than those of trypsin II, as
shown by the significant increase of c-Fos IR at all pancreatic
spinal levels in the superficial laminae of the dorsal horn of
T8 –T10 compared with vehicle (Fig. 2, A and B). These effects
were dramatically blocked by MGT pretreatment, suggesting a
major role of inhibitor-resistant trypsin isoforms in mediating
pancreatic pain. There were no effects on c-Fos IR in the spinal
neurons of T6 or T12, indicating that effects were confined to
the regions of the spinal cord that receive input from pancreatic
sensory neurons (Fig. 2A). Thus a “threshold dose” of trypsin
II was identified, at which activation of nociceptive pathways
is measurable but effects on inflammatory end points are not
significant. This same dose of inhibitor-resistant isoforms tryp-
sin IV and P23 caused pancreatitis and pain in this model,
suggesting that these trypsin isoforms could be important
mediators of protease-induced pancreatic inflammatory pain.
Effects of intraductal ENK on pancreatitis. To determine
whether a sufficient reservoir of activatable trypsinogens exists
in vivo to produce the effects we observed with intraductal
infusion of exogenous trypsins, we experimentally reproduced
the unregulated activation of pancreatic endogenous trypsins
by injecting ENK into the pancreatic duct. We measured serum
amylase and pancreatic MPO activity and assessed pancreatic
histology as indexes of pancreatitis. To confirm that the ob-
served effects were due to trypsin release, we measured pan-
creatic concentration of TAP, a trypsinogen cleavage by-
product and index of trypsin activation. Injection of ENK into
the pancreatic duct significantly increased TAP levels in the
pancreas compared with vehicle controls (Fig. 3A), thereby
confirming activation of pancreatic trypsinogens. In addition,
intraductal injection of ENK caused inflammation as shown by
increased serum amylase (Fig. 3B), pancreatic MPO activity
(Fig. 3C), and increased HSS (Fig. 3D). Histological findings
in ENK-treated tissues included edema, a paucity of zymogen
granules, polymorphonuclear leukocyte infiltration, mild ne-
crosis, and hemorrhage (Fig. 4D). The vehicle had no effect.
Thus administration of ENK into the pancreatic duct induces
activation of trypsinogens within the pancreas and also causes
inflammation.
To evaluate whether the proinflammatory effects of intra-
ductal ENK depend on activation of endogenous trypsinogen,
we determined whether inhibition of trypsins would ameliorate
ENK-induced pancreatitis. We used three different trypsin
inhibitors because of their differing sensitivities to trypsin
isoforms: STI, a reversible, semiselective trypsin inhibitory
peptide; TPI, a specific trypsin inhibitor; and MGT, a serine
protease inhibitor that also inhibits trypsin IV and P23 (see
Table 1). Administration of each inhibitor into the peritoneal
cavity 1 h before ENK infusion did not affect serum amylase
activity (Fig. 4A) but reduced pancreatic inflammation, as
shown by a marked decrease in MPO activity and HSS (Fig. 4,
BD). Both STI and TPI improved pancreatic histological
Fig. 2. Effects of intraductal injection of
trypsin isoforms on pancreatic spinal nocice-
ptive activation. Trypsin, trypsin IV, P23
(0.01 mg/ml in 250 l of saline), or vehicle
(250 l of saline) was injected into the pan-
creatic duct. Some rats were pretreated with
MGT (0.188 mg/kg ip) 1 h before the sur-
gery. After 2.5 h, c-Fos immunoreactivity
(IR) was localized in the superficial laminae
I/II of the dorsal horn of the spinal cord
(T6 –T12) and the number of c-Fos IR-posi-
tive nuclei per spinal section was determined.
Trypsin II produced c-Fos IR in segment T9,
whereas the inhibitor-resistant trypsin IV and
P23 robustly increased c-Fos IR in all pan-
creatic segments (T8 –T10). This effect was
completely reversed by MGT pretreatment.
In control mice, vehicle had no effect. The
c-Fos IR did not increase significantly in the
internal control spinal levels T6 or T12. *P
0.05 vs. Veh; ***P 0.001 vs. Veh; ^P
0.01 vs. same treatment without MGT pre-
treatment; ^^^P 0.001 vs. same treatment
without MGT pretreatment; n 4–6.
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injury scores compared with controls (Fig. 4D); in particular,
they decreased neutrophil infiltration in the tissue and necrosis
and vacuoles in the acinar cell. In contrast, MGT did not
improve the HSS. Instead, there were increased vacuoles in
acinar cells and increased necrosis present in the parenchyma.
However, this result was not statistically significant compared
with ENK treatment alone. Thus trypsin inhibitors reduce
ENK-induced pancreatitis, but with variable effectiveness.
These results indicate that ENK-induced pancreatitis is medi-
ated by intrapancreatic generation of trypsins.
To better understand the effect of the inhibitors used in these
experiments we also looked at their efficacy to inhibit various
purified trypsins in vitro. As a convenient readout, we deter-
mined their IC
50
values and converted these in K
ic
to be able to
compare the different enzymes. To mimic an immediate effect
in vivo, we did not extensively preincubate enzyme and inhib-
itor, but started the reaction by addition of the substrate within
5 min. The results can be found in Table 1.
Effects of intraductal ENK on pancreatic pain. To investi-
gate the effects of endogenous trypsins on spinal nociceptive
activation, we collected samples from spinal cord levels (T6
T12) 2.5 h after pancreatic intraductal injection of ENK and
assessed c-Fos IR in neurons of the superficial laminae I/II of
the dorsal horns. ENK injection caused a robust increase in
c-Fos IR, suggesting activation of pancreatic nociceptive path-
ways (Fig. 5A). This pronounced effect was reversed to differ-
ent extents by all trypsin inhibitors (Fig. 5B). STI diminished
c-Fos IR in pancreatic spinal levels (T8 –T10) to a greater
extent than TPI, whereas MGT was the most effective of all.
Thus ENK promoted premature activation of trypsinogen and
increased trypsin activity in the pancreas, thereby causing
pancreatic pain. Inhibition of trypsins, especially of the inhib-
itor-resistant trypsin isoforms, blocked spinal nociceptive ac-
tivation induced by intrapancreatic infusion of ENK.
Inhibition of inhibitor-resistant trypsin isoforms ameliorates
inflammation and pain in cerulein-induced pancreatitis. Since
injection of the inhibitor-resistant trypsin IV and P23 resulted
in pronounced pancreatic inflammation and activation of noci-
ceptive neurons, we evaluated their contribution to inflamma-
tory pain during experimental pancreatitis. To do so, we
assessed the effects of pretreatment with MGT, which inhibits
the serine proteases thrombin and trypsin II, and also the
inhibitor-resistant isoforms trypsin IV and P23, on acute pan-
creatitis and pancreatic pain induced by supramaximal doses of
the acinar cell secretagogue cerulein. As expected, cerulein
caused a marked increase in serum amylase and MPO activity
(Fig. 6, A and B) and a 5-fold increase in HSS (Fig. 6, C and
F). Histological characteristics of cerulein-induced pancreatitis
included changes in all of the end points examined, most
strikingly in intra- and interlobular edema, neutrophil infiltra-
tion, and vacuolization. MGT significantly reduced MPO ac-
tivity but did not ameliorate amylase activity or HSS. MGT
pretreatment did not alter vacuole formation in acinar cells.
However, decreased neutrophil infiltration occurred, and this
result is consistent with our finding of diminished MPO activ-
ity in the tissue. Cerulein treatment resulted in a twofold
increase in c-Fos IR in positive neurons in the dorsal horn of
the pancreatic spinal level T9, but not T6 or T12, indicative of
activation of pain pathways originating from the inflamed
pancreas and surrounding tissues (Fig. 6D). MGT robustly
decreased c-Fos IR (Fig. 6D). These results were corroborated
by behavioral experiments where nocifensive behavior was
measured by applying different sizes of calibrated VFFs to the
abdomen of rats. Once again, cerulein treatment resulted in a
robust nocifensive behavior, which was partially reversed by
pretreatment with MGT (Fig. 6E). Thus MGT strongly de-
creased c-Fos IR and nocifensive behavior, suggesting that
inhibitor-resistant isoforms of trypsin have a major role in
mediating pancreatitis pain.
DISCUSSION
Trypsin IR in the serum increases early in the course of acute
pancreatitis in humans (30), and urinary trypsinogen-2 levels
are now used to diagnose acute pancreatitis (1); however, the
role of trypsins in the pathogenesis of pancreatic inflammation
has been difficult to characterize because of the concomitant
secretion of trypsin inhibitors that rapidly degrade trypsin II,
the most ubiquitous species of trypsin. Trypsin has been
reported to mediate inflammatory pain in acute pancreatitis via
PAR
2
activation, but its role remains controversial (14, 20, 26,
29, 31, 42). The pancreas has abundant endogenous stores of
trypsin inhibitors (8), and trypsin inhibition is an important
protective mechanism against pancreatitis since cohorts of
patients with recurrent acute pancreatitis have been found to
have mutations in the serine protease inhibitor SPINK1 (2).
Our findings suggest that minor isoforms of trypsin that are
resistant to degradation by endogenous inhibitors could lead to
Fig. 3. Effects of intraductal injection of enterokinase (ENK) on pancreatic
inflammation. ENK (100 U/kg in 250 l of 0.1 M PBS) or vehicle (0.1 M PBS
250 l) was injected into the pancreatic duct of rats. After 2.5 h, trypsin-
associated peptide (TAP) concentration (A), serum amylase, pancreatic MPO
activity (B and C), and HSS (D) were measured. Intraductal ENK increased
TAP concentration (A) and index of trypsinogen cleavage and caused pancre-
atic inflammation (BD). *P 0.05 vs. Veh; **P 0.005 vs. Veh; n 6 –7.
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sustained protease signaling and thereby promote pancreatic
inflammation and pain.
The pancreas has abundant endogenous stores of trypsin
inhibitors (8), but inhibitor-resistant isoforms of trypsin,
namely trypsin IV and P23, have been identified, whose role in
pancreatitis-induced inflammatory pain has not been investi-
gated.
In this study, we investigated for the first time the contribu-
tion of trypsin IV and P23 on pancreatic nociception. We show
the important role of human trypsin IV and rat P23 in causing
pancreatic inflammation and nociception. First, we observed
that intraductal injection of a subinflammatory dose of exoge-
nous trypsin II caused nociception with no evidence of inflam-
mation, in accord with a previous study (16). Second, we
injected the same subinflammatory dose of the proteinaceous
inhibitor-resistant trypsin IV and rat P23 and observed that
they caused pancreatic inflammation and robust nociception,
which were blocked by MGT pretreatment, the only serine
protease inhibitor that we show binds with affinity to trypsin IV
and P23, as well as trypsin (Table 1). Third, we investigated
the contribution of endogenous trypsin isoforms by pancreatic
intraductal injection of ENK after pretreatment with trypsin
inhibitors (STI, TPI, and MGT) with differing specificities to
trypsin isoforms. Finally, we showed that pretreatment with
MGT significantly decreased nociception and mechanical vis-
ceral hyperalgesia induced by acute pancreatitis, suggesting the
importance of trypsin IV and rat P23 during pathological
conditions. The present study supports the postulate that pre-
Fig. 4. Effects of trypsin inhibitors on ENK-in-
duced pancreatic inflammation. Trypsins were in-
hibited with either soybean trypsin inhibitor type
II-S (STI; 8 mg/kg ip) or trypsin inhibitor type I-P
(TPI; 7 mg/kg ip) or MGT (0.188 mg/kg ip) 1 h
before induction of pancreatic inflammation by
ENK intraductal injection (100 U/kg in 250 lof
0.1 M PBS). After 2.5 h, serum amylase, pancreatic
MPO activity (A and B) and HSS (C and D) were
measured. Intraductal ENK caused neutrophil infil-
tration (arrows) and hemorrhage (arrowheads) as
well as edema and mild tissue necrosis. All trypsin
inhibitors reduced MPO (B). **P 0.005 vs. Veh;
***P 0.001 vs. Veh; n 6.
Table 1. K
ic
of trypsin inhibitors MGT and STI against
recombinant rat P23, recombinant human trypsin IV, and
pancreatic trypsin
Inhibitor
Recombinant Rat
P23 K
ic
,nM
Recombinant Human
Trypsin IV K
ic
,nM
Human Pancreatic
Trypsin K
ic
,nM
MGT 17 7.3 4.5
STI 230 1,700 3.7
K
ic
, inhibition constant; MGT, melagatran; STI, soybean trypsin inhibitor.
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mature trypsin activation plays a major role in the pathogenesis
of inflammation and pain during acute pancreatitis. Our find-
ings are an important extension of previous work that suggests
the major contribution of proteinaceous inhibitor-resistant tryp-
sin isoforms in mediating pancreatic nociception and inflam-
mation (43). Given the pharmacological profile of MGT, which
also inhibits thrombin and plasmin together with trypsins (10,
11), we cannot rule out the possibility that our results may be
partially due to its effect on inhibiting thrombin-induced acti-
vation of PAR
1
and PAR
4
. Further investigation is needed to
clarify this possibility. However, all of the trypsin inhibitors
used in this study can inhibit thrombin and other serine pro-
teases (24, 37). Thus we took advantage of their different
selectivities to assess the relative role of various proteases.
Premature intracellular activation of trypsinogen in the pan-
creas leads to acinar cell injury accompanied by necrosis (9,
32). This unregulated trypsin activation is believed to occur
when intracellular protective mechanisms that function to pre-
vent trypsinogen activation or reduce trypsin activity are over-
whelmed (45). Available trypsin then triggers a localized
inflammatory response mediated by proinflammatory cyto-
kines, platelet-activating factor, and substance P. Some of
these inflammatory mediators, including prostanoids, brady-
kinin, tachykinins, serotonin, and other biological factors, are
considered noxious substances that can activate or sensitize
nociceptors (16). Trypsin has been demonstrated to play an
important role in mediating not just the inflammatory aspects
of pancreatitis, but also nociception, via PAR
2
activation on
peripheral nociceptive sensory nerve endings leading to the
central release of substance P and calcitonin gene-related
peptide in the spinal cord (16, 17). The resulting excitation of
second-order neurons in the dorsal horn activates ascending
pathways to the brain and leads to increased afferent signaling,
central sensitization, and potential amplification and persis-
tence of pain. The isoforms of trypsin that mediate these effects
are unknown. We hypothesized that this pathway involves not
only trypsin II, but also its proteinaceous inhibitor-resistant
isoforms, trypsin IV and P23.
Human trypsinogen IV is a splice variant of mesotrypsino-
gen (47). The NH
2
-terminal sequences of trypsinogen IV and
mesotrypsinogen derive from different exons (38, 43, 47).
However, when these zymogens are cleaved by ENK, the
active COOH-terminal products, trypsin IV and mesotrypsin,
are identical. In the rat, P23 is the corresponding protein and
represents a minor trypsinogen isoform within the pancreas (7,
40). The main characteristics of these minor isoforms are not
only the resistance to endogenous polypeptide inhibitors, but
also the ability to degrade and inactivate such inhibitors,
endowed by the evolutionary selection of Arg
198
(43). The
physiological and pathological functions of inhibitor-resistant
isoforms are still largely unknown. It has been suggested that
inappropriate activation of human trypsinogen IV/mesot-
Fig. 5. Effects of intraductal injection of ENK,
with or without pretreatment with trypsin inhib-
itors, on pancreatic spinal nociceptive activation.
ENK (100 U/kg in 250 l of 0.1 M PBS) or
vehicle (0.1 M PBS 250 l) was injected into the
pancreatic duct of rats. In 3 experimental groups,
trypsins were inhibited with either STI (8 mg/kg
ip), TPI (7 mg/kg ip), or MGT (0.188 mg/kg ip)
1 h before induction of pancreatic inflammation
by ENK intraductal injection (100 U/kg in 250 l
of 0.1 M PBS). After 2.5 h, c-Fos IR was local-
ized in the dorsal horn of the spinal cord (T6
T12) and the number of c-Fos IR-positive nuclei
per spinal section was determined. ENK, but not
its vehicle, produced a robust increase in c-Fos
IR (A) in the superficial laminae I/II of the spinal
cord that was diminished by all trypsin inhibi-
tors. MGT, which is more selective for the in-
hibitor-resistant trypsin isoforms, was the most
effective in blocking spinal nociceptive activa-
tion. ^^P 0.005 vs. Veh; ^^^P 0.001; vs.
Veh; ***P 0.001 vs. ENK; n 6.
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rypsinogen in the pancreas could degrade available trypsin
inhibitor, thereby reducing its protective effect and promoting
pancreatitis (43). The lysosomal protease cathepsin B activates
trypsinogen IV at a higher rate than human cationic or anionic
trypsinogens during pancreatitis (43). Finally, in rats with acute
pancreatitis induced by the secretagogue cerulein, P23 is up-
regulated 14-fold, which represents the largest incremental
change of any protein in the rat exocrine pancreas (7).
Our findings, that intrapancreatic infusion of the protein-
aceous trypsin inhibitor-resistant isoforms trypsin IV and rat
Fig. 6. Effects of MGT on cerulein-induced acute pancreatitis and spinal nociceptive activation. MGT was injected (0.188 mg/kg ip) 1 h before pancreatic
inflammation and pain were induced by cerulein (200 g·kg
1
·h
1
sc; 6 h). After 6 h, serum amylase and pancreatic MPO activity (A and B) and HSS (C and
F) were measured; c-Fos IR (D) was localized in the dorsal horn of the spinal cord (T6–T9 –T12) and the number of c-Fos IR-positive nuclei per spinal section
was determined. In behavioral experiments, after 6 h rats were tested for nocifensive behavior by probing their abdomen with von Frey filaments (VFFs; E). MGT
did not affect amylase activity or HSS but significantly decreased MPO activity in pancreas (B). MGT also robustly diminished spinal c-Fos activation in T9
(D). There were no significant changes in c-Fos IR in T6 or T12. Finally, MGT strongly ameliorated nocifensive behavior (E). n 4–6; **P 0.01 vs. Veh;
***P 0.001 vs. Veh. ^P 0.05 vs. Cer; ^^^P 0.001 vs. Cer.
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P23 caused robust nociception and inflammation, which were
blocked by pretreatment with MGT, support our previous work
in which we found that trypsin IV and P23 induced inflamma-
tion and hyperalgesia by cleaving PAR
2
at lower doses than
pancreatic trypsin (22). The magnitude of spinal nociception
activation and pancreatic inflammation induced by trypsin IV
and P23 in the present study was larger than that seen with
trypsin II, which did not cause inflammation at the selected
dose. It is possible that resistance to degradation may potenti-
ate their effect by prolonged signaling. In addition, trypsin IV
and P23 may stimulate the generation of other proteases that
can activate PARs, thereby amplifying the inflammatory and
nociceptive responses. Additional studies are required to in-
vestigate these possibilities.
The results of our experiments using ENK injection into the
pancreatic duct support the importance of release of pancreatic
trypsins in both inflammation and pain in the pancreas. ENK
caused release of activated trypsin as reflected by pancreatic
TAP levels, and the resulting inflammatory pain was due to
trypsin species since both inflammation and pain were inhib-
ited by trypsin inhibitors. MGT pretreatment, which blocks
trypsin IV and P23 activity as well as that of trypsin II, had a
more profound inhibitory effect on pain than did either of the
trypsin II inhibitors STI or TPI, suggesting that the difference
may be due to inhibition of the proteinaceous trypsin inhibitor-
resistant isoforms in the pancreas.
Finally, we evaluated the effects of pretreatment with MGT
on acute pancreatitis induced by supramaximal doses of the
acinar cell secretagogue cerulein. This model of acute pancre-
atitis has largely been used for investigating the involvement of
trypsin in pain and inflammation (12, 15, 19, 20, 23, 29).
Cerulein treatment caused pancreatitis, as shown by the in-
crease in serum amylase and MPO activity and HSS; pancre-
atic spinal nociception activation, as confirmed by c-Fos IR
increase; and behavioral visceral hyperalgesia, as measured
with VFFs. MGT pretreatment reduced MPO activity, but had
no effect on serum amylase activity and pancreas morphology.
MGT strikingly reduced spinal nociceptive activation, by de-
creasing c-Fos IR, and nocifensive behavior, by decreasing
withdrawal responses to VFFs. Thus MGT ameliorated pan-
creatitis, and especially pancreatic pain, suggesting that inhib-
itor-resistant isoforms of trypsin play a major role in mediating
inflammatory pain. These findings support other reports that
suggest the importance of trypsin IV/mesotrypsin and P23 in
the pathogenesis of acute pancreatitis (7, 43).
The effect of MGT on pancreas histology was similar to that
reported in a rat model of endotoxemia in which MGT pre-
treatment, at higher doses than the one we used in our study,
improved liver and kidney function but not histology (33).
Additional studies are required to determine the exact mecha-
nism by which MGT improved pancreatic inflammation and
pain, but not histological damage score. It is possible that
MGT-induced inhibition of thrombin could have resulted in
PAR-mediated endothelial dysfunction. In conclusion, endog-
enous trypsins, which are prematurely activated in the early
course of acute pancreatitis, play an important role in promot-
ing pancreatic inflammatory pain. In particular, inhibitor-resis-
tant isoforms of trypsin, human trypsin IV and rat P23, may be
important in mediating prolonged pancreatic inflammatory
pain in pancreatitis.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
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    • "These agents included formalin , which can activate the TRPA1 ion channel on sensory nerves (McNamara et al., 2007), and bradykinin and a PAR2- selective activating peptide (PAR2-AP), which can activate GPCRs on sensory nerves (Vergnolle et al., 2001). We found that formalin, bradykinin and PAR2-AP activated proteases in paw tissues that were inhibited by the serine protease inhibitor , melagatran (Gustafsson et al., 1998), but not by soybean trypsin inhibitor (SBTI), consistent with activation of trypsin IV-like protease (Ceppa et al., 2011). In common with human trypsin IV, mouse trypsin 4 was inhibited by melagatran, degraded SBTI, and activated PAR2. "
    [Show abstract] [Hide abstract] ABSTRACT: Background and purpose: Although serine proteases and agonists of protease-activated receptor 2 (PAR2) cause inflammation and pain, the spectrum of proteases that are activated by proinflammatory and algesic stimuli and their contribution to inflammatory pain are uncertain. Experimental approach: Enzymic assays and selective inhibitors were used to characterize protease activity in mice after intraplantar injections of formalin, bradykinin, PAR2 activating peptide (AP) or vehicle. The capacity of these proteases and of recombinant mouse trypsin 4 to cleave fragments of PAR2 and to activate PAR2 in cell lines was determined. Protease inhibitors and par2 (-/-) mice were used to assess the contributions of proteases and PAR2 to pain and inflammation. Key results: Intraplantar injection of formalin, bradykinin or PAR2-AP led to the activation of proteases that were susceptible to the serine protease inhibitor melagatran but resistant to soybean trypsin inhibitor (SBTI). Melagatran inhibited mouse trypsin 4, which degraded SBTI. Proteases generated in inflamed tissues cleaved PAR2-derived peptides. These proteases and trypsin 4 increased [Ca(2+) ]i in PAR2-transfected but not in untransfected cells, and melagatran suppressed this activity. Melagatran or PAR2 deletion suppressed oedema and mechanical hypersensitivity induced by intraplantar formalin, bradykinin and PAR2-AP, but had no effect on capsaicin-induced pain. Conclusions and implications: Diverse proinflammatory and algesic agents activate melagatran-sensitive serine proteases that cause inflammation and pain by a PAR2-mediated mechanism. By inducing self-activating proteases, PAR2 amplifies and sustains inflammation and pain. Serine protease inhibitors can attenuate the inflammatory and algesic effects of diverse stimuli, representing a useful therapeutic strategy.
    No preview · Article · Apr 2014 · British Journal of Pharmacology
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    • "SP activity deregulation is generally due to failures of the signaling system and leads to severe disorders which must be therefore treated by supplementing the patients with the defective protease or specific inhibitor123. Consequently, proteases have emerged as promising targets for drug discovery for a wide variety of human diseases, including cancer, neurodegeneration, ischemic diseases, inflammation and infectious diseases456 . Very wellknown examples of therapeutic application of SPs are the intravenous administration of tissue plasminogen activator (t-PA) to improve clinical outcomes in patients with acute ischemic stroke [7] , and intravenous infusion of recombinant Factor IX (FIX) for the prophylaxis and treatment of hemophilia B patients [8]. "
    [Show abstract] [Hide abstract] ABSTRACT: Anomalous protease activities are associated with many diseases. Great efforts are paid for selecting specific protease modulators for therapeutic approaches. We have selected new modulators of enzyme activity by an homogeneous assay based on a doubly labeled small peptide used as substrate of trypsin. The substrate incorporates the fluorophore 5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid (EDANS) at one end and an EDANS-quenching moiety (Dabcyl, (4-(4-dimethylaminophenylazo)-benzoic acid)) on the other end. Following cleavage by trypsin, the peptide-EDANS product is released interrupting the fluorescence resonance energy transfer effect and yielding bright fluorescence, which can be detected using excitation wavelengths at 335-345 nm and emission wavelengths at 485-510 nm. The method optimized, tested by detecting the strong inhibiting effect of α1-antitrypsin on trypsin activity, has been developed on 384 multi-well plates in a volume of 10 μL, using an automated platform. From the screening of a chemical library, four compounds that inhibit trypsin activity with IC(50)s in the micromolar range have been identified. Interestingly, the most active compound (M4) shows a chemical structure recapitulating that of other more potent inhibitors with thiourea and halogenated centers. Molecular docking studies show that M4 is a competitive inhibitor recognizing most residues within or nearby the catalytic pocket.
    Full-text · Article · Jun 2012 · Molecular Biotechnology
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  • [Show abstract] [Hide abstract] ABSTRACT: Bile acids are the initiating factors of biliary acute pancreatitis. Bile acids can induce the activation of intracellular zymogen, thus leading injury in pancreatic acinar cells. Pathological zymogen activation in pancreatic acinar cells is a common feature of all types of acute pancreatitis. The proteins expressed in pancreatic acinar cells during the activation of zymogen may determine the severity of acute pancreatitis. The present study aims to determine the differentially expressed proteins in taurolithocholic acid 3-sulfate-stimulated pancreatic acinar cells as an in vitro model for acute pancreatitis. Rat pancreatic acinar AR42J cells were treated with taurolithocholic acid 3-sulfate for 20 min. Laser confocal scanning microscopy and flow cytometry were used to detect activated trypsinogen in pancreatic acinar AR42J cells. After the determination of trypsinogen activation, proteome analysis was performed to identify the proteins differentially expressed in taurolithocholic acid 3-sulfate-treated cells and non-treated cells. After treatment with taurolithocholic acid 3-sulfate for 20 min, the activation of trypsinogen in AR42J cells was concurrent with changes in the protein expression profile. Thirty-nine differentially expressed proteins were detected; among these, 23 proteins were up-regulated and 16 proteins were down-regulated. KEGG analysis indicated that these proteins are involved in cellular metabolic pathways, cellular defensive mechanisms, intracellular calcium regulation and cytoskeletal changes. The expression of proteins in the pancreatic acinar cell changes at the early stage of biliary acute pancreatitis. These differentially expressed proteins will provide valuable information to understand the pathophysiologic mechanism biliary acute pancreatitis and may be useful for prognostic indices of acute pancreatitis.
    No preview · Article · May 2012 · Pancreatology
    Zhituo Li Zhituo Li Ming Lu Ming Lu Jiangtao Chu Jiangtao Chu +4 more authors... Xin Qiao Xin Qiao
    0Comments 5Citations
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