From the Medizinische Klinik B and
§ Institut für Sportmedizin, Westfälische
Wilhelms-Universität, 48129 Münster, Germany,
Cell
Communication Group, Department of Applied Biology, University
of Central Lancashire, Preston PR12HE, United Kingdom, and
** Division of Medical Biology, Department of Pathology,
University of Rostock, 18057 Rostock, Germany
Received for publication, July 24, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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Intracellular
Ca2+-changes not only participate in important
signaling pathways but have also been implicated in a number of disease
states including acute pancreatitis. To investigate the underlying
mechanisms in an experimental model mimicking human gallstone-induced
pancreatitis, we ligated the pancreatic duct of Sprague-Dawley rats and
NMRI mice for up to 6 h and studied intrapancreatic changes
including the dynamics of [Ca2+]i in isolated
acini. In contrast to bile duct ligation, pancreatic duct obstruction
induced intra-pancreatic trypsinogen activation, leukocytosis,
hyperamylasemia, and pancreatic edema and increased lung
myeloperoxidase activity. Although resting [Ca2+]i in isolated acini rose by 45% to
205 ± 7 nmol, the acetylcholine- and cholecystokinin
(CCK)-stimulated calcium peaks as well as the amylase
secretion declined, but neither the
[Ca2+]i-signaling pattern nor the amylase output
in response to the Ca2+-ATPase inhibitor thapsigargin nor
the secretin-stimulated amylase release were impaired by pancreatic
duct ligation. On the single cell level pancreatic duct ligation
reduced the percentage of cells in which submaximal secretagogue
stimulation was followed by a physiological response (i.e.
Ca2+ oscillations) and increased the percentage of cells
with a pathological response (i.e. peak plateau or absent
Ca2+ signal). Moreover, it reduced the frequency and
amplitude of Ca2+ oscillation as well as the capacitative
Ca2+ influx in response to secretagogue stimulation. Serum
pancreatic enzyme elevation as well as trypsinogen activation was
significantly reduced by pretreatment of animals with the calcium
chelator BAPTA-AM. These experiments suggest that pancreatic duct
obstruction rapidly changes the physiological response of the exocrine
pancreas to a Ca2+-signaling pattern that has been
associated with premature digestive enzyme activation and the onset of
pancreatitis, both of which can be prevented by administration of an
intracellular calcium chelator.
Alterations in intracellular calcium signaling have previously
been reported from an experimental animal model of acute pancreatitis that employs supramaximal secretagogue stimulation for the disease induction (1). Moreover, the spatial and temporal distribution of
intracellular calcium signals in response to either physiological or
pathological stimuli has been found to be directly related to premature
digestive enzyme activation in the pancreas and to acinar cell injury
(2-5). Intracellular activation of trypsinogen on the other hand can
be completely prevented with agents that interfere with either the
uptake of calcium, the maintenance of a calcium gradient across the
plasma membrane, or the rapid release of calcium from apical
intracellular stores (4, 6). All of these studies indicate clearly that
the spatial and temporal distribution of intracellular calcium signals
plays a critical role in the early cellular events that precede the
onset of pancreatitis. The mechanism, however, that was used to induce
acinar cell injury in these studies or in response to which
intracellular calcium changes were characterized, supramaximal
secretagogue stimulation, is not necessarily an event that is generally
involved in the pathogenesis of clinical pancreatitis.
In many parts of the world the most common etiological factor
associated with acute pancreatitis is gallstone disease. Experimental (7) as well as clinical studies (8, 9) suggest that the onset of
gallstone-induced pancreatitis requires migration of the offending
stone through the biliary tract and its impaction at the duodenal
papilla. Here, at the junction of the common bile duct and the
pancreatic duct, the stone can impair the flow of pancreatic secretion
or lead to a complete blockage of the pancreatic duct. It is now
generally accepted that this impairment of pancreatic secretion (7, 10,
11), rather than a potential reflux of bile into the pancreas (13),
represents the critical pathophysiological event for the development
gallstone-induced pancreatitis. To investigate whether this clinically
relevant mechanism for the onset of pancreatitis is, in analogy to the
secretagogue-induced animal models, associated with intracellular
calcium changes and premature protease activation, we ligated the
pancreatic duct of laboratory animals for up to 6 h. The results
from our experiments indicate that pancreatic duct obstruction is
indeed followed by rapid changes in the response of acinar cells to
secretory stimuli, leads to complex pathological alterations in the
intracellular Ca2+-signaling pattern, and induces premature
digestive enzyme activation. In vivo administration of an
intracellular Ca2+ chelator on the other hand can largely
prevent hyperamylasemia, hyperlipasemia, and intrapancreatic
trypsinogen activation.
Materials--
Fura-2-acetoxymethyl ester (AM) was from
Molecular Probes (Eugene, Oregon). Cell-Tak was obtained from
Collaborative Research Inc., Bedford, MA. Rhodamine-labeled phalloidin
was obtained from Molecular Probes (Junction City, OR). All other
reagents were of the highest purity available and were purchased from
Sigma unless indicated otherwise.
Surgical Procedure--
All animal experiments were approved by
and conducted under the guidelines of the Animal Use and Welfare
Committee of the University of Münster. Male Sprague-Dawley rats
weighing between 250 and 350 g and adult male white NMRI mice
weighing between 30 and 35 g (both species from Charles River,
Sulzfeld, Germany) were kept in Nalgene shoebox cages in a 12-h/12-h
light/dark cycle with unlimited access to standard chow and water. All
animals were adjusted to laboratory conditions over the course of 1 week before the experiments. After a 12-h fast with free access to drinking water and after anesthesia with sodium pentobarbital (72 mg/kg
of body weight), the abdomen was opened, and the pancreas was exposed.
Above the head of the pancreas the common bile duct was ligated in all
animals (bile duct controls and treatment group), and subsequently, the
main pancreatic duct was ligated without affecting the splenic vessels
in the treatment group. In addition to sham-operated controls for most
experiments (laparotomy only) a bile duct-ligated control group was
used for several reasons as follows. (a) Bile duct ligation
alone is known to raise circulating cholecystokinin levels in the blood
and could, by itself, exert a stimulatory effect on the pancreas (14).
(b) It has been suggested that bile duct obstruction could
be a confounding or aggravating factor for the events involved
pancreatic duct obstruction-induced pancreatitis (15, 16).
(c) Ligation of the pancreatic duct system in mice without
compromising the unimpaired flow of bile is technically more prone to
surgical complications than ligation of both ducts and the latter is a
more likely to reflect the situation that arises when a gallstone is
impacted at the papilla. At time intervals of up to 6 h the
anesthetized animals were killed by cervical dislocation, venous blood
was collected from the right ventricle, and the pancreas was rapidly
removed for either morphology, enzyme activity measurements, or fresh
acinar cell isolation. In a series of experiments that were performed
to modify the pathological calcium-signaling pattern in pancreatic
acinar cells that was associated with pancreatic duct ligation we used
the calcium chelator BAPTA-AM in in vivo experiments.
BAPTA-AM was administered intraperitoneally in mice (10 µg/kg of body
weight in 180 µl of propylene glycol as a solvent) by 2 single
injections at 30 min before the operation and at the time of
laparotomy. Controls received only propylene glycol vehicle
intraperitoneally. Serum activities of amylase and lipase as well as
trypsinogen activation peptide in the pancreas were measured 4 h
after pancreatic duct ligation. After sacrifice pancreatic tissue was
either fixed for morphological studies, snap-frozen for the assays
outlined below, or desiccated for 12 h at 160 °C to determine
the dry/wet weight ration as an indicator of pancreatic edema formation.
Cell Isolation Procedures--
The reason for using rats as well
as mice for our studies was 2-fold. One reason was to test our
hypotheses in different species of laboratory animals, and the second
was the observation that rat pancreatic acini preparations permit a
greater yield, whereas mouse pancreatic acinar cells are better suited
for single cell microfluorometry. In the following we briefly outline
the somewhat different protocols for acinar cell preparations from
mouse and rat pancreas. The rat pancreas was immediately transferred to iced medium (preoxygenated for 10 min) composed of modified
Krebs-Ringer-HEPES buffer containing NaCl (130 mM), KCl (5 mM), HEPES (20 mM),
KH2PO4 (1.2 mM), MgSO4
(2 mM), glucose (10 mM), CaCl2 (1 mM), soybean trypsin inhibitor (0.1 mg/ml), bovine serum
albumin, 0.2% (w/v) at pH 7.4. The tissue was rapidly minced with
scissors, and acinar cells were dissociated by incubation in
calcium-free medium containing collagenase type V (268 units/ml) for 25 min at 37 °C in a shaking water bath (17, 18). The collagenase
solution was discarded, and the cell suspension was washed 3 times with
calcium-free medium, filtered through a nylon mesh, and centrifuged
with medium containing 5% bovine serum albumin. Finally the cell
suspension was centrifuged and resuspended in Krebs-Ringer-HEPES medium.
For single cell studies mouse pancreas was rapidly removed, minced into
small pieces on ice, and placed into buffer (pH 7.4) containing NaCl
(130 mM), KCl (5 mM), HEPES (10 mM), KH2PO4 (1.2 mM),
CaCl2 (1 mM), MgSO4 (1 mM), glucose (10 mM), and collagenase (100 units/ml Type V). After 10 min of incubation at 37 °C under continuous shaking (120 cycles/min) the digested tissue was washed 3 times in 10 ml of Krebs-Ringer-HEPES buffer without collagenase and
again shaken 10 times to dissociate acini. Acini were then filtered
through muslin gauze, centrifuged at 400 rpm for 3 min, and washed
twice more in buffer containing 4% bovine serum albumin. After the
second wash, acini were suspended in 8 ml of Krebs-Ringer-HEPES buffer
containing soybean trypsin inhibitor (0.1 mg/ml) and bovine serum
albumin (0.2% w/v). A stock suspension of acini was kept on ice for up
to 4 h without significant reduction in cell viability (>95%) as
assessed by trypan blue exclusion.
Enzyme Activity and Activation Measurements--
Pancreatic
acini were transferred to 5-ml vials with aliquots of
Krebs-Ringer-HEPES medium and incubated with secretagogue at 37 °C
and constantly agitated for 5 min. After centrifugation at 50 × g for 5 min, amylase activity was measured in the
supernatant by on-line fluorometry using amylopectin anthranilate as a
substrate (19, 20). Amylase measurements were expressed as units/ml supernatant/100 mg of acinar cells. The values obtained were calibrated against an Acinar Cell Morphology--
After removal of the pancreas of
sham operated, bile duct-ligated and pancreatic duct-ligated animals
small tissue sections of 2 mm were immediately fixed in an iced
solution of 2% formaldehyde, 2% glutaraldehyde, embedded in Epon,
post-fixed, and contrasted with osmium, uranyl, and lead. Semithin
section were studied by light microscopy and thin sections evaluated on
a Philips EM10 electron microscope as previously reported (24). To
study the acinar cell microfilament network after pancreatic duct
ligation, tissue was snap-frozen in liquid nitrogen, and 5-µm
cryosections were placed on poly-L-lysine-coated
coverslips, fixed with 4% formaldehyde in PBS, and incubated with
rhodamine-labeled phalloidin for 20 min in the dark. The micrographs
shown in Fig. 3 were taken by electron microscopy or
fluorescence microscopy and are representative for four or more
individual experiments.
Measurements of [Ca2+]i--
Intracellular
Ca2+ concentrations were determined using the
calcium-sensitive fluorescent dye fura-2. For acinar cell suspension experiments rat acinar cells were loaded with 1 µM
fura-2/AM for 25 min at room temperature. After washing the cells were
incubated for another 20 min in Krebs-Ringer-HEPES medium to permit
complete de-esterification of the probe. Measurements were performed in a PerkinElmer Life Sciences LS-50B spectrofluorimeter using excitation wavelengths of 340 and 380 nm, and emitted light was collected at 510 nm. After each experiment the signals were calibrated according to
Grynkiewicz et al. (25) by incubating cells in either
calcium-free medium (1 mM EGTA) or calcium-containing
buffer in the presence of 100 µM digitonin for complete permeabilization.
For microfluorometric studies of [Ca2+]i dynamics
mouse acini were loaded with the AM-ester or fura-2 (5 µM) at room temperature for 20 min. Fura-2-loaded cells
were plated onto glass coverslips coated with Cell-Tak (1 µl/cm2) and kept at room temperature for 30 min before
they were mounted in a perfusion chamber with an internal volume of
0.28 ml and placed on the stage of a Nikon Diaphot-TMD inverted
microscope equipped with a Fluor ×100 oil immersion objective.
Acini were continuously perfused with medium at a flow rate of 3 ml/min
at room temperature. Fluorochrome excitation (340 and 380 nm for fura-2) was achieved using a xenon lamp in series with two
chopper-linked monochromators that were coupled to the microscope via
fiber optics. Emitted light was collected behind a band-pass filter
(509 nm for fura-2) by a photomultiplier. In unloaded controls
background fluorescence was found to be negligible.
Ratios obtained from the dual excitation wavelengths probe fura-2 were
converted to calcium concentrations as previously reported (25, 26)
using the formula [Ca2+]i = (R Statistical Analysis--
Data shown in the figures are
representative of six or more individual experiments in each group and
indicate the means ± S.D. or S.E. Differences between groups were
compared using Student's t test with Bonferroni correction
where more than two groups were compared. Asterisks in the
figures indicate statistically significant differences when the
p values were <0.05.
Acinar Cell Function and Acinar Cell Damage--
In the rat bile
duct ligation alone had no effect on either serum amylase activity,
acinar cell damage, or the basal and secretagogue-stimulated amylase
secretion (Fig. 1, A and
B). Over the 6-h course of duct ligation in rats no
difference between the basal amylase release from acini of bile
duct-ligated (16.47 ± 3.20 units/ml/100 mg; n = 23) and pancreatic duct-ligated animals (14.34 ± 3.01 units/ml/100 mg; n = 19) was found. In contrast, the
acetylcholine (Ach; 10
When we characterized pancreatic duct ligation-induced pancreatic
damage in the mouse, serum amylase activity rose just as significantly
as in the rat model (Fig. 2A).
Moreover, a premature and intrapancreatic activation of trypsinogen
could be demonstrated by increased TAP levels in pancreatic tissue and
the urine of pancreatic duct-ligated mice at 4 h (Fig. 2,
B and C). On ultrastructural morphology bile duct
ligation alone had no effect on either pancreatic duct cell (Fig.
3A) or exocrine cell
morphology (Fig. 3B). The latter preserved their
characteristic structure with zymogen granules grouped at the apical
pole and around the lumen. After 4 h of combined pancreatic and
bile duct ligation the ducts were found to be largely distended
(arrow in Fig. 3C), and some duct-lining cells
had damaged cell membranes facing the lumen. Acinar cells on the other
hand had not undergone any necrotic changes at this time; their lumen
was only marginally distended (asterisk in Fig. 3C), and the most prominent intracellular change was a
dilatation of the endoplasmic reticulum (Fig. 3D). To study
the actin microfilament network, which is known to be critically
involved in fusion/fission events of zymogen granule exocytosis, we
used phalloidin-labeled cryosections. In untreated control animals
(Fig. 3E) or animals after bile duct ligation (Fig.
3F) F-actin labeling was found at the terminal microfilament
web surrounding the acinar lumen (asterisks) as well as
along the lateral and basal membrane (arrows). After
pancreatic duct ligation (Fig. 3G) F-actin labeling remained unchanged at the apical locations, whereas it was redistributed from
the basal acinar cell membrane to the basal cytosol surrounding the
nuclei (circles in Fig. 3G). This suggests that
the apical microfilament web involved in zymogen granule fusion/fission
events remains unaffected by pancreatic duct ligation.
Systemic and Inflammatory Response to Pancreatic Duct
Ligation--
To permit a comparison between pancreatic duct ligation
in rodents with other models of experimental pancreatitis we determined a variety of parameters that are known to reflect disease severity or
systemic inflammatory response. As shown in Fig.
4 and in contrast to bile duct ligation,
pancreatic duct obstruction was followed by a significant increase in
circulating leukocytes (Fig. 4A), pancreatic edema (Fig.
4B), and lung myeloperoxidase activity, all of which have
been reported to a comparable degree in other experimental models of
acute pancreatitis, including the secretagogue-induced variety
(23).
Ca2+ Signaling after Pancreatic and Bile Duct
Ligation--
To study the effect of a calcium-mobilizing secretagogue
on the response of acini from bile and pancreatic duct-ligated animals we stimulated them with acetylcholine (10
During the rapid peak phase after acetylcholine stimulation the
increase in [
When we investigated other calcium-mobilizing agents in comparison to
acetylcholine we found that the response to physiological concentrations of cholecystokinin (10 Single Cell Microfluorometry of Calcium Signaling--
On the
single cell level pancreatic acinar cells are known to respond with a
characteristic [Ca2+]i pattern in response to
different concentrations of secretagogue, and pathological signaling
patterns have been associated with the onset of pancreatitis (3, 27,
28). Submaximal concentrations of either acetylcholine of
cholecystokinin evoke repetitive Ca2+ peaks, so-called
Ca2+ oscillations, whereas maximal or supramaximal
concentrations induce an initial peak that is followed by a subsequent
plateau phase. When we stimulated single acinar cells from the pancreas of control mice with submaximal concentrations of cholecystokinin (20 pM) almost 90% of the cells responded with
Ca2+ oscillations, whereas 4% responded with a
peak/plateau Ca2+ pattern. After 4 h of pancreatic
duct ligation the percentage of cells that responded to the submaximal
20 pM cholecystokinin concentration with a peak/plateau
Ca2+-pattern increased to 10% (Fig.
7A). The percentage of cell
that had no [Ca2+]i response at all on the other
hand increased from 4% in bile duct-ligated controls to 27% after
pancreatic duct obstruction. The physiological pattern of
Ca2+ oscillations that predominated in most cells (~90%)
from control pancreas was found in only 63% of cells after ligation of
the pancreatic duct (Fig. 7A).
When we studied the frequency and amplitude of Ca2+
oscillations by single cell microfluorometry we confirmed that
individual resting [Ca2+]i levels were indeed
increased after mouse pancreatic duct ligation (to 95 ± 5 nM versus 76 ± 5 nM in bile
duct ligation controls). In terms of Ca2+ oscillations
pancreatic duct ligation shifted the signaling pattern toward lower
frequencies and lower amplitudes. Although the mean period of an
oscillation was 76 ± 3 s (oscillatory frequency 0.79/min) in
controls, it increased to 127 ± 10 s (frequency 0.47/min)
after 4 h of pancreatic duct obstruction. Not only was the
percentage of cells in the lower frequency band substantially increased
(Fig. 7B), but also the amplitude of individual oscillations
decreased from a mean of 491 ± 37 nM to a mean of
459 ± 50 nM after pancreatic duct ligation.
Transmembranous Calcium Fluxes--
The fact that the
secretagogue-stimulated [Ca2+]i plateau phase as
well as the frequency of Ca2+ oscillations is affected
after pancreatic duct obstruction could be explained by changes in the
calcium influx pathway (26). We, therefore, performed calcium influx
experiments in which we replaced Ca2+ in acinar cell
perfusion medium with manganese. In unstimulated control cells
Ca2+ substitution by Mn2+ resulted in a basal
decrease of the fura-2 signal at 360 nm, which reflects the leak influx
of Mn2+. This basal rate of Mn2+ entry was
comparable in acinar cell from control animals (bile duct ligation) and
in those after pancreatic duct ligation (Fig. 8A). In contrast, in cells
after pancreatic duct ligation the Mn2+ entry in response
to acetylcholine stimulation, which reflects Mn2+ influx
via a receptor-operated Ca2+ channel, was reduced to
~65% that found in controls (Fig. 8B).
Effect of Intracellular Calcium Chelation--
The association of
pathological calcium-signaling patterns with premature digestive
protease activation and the onset of duct ligation-induced pancreatitis
would predict that an interference with calcium signaling would prevent
or ameliorate the experimental disease course. To test whether a causal
relation exists between calcium signaling and the onset of duct
ligation-induced pancreatitis, we treated animals in vivo
with the AM ester of the calcium chelator BAPTA. This compound has been
shown to bind intracellular calcium with high affinity in pancreatic
acinar cells (3). In mice that had received two intraperitoneal
injections of BAPTA-AM before pancreatic duct ligation at a non-toxic
concentration of 10 µg/kg, the pancreatitis-induced rise of serum
amylase and lipase as well as the premature activation of trypsinogen
in the pancreas were reduced by more than 50% (Fig.
9, A-C). Higher
concentrations of BAPTA-AM that could potentially have prevented
pancreatitis completely could not be employed because of unacceptable
systemic toxicity.
Under physiological resting conditions most cell types, including
the acinar cells of the exocrine pancreas, maintain a Ca2+
gradient across the plasma membrane with low intracellular (nanomolar range) facing high extracellular (millimolar range) Ca2+
concentrations. A rapid Ca2+ release from intracellular
stores in response to external and internal stimuli is used by many of
these cells as a signaling mechanism that regulates such diverse
biological events as growth and proliferation, locomotion and
contraction, and the regulated secretion of exportable proteins. A
disturbance of the cellular Ca2+ balance and gradient has
long been suspected to play a role in the pathophysiology of acute
pancreatitis, because elevated extracellular Ca2+
concentrations represent a distinct risk factor for developing the
disease. This association has been established for endocrine disorders
associated with hypercalcemia (27) and has also been reported for
patients who develop pancreatitis after open heart surgery because of
the supraphysiological Ca2+ concentrations in the solutions
used for extracorporeal circulation (28). In laboratory animals
experimental hypercalcemia has been shown to either decrease the
threshold level for the induction of pancreatitis or to induce
morphological alterations that parallel those seen in pancreatitis (29,
30). In a model of experimental pancreatitis in which the disease
induction is achieved by injection of supraphysiological secretagogue
concentrations a progressive disruption of intracellular
Ca2+ signaling was reported (1). Accordingly, it has been
proposed that an elevation of acinar cell cytosolic-free ionized
calcium represents the most probable common denominator for the onset of various clinical varieties of pancreatitis (31). Support for this
hypothesis came from studies in which calcium chelators were found to
prevent an intracellular activation of pancreatic digestive enzymes in
isolated acini (3, 6, 32).
In the present study we have investigated the intracellular
Ca2+ dynamics in acinar cells using a pancreatitis model
that is induced by pancreatic duct ligation, and we have chosen this
approach for several reasons as follows. (a) The disease
phenotype, although associated with premature intrapancreatic digestive
enzyme activation, hyperamylasemia, pancreatic edema, and a systemic
inflammatory response, is associated with only a moderate extent of
acinar cell injury that does not preclude the investigation of
signaling events in freshly isolated cells. (b) The
mechanism of disease induction, surgical pancreatic duct ligation, does
not involve an non-physiological stimulus that is inherently
coupled to intracellular Ca2+ release like that in the
secretagogue-induced pancreatitis models. (c) The triggering
event of pancreatic duct obstruction represents, unlike supramaximal
cholecystokinin stimulation, a common factor in the clinical disease
variety of gallstone-induced pancreatitis (33).
We found that short term ligation of the pancreatic duct in the rat or
mouse leads, in analogy to the common secretagogue-induced model of
pancreatitis, to pancreatic edema, elevated serum pancreatic enzymes, a
systemic inflammatory response, and premature intrapancreatic digestive
zymogen activation. Using TAP levels as a measure of comparison,
intrapancreatic trypsinogen activation is ~30% higher in the duct
obstruction model versus the secretagogue model, although in
neither model trypsinogen activation exceeds ~1% of the total cellular trypsinogen content. On the ultrastructural level duct ligation-induced pancreatitis is of considerably less severity than the
secretagogue-induced model, and neither involves the massive formation
of cytoplasmic vacuoles nor an impairment of the actin microfilament
web along the acinar lumen. The pathophysiological consequences of an
F-actin redistribution from the basal plasma membrane of acinar cells
observed after pancreatic duct ligation is harder to determine at
present but does not seem to involve the fusion/fission events involved
in zymogen granule exocytosis.
In terms of acinar cell function after pancreatic duct obstruction we
found a profound disturbance of the intracellular Ca2+
homeostasis under both basal and hormone-stimulated conditions, which
suggests a progressive disruption of
Ca2+-dependent signaling processes. These
included a progressively increased basal [Ca2+]i
level that is generally regarded as an imbalance between Ca2+ influx, Ca2+ release from intracellular
stores, and active Ca2+ export from the cytosol. Such an
impaired cellular capacity to maintain the Ca2+ gradient
across the plasma membrane has previously been identified as a common
pathophysiological characteristic of vascular hypertension (34, 35),
malignant tumor growth (36, 37), and cell damage in response to toxins
(38). It has also been observed in the secretagogue-induced model of
acute pancreatitis (1, 39). Our manganese influx studies suggest that
the increased [Ca2+]i is not caused by an
impaired integrity of the plasma membrane because the basal or
"leak" manganese influx remained unchanged after pancreatic duct
ligation. We therefore suggest that an impairment of the calcium pumps
accounts for the increased basal [Ca2+]i level
and non-physiological gradient. One mechanism that is likely to be
involved in this process is the generation of oxygen radical species,
which are not only released in abundance by acinar cells during
pancreatitis but are also known to inhibit calcium pumps (40, 41,
42).
Another characteristic of pancreatic duct obstruction in our study was
the rapidly decreasing Ca2+ response of pancreatic acinar
cells to stimulation with acetylcholine and cholecystokinin. These
results further indicate a differential intracellular calcium
sequestration because the mobilization of calcium from acetylcholine
and cholecystokinin-sensitive stores was greatly impaired, whereas
neither the amylase secretion nor the Ca2+ release in
response to thapsigargin was negatively affected. This suggests that,
in addition to raised basal calcium levels, a preferential refilling of
thapsigargin-sensitive stores as opposed to Ach- or CCK-sensitive
stores, is a consequence of short term pancreatic duct obstruction. As
previously demonstrated by Cancela et al. (43), Ach and CCK
address through different mechanisms a common Ca2+
oscillatory unit that most probably consists of closely clustered inositol 1,4,5-trisphosphate and ryanodine channels (43). The increased number of cells after pancreatic duct ligation that responded
with an irregular Ca2+-signaling pattern (i.e.
peak plateau instead of oscillations) allows for the possibility that
this common oscillatory element is disrupted or impaired. The fact that
both the maximal peak Ca2+ response after secretagogue
stimulation as well as the sustained plateau phase of
[Ca2+]i were affected by duct ligation indicates
that not only the rapid release of Ca2+ from intracellular
stores but also the Ca2+ influx across the plasma membrane
was impaired. An involvement of the latter event was further confirmed
by our manganese experiments, which clearly demonstrated a reduced
Ca2+ influx after duct ligation and, thus, provided an
explanation for the reduced refilling of intracellular Ca2+ stores.
Finally, the observation that neither the calcium release nor amylase
secretion in response to the ATPase inhibitor thapsigargin was reduced
after pancreatic duct ligation nor that the enzyme secretion elicited
via cAMP-dependent and secretin-stimulated pathways was
impaired indicates that the secretory machinery required for enzyme
exocytosis, which includes the fusion/fission events between secretory
vesicles and the apical plasma membrane, were not disturbed by
pancreatic duct obstruction. Taken together these data further refute
the hypothesis that the secretory impairment after pancreatic duct
obstruction is solely the result of a mechanical blockade of fluid
secretion (12).
All the above data would support the conclusion that pancreatic duct
obstruction is paralleled by very early and characteristic changes in
intracellular Ca2+ signaling but provide no direct evidence
that irregular Ca2+-signaling patterns are involved in the
onset of the disease. When we used an intracellular Ca2+
chelator to interfere with intrapancreatic Ca2+ signaling
we found that two characteristic parameters of early duct
obstruction-induced pancreatitis, specifically elevated serum pancreatic enzyme levels and premature zymogen activation in the pancreas, were significantly reduced in vivo. This indicates
that Ca2+-signaling alterations are directly involved in
the onset of experimental pancreatitis and provide the first direct
in vivo evidence for this causal relation.
We conclude that in a model system that mimics the pathophysiological
events involved in the onset of gallstone-induced pancreatitis we found
that the Ca2+ signaling in isolated pancreatic acinar cells
is markedly disturbed, that an increased resting
[Ca2+]i is not only paralleled by irregular
Ca2+ responses to secretagogue stimulation but also by a
reduced frequency and amplitude of Ca2+ oscillation as well
as a decreased capacitative Ca2+ influx across the acinar
cell membrane. These characteristic changes in intracellular
Ca2+ signaling have been associated with and are indeed
paralleled by a blockage of digestive enzyme secretion and premature
digestive protease activation in the pancreas. They may, therefore,
provide the first insight on the cellular level by what underlying
mechanisms gallstone-induced pancreatitis is triggered. By systemic
administration of the intracellular Ca2+ chelator BAPTA-AM,
a novel approach to interfere with intrapancreatic Ca2+
signaling in vivo, we could demonstrate for the first time
that [Ca2+]i is indeed a critical mediator in the
initiating phase of a clinically relevant model of acute pancreatitis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase activity standard. To determine the extend of
premature and intracellular trypsinogen activation, a parameter that
has been found to closely parallel the severity of pancreatitis (21),
we measured the generation and excretion of trypsinogen activation
peptide (TAP)1 in urine and
pancreatic tissue of mice after pancreatic duct ligation using a
standardized enzyme-linked immunosorbent assay (Biotrin, Dublin,
Ireland) as previously reported (22, 23). Myeloperoxidase activity in
lung tissue was determined as previously described in detail (23) using
purified myeloperoxidase (Calbiochem) as a standard.
Rmin)/(Rmax
R) × Kd
(Iion-free/Iion-saturated)380 nm, in which Rmin and Rmax
are the minimal and maximal ratios obtained in either ion-free or
ion-saturated solutions, respectively, Kd is the
dissociation constant of fura-2 for calcium, and I is the fluorescence intensity at the 380-nm wavelength of the free dye divided
by the saturated ion-dye complex. All calibration parameters were
obtained in separate experiments.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
7 mol/liter)-induced
amylase secretion from isolated acini decreased to a nadir of ~16%
of bile duct controls, whereas serum amylase activity increased in
parallel. These data indicate that acinar cell damage (Fig.
1A) and acinar cell secretory dysfunction (Fig. 1B), respectively, can already be detected as early as
2 h after pancreatic duct ligation.
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Fig. 1.
Effect of pancreatic and bile duct ligation
in the rat on serum amylase activity in vivo
(A) and stimulated amylase secretion from
isolated pancreatic acini (B). After surgical
bile duct ligation in control animals (open triangles) and
pancreatic duct ligation in the treatment group (filled
circles) serum amylase activity was measured over intervals of up
to 6 h (A) and compared with sham-operated animals
(open squares). Alternatively, isolated acini were prepared
by collagenase digestion (B) and stimulated with
acetylcholine (10 7 mol liter
1) as described
under "Experimental Procedures." Although bile duct ligation alone
had no affect on either serum amylase activity or stimulated amylase
secretion, pancreatic duct ligation rapidly induced increased serum
levels and reduced secretion of amylase from acini. Data represent
means ± S.E., and asterisks indicate significant
differences at the 5% level compared with controls. U,
unit(s).
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Fig. 2.
Effect of pancreatic and bile duct ligation
on serum amylase activity and TAP concentrations in the mouse.
NMRI mice underwent either surgical bile duct ligation (gray
bars), pancreatic duct ligation (hatched bars), or sham
operation (op; white bars) and were sacrificed
after 4 h. Serum amylase activity (A), TAP levels in
pancreatic tissue homogenate (B), and urine (C)
were measured as described under "Experimental Procedures."
Although bile duct ligation alone had no effect on either serum amylase
activity or trypsinogen activation, pancreatic duct ligation was
followed by significant increases in both parameters. Data represent
means ± S.E., and asterisks indicate significant
differences at the 5% level compared with controls. U,
unit(s).
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Fig. 3.
Effect of pancreatic and bile duct ligation
on pancreatic morphology. Plastic-embedded thin sections of mouse
pancreatic tissue after surgical bile duct ligation (A and
B) and pancreatic duct ligation (C and
D) were contrasted with osmium, uranyl, and lead, and random
fields were photographed at a standardized ×1800 magnification. Images
shown are representative for four or more samples from each group. Bile
duct ligation alone had no discernible effect on the ultrastructural
morphology of acinar cells and their luminal surface
(asterisks in A and B) or on the
epithelial cells lining pancreatic ducts (arrows). Up to
4 h of pancreatic duct ligation led to apical formation of small
vacuoles in acinar cells (C) and dilatation of the
endoplasmic reticulum (D) but not to a significant
distension of the acinar lumen (asterisks). The latter
effect was prominent in the lumen of pancreatic ducts (arrow
in C). Bars indicate 10 µm. On
phalloidin-labeled cryosections of pancreatic tissue F-actin was
localized prominently in the apical microfilament web surrounding the
acinar lumen (asterisks) as well as along the basal and
lateral plasma membrane (arrows) of pancreatic acinar cells.
This distribution was identical in the pancreas of sham operated
(E) and bile duct-ligated (F) animals. In the
pancreas, after 4 h of pancreatic duct ligation (G) the
apical and lateral F-actin distribution remained unchanged, whereas
that at the basal plasma membrane was greatly reduced and redistributed
to the basal cytosol, where it surrounded the acinar cell nuclei
(circles).
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Fig. 4.
Parameters of severity and inflammation.
Rats underwent surgery or sham operation as described under
"Experimental Procedures." Compared with sham-operated animals
(empty bars) and bile duct-ligated controls (gray
bars) 4 h of pancreatic duct ligation (hatched
bars) led to significant leukocytosis (A), the
formation of pancreatic edema (B), and an increased
myeloperoxidase (MPO) activity in the lungs (C).
Data represent means ± S.E., and asterisks indicate
significant differences at the 5% level compared with sham-operated
controls. mU, milliunit(s);
T/µl,
thousand/microliter.
5
M). In suspensions of acini this typically resulted in a
biphasic response of [Ca2+]i that begins with a
rapid release of Ca2+ from intracellular stores (peak
phase) and is followed by a sustained Ca2+ influx (plateau
phase, Fig. 5A). Because the
absolute resting levels of [Ca2+]i were already
changed dramatically in acini from the pancreatic duct-ligated group
(see Fig. 5B), we compared secretagogue-induced changes in
[Ca2+]i as increases over basal levels
(
[Ca2+]i) in either the peak-phase (maximum
[Ca2+]i) or in the plateau-phase
(
[Ca2+]i between 60 and 100 s after the
peak). When we studied basal intracellular calcium concentrations in
acini from control rats they were in the low nanomolar range (140 ± 5 nmol). In bile duct-ligated control animals the resting
[Ca2+]i was slightly decreased in comparison to
sham-operated controls but remained constant at this level throughout
the 6-h observation period (Fig. 5B). Ligation of the
pancreatic duct on the other hand induced a gradual and consistent
increase of resting [Ca2+]i in acinar cells to a
maximum of 205 ± 7 mM after 6 h (Fig.
5B).
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Fig. 5.
Intra-acinar cell calcium transients after
pancreatic and bile duct ligation. Isolated acini were generated
from rat pancreas by collagenase digestion after animal sacrifice at
intervals of up to 6 h after either surgical bile duct ligation
(triangles), combined pancreatic and bile duct ligation
(filled circles), or sham operation (squares).
[Ca2+]i was measured spectrofluorometrically as
basal levels or after acetylcholine (10 7 mol
liter
1) stimulation. Acetylcholine evokes a biphasic
Ca2+ signal including a peak phase and a plateau phase
(A), and therefore, both phases in acini from different
treatment groups were analyzed separately in panels C and
D in addition to the basal [Ca2+]i
shown in panel B. Although basal
[Ca2+]i in acini from control pancreas (140 ± 5 nM) was not affected by bile duct ligation of up to
6 h (B, triangles) pancreatic duct ligation
led to a continuous increase in basal [Ca2+]i
(B, filled circles). Conversely, the
Ca2+ response during the peak (C) as well as the
plateau phase (D) of the Ca2+ signal after
acetylcholine stimulation was significantly reduced after pancreatic
duct ligation. Note that Ca2+ response in C and
D is shown as
[Ca2+]i = [Ca2+]i peak/plateau
[Ca2+]i basal to correct for the different basal
Ca2+ levels shown in panel B. Data represent the
means ± S.E., and asterisks indicate differences at
the 5% level.
Ca2+]i in acini from bile
duct-ligated animals remained roughly in the range of control animals,
whereas it decreased significantly and continuously in acini from the
pancreatic duct-ligated group (Fig. 5C). The differences
between the groups were even more pronounced during the subsequent
plateau phase because
[Ca2+]i declined in
acini from pancreatic duct-ligated animals and, at least in the last
phase of up to 6 h, increased in the bile duct-ligated group (Fig.
5D).
8 M) was
similarly impaired and consisted in a reduced amylase secretion by 75%
(Fig. 6A) as well as an equal
reduction in either the peak or the plateau
[Ca2+]i after stimulation (Fig.
6C). In contrast, when we used thapsigargin
(10
6 M), an inhibitor of
Ca2+-ATPases that releases [Ca2+]i by
a different, not receptor- or G-protein-coupled mechanism, we found
that the subsequent amylase secretion was unimpaired in the acini from
pancreatic duct-ligated animals (Fig. 6A). This increase in
[Ca2+]i, in response to thapsigargin, which does
not follow a peak/plateau pattern (3) (these two phases are, therefore, not distinguished in Fig. 6C), was even somewhat higher in
the pancreatic duct-ligated animals. When we investigated amylase secretion in response to secretin, a cAMP-mediated stimulus, we found
it, in analogy to the thapsigargin experiments, not to be impaired by
pancreatic duct ligation (Fig. 6B). These data indicate that
the observed impairment of calcium-mediated secretion after pancreatic
duct ligation affects, indeed, only calcium-mediated pathways and is
not due to cellular damage in general. To further rule out that a
disproportionate rate of acinar cell necrosis would account for some of
the functional changes, we measured lactate dehydrogenase release from
resting and stimulated acini after either bile or pancreatic duct
ligation. Lactate dehydrogenase release did not exceed 1.5-2.3% of
the total cellular lactate dehydrogenase in any of the groups or under
any experimental conditions, and no differences between
sham-operated, bile duct-ligated and pancreatic duct-ligated animals
were found (data not shown), thus excluding significant necrosis or
cellular leakage as a confounding factor for acinar cell
impairment.
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Fig. 6.
Effect of different calcium-mobilizing agents
on Ca2+ transients and acinar cell amylase secretion after
pancreatic and bile duct ligation. Pancreatic acini from rat
pancreas were prepared by collagenase after 4 h of either sham
operation (white bars), bile duct ligation (gray
bars), or pancreatic duct ligation (hatched bars). In
panel A amylase secretion from acini in response to Ach
(10 7 mol liter
1), CCK (10
8
mol liter
1), and the ATPase inhibitor thapsigargin
(10
6 mol liter
1) is shown, and in
panel C the corresponding peak and plateau of
[Ca2+]i are indicated for the respective
secretagogues. Panel B shows the cAMP-dependent
and secretin-stimulated (0.1 µM) amylase secretion. In
contrast to the biphasic Ca2+ response after Ach and CCK,
thapsigargin induced only a monophasic Ca2+ transient.
Although the Ach- and CCK-stimulated Ca2+ response as well
as amylase secretion were greatly reduced after pancreatic duct
obstruction, the thapsigargin-induced amylase secretion remained
unchanged, and the according Ca2+ response was even
somewhat increased. No difference between the groups in
secretin-stimulated amylase secretion was found (panel B).
Data represent the means ± S.E., and asterisks
indicate differences at the 5% level. U, unit(s).
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Fig. 7.
Single cell [Ca2+]i
changes after pancreatic and bile duct ligation. Isolated acini
from mouse pancreas were prepared by collagenase digestion 4 h
after ligation of the pancreatic or bile duct in mice. Stimulation with
submaximal concentrations of CCK (20 pmol liter 1)
elicited rhythmic Ca2+ oscillations (cross-hatched
bars) in 96% of cells, a peak/plateau pattern (white
bars) in 2%, and no response (black bars) in 2% of
cells. After pancreatic duct ligation (right set of
columns), the number of cell without Ca2+ response
rose to 24%, that with a peak/plateau pattern rose to 10%, and the
proportion of cells with physiological Ca2+ oscillations
declined to 62%. The numbers above the respective columns
indicate the total number of cells in each group. In panel B
the oscillation patterns in single acinar cells are shown after 4 h of bile duct ligation (straight line) or pancreatic duct
ligation (dotted line). Oscillation periods are indicated on
the horizontal axis in 10-s intervals, and the number of
cells with oscillations lasting for the respective period was blotted
on the vertical axis. The mean duration of oscillations was
75 s for acinar cells after bile duct ligation (oscillation
frequency of ~0.8/min), whereas it was 130 s (~0.5/min) after
pancreatic duct ligation. The inset in panel B
represents the regression curves derived from the data below in
B.
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Fig. 8.
Effect of pancreatic and bile duct ligation
on the calcium influx pathway. The representative recordings at
360 nm (the isosbestic line of fura-2) in panel A gives an
example of the manganese influx measurements. After stimulation of
acinar cells with Ach (10 7 mol liter
1), a
small signal increase is followed by a precipitous decline at the time
when Mn2+ is added, which indicates quenching of the dye by
Mn2+ influx into the cell via the receptor-operated calcium
channel. This Mn2+ influx is an indicator of
Ca2+ influx and is reduced in acini from pancreatic
duct-ligated animals (dotted line) in comparison to those
from bile duct-ligated animals (continuous line). The
histograms in panel B summarize the results from 35 experiments and indicate that Mn2+ quenching of fura-2 in
unstimulated acinar cells (basal) was identical in acini
from bile duct-ligated (gray bars) and pancreatic
duct-ligated (hatched bars) animals, which indicates an
unchanged Ca2+ leak influx under resting conditions.
Mn2+ influx after acetylcholine (10
7 mol
liter
1) stimulation on the other hand was found to be
significantly reduced in acini after pancreatic duct ligation. Data
represent the means ± S.E., and asterisks indicate
differences at the 5% level.
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Fig. 9.
Effect of the intracellular
Ca2+-chelator BAPTA-AM on intrapancreatic trypsinogen
activation and serum pancreatic enzyme activities. NMRI mice
underwent pancreatic duct ligation or sham operation (sham
op) and were sacrificed after 4 h. Serum amylase activity
(A), serum lipase activity (B), and TAP levels in
pancreatic tissue homogenate (C) were measured as described
under "Experimental Procedures." Animals with pancreatic duct
ligation received 2 intraperitoneal injections (30 min before and at
the time of laparotomy) of either BAPTA-AM (10 µg/kg of body weight
in 180 µl of propylene glycol as vehicle hatched bars) or
vehicle alone (gray bars). Treatment with the
Ca2+ chelator BAPTA-AM reduced serum pancreatic enzyme
activities as well as intrapancreatic trypsinogen activation
significantly. Complete inhibition, which arguably could have been
obtained with higher BAPTA-AM concentrations, was not achievable
because of systemic toxic effects at higher doses of the chelator. Data
represent the means ± S.E., and asterisks indicate
significant differences at the 5% level compared with vehicle-treated
animals. U, unit(s).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
---|
We thank U. Breite for expert technical assistance.
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FOOTNOTES |
---|
* This work was supported by Interdisziplinäres Zentrum für Klinische Forschung Münster Grants H3 and D21 and Deutsche Forschungsgemeinschaft Grants KR 1274/2-2, Le 625/5-2, and SFB 293 B7.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ These authors were equal contributors.
To whom correspondence should be addressed: Dept. of Medicine
B, Westfälische Wilhelms-Universität,
Albert-Schweitzer-Str. 33, 48129 Münster, Germany. Tel.:
49-251-8347559; Fax.: 49-251-8349504; E-mail:
markus.lerch@uni-muenster.de.
Published, JBC Papers in Press, January 8, 2002, DOI 10.1074/jbc.M207454200
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ABBREVIATIONS |
---|
The abbreviations used are: TAP, trypsinogen activation peptide; Ach, acetylcholine; CCK, cholecystokinin; BAPTA-AM, acetoxymethyl ester (AM) of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid.
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REFERENCES |
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