From the Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215 and Harvard Medical School, Boston, Massachusetts 02215
Received for publication, October 24, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Prior stress ameliorates caerulein-induced
pancreatitis in rats. NF- Doses of caerulein, the decapeptide analog of cholecystokinin, in
excess of those that elicit a maximal rate of digestive enzyme
secretion from the rat pancreas, elicit a reversible form of acute
interstitial pancreatitis. This model of pancreatitis, referred to as
secretagogue-induced pancreatitis, is associated with and possibly
brought about by intra-acinar cell activation of digestive enzyme
zymogens including trypsinogen (1). Supramaximal stimulation of freshly
prepared but otherwise normal pancreatic acini with caerulein also
results in intra-acinar cell activation of trypsinogen and,
subsequently, evidence of acinar cell injury in vitro
(2).
We have shown recently that prior water immersion stress, under
conditions that result in expression of heat shock protein 60 (HSP60),1 prevents
caerulein-induced in vivo activation of trypsinogen in
acinar cells and protects against this form of secretagogue-induced pancreatitis (3). Pancreatic acini prepared from prior water-immersed rats, which contain increased amounts of HSP60, are also protected against caerulein-induced in vitro activation of trypsinogen
as well as caerulein-induced in vitro injury. The
mechanism(s) responsible for this stress-induced protection against
caerulein-induced injury has not been established.
Nuclear factor- Pancreatic acinar cell NF- In the present communication, we report studies that have evaluated the
effects of prior water immersion stress, under conditions associated
with up-regulated HSP60 expression, on the activation of pancreatic
NF- Male Wistar rats weighing 100-200 g (Charles River
Laboratories, Wilmington, MA) were used in all experiments. The animals were housed in temperature-controlled (23 ± 2 °C) rooms with a 12-h light/dark cycle, fed standard laboratory chow, and allowed to
drink ad libitum. Caerulein was purchased from Research Plus (Bayonne, NJ), rat recombinant TNF- In Vivo Experiments--
Rats were exposed to water immersion
stress as described previously (3). In brief, the animals were placed
in restriction cages and vertically immersed to the xiphoid process.
The duration of water immersion was 6 h in all experiments unless
otherwise specified. Caerulein (50 µg/kg) and rat TNF- In Vitro Experiments--
Dispersed rat pancreatic acini were
prepared by collagenase digestion from normal rats or rats after 6 h of water immersion and studied as described previously (10-12). The
acini were allowed to equilibrate for 5 min at 37 °C and then were
stimulated with either caerulein (0.1 µM) or TNF- Assays--
For evaluation of NF- Electrophoretic Mobility Shift Assay (EMSA)--
Reaction
mixtures (25 µl, pH 7.5) contained 7.5-10.0 µg of nuclear protein,
5 mM Tris, 100 mM NaCl, 1 mM
dithiothreitol, 1 mM EDTA, 4% (v/v) glycerol, and 0.08 mg/ml salmon sperm DNA. The oligonucleotide probe (5'-AGT TGA GGG GAC
TTT CCC AGG C-3', Promega, Madison, WI) containing the Western Blot Analysis--
Equal amounts of cytoplasmic protein
extracts (5-10 µg) were diluted in Laemmli sample buffer with 5%
mercaptoethanol. After boiling, the samples were resolved in 10%
polyacrylamide gels in Tris-glycine-SDS buffer. The gels were
transferred to nitrocellulose membranes, blocked in 5% nonfat dry milk
in phosphate-buffered saline (PBS), pH 7.5, containing 0.1% (v/v)
Tween 20 (PBST-milk). Blots then were incubated with polyclonal rabbit
anti-I Analysis of Data--
The results reported in this communication
represent means ± S.E. of the mean values obtained from three or
more separate experiments. In all figures, vertical bars
denote S.E. values. Statistical evaluation of data was accomplished by
analysis of variance, and p values of less than 0.05 were
considered significant. All EMSA and Western blot gels shown are
representative of at least three such gels prepared from independent experiments.
Effects of Water Immersion Stress on NF-
As shown in Fig. 1, prior water immersion
profoundly inhibits the NF-
The observation that caerulein-induced NF- Effects of Prior Water Immersion on NF- Effects of Chelating [Ca2+]i on
Caerulein- and TNF- Effects of Prior Water Immersion on Caerulein-induced Changes in
[Ca2+]i--
Freshly prepared acini were
loaded with Fura-2/AM, washed, and then incubated with a supramaximally
stimulating concentration of caerulein. As shown in Fig.
7, when those acini were prepared from
control animals, caerulein causes a large but transient rise in
[Ca2+]i, which is followed by a sustained but
lesser elevation of [Ca2+]i that persists
throughout the period of observation. The resting
[Ca2+]i in acini prepared from water-immersed
animals is lower than that observed in acini prepared from the control
group, and both the peak and the sustained increases in
[Ca2+]i noted after caerulein addition are
attenuated profoundly.
Otaka et al. (17), in 1994, reported the results of
studies that indicated that HSP60 expression in the rat pancreas was up-regulated by prior water immersion stress and that water immersion stress protected those animals from subsequent caerulein-induced pancreatitis. Recently, in studies designed to explore the mechanism(s) responsible for the protective effect of water immersion on
pancreatitis, we found that caerulein-induced intrapancreatic
trypsinogen activation, an early event in secretagogue-induced
pancreatitis, was prevented by prior water immersion (3). We noted that
this prevention of trypsinogen activation was correlated temporally
with the up-regulation of HSP60 expression. Furthermore, we found that
the effects of prior water immersion could be detected in acini studied
in vitro. That is, intra-acinar cell activation of
trypsinogen and cell injury were observed when acini from
non-water-immersed animals were exposed to a supramaximally stimulating
concentration of caerulein in vitro, but neither trypsinogen
activation nor cell injury was observed when acini prepared from
water-immersed animals were exposed in vitro to a
supramaximally stimulating concentration of caerulein (3). The
currently reported studies were designed to examine further the
mechanisms responsible for the protective effect of water immersion on
secretagogue-induced pancreatitis.
NF- Pancreatic acinar cell NF- The currently reported studies indicate that the activation of
pancreatic acinar cell NF- Although the role of HSP60 in the events triggered by water immersion
stress remains uncertain, the studies reported in this communication
still provide some insights into the mechanisms by which water
immersion stress affects NF- In summary, our studies indicate that supramaximally stimulating doses
of caerulein and TNF-B is a proinflammatory transcription factor
activated during caerulein pancreatitis. However, the effects of prior
stress on pancreatic NF-
B activation are unknown. In the current
study, the effect of prior water immersion stress on caerulein and
tumor necrosis factor-
(TNF-
)-induced NF-
B activation in the
pancreas was evaluated. Water immersion of rats for up to 6 h
prevents supramaximal caerulein-induced pancreatic I
B-
degradation and NF-
B activation in vivo. NF-
B
activity is also inhibited in vitro in pancreatic acini
prepared from water-immersed animals. TNF-
-induced NF-
B
activation in pancreas or in pancreatic acini is unaffected by prior
water immersion. Chelation of intracellular Ca2+ by
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate/acetoxymethyl ester has similar effects to water immersion in preventing
caerulein but not TNF-
-induced NF-
B activation in pancreas. Both
the spike response and the sustained rise in
[Ca2+]i in response to supramaximal caerulein
stimulation are reduced markedly in acini prepared from water-immersed
animals as compared with normal animals. Our findings indicate that, in addition to Ca2+-dependent mechanisms,
Ca2+-independent signaling events also may lead to NF-
B
activation in pancreatic acinar cells. Water immersion stress prevents
supramaximal caerulein-induced NF-
B activation in pancreas in
vivo and in vitro by affecting intracellular
Ca2+ homeostasis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B (NF-
B) is a family of widely expressed
transcription factors that acts to modulate inflammatory processes. Activation of NF-
B involves an intracytoplasmic kinase cascade that
culminates in the phosphorylation of I
B proteins leading to their
dissociation from NF-
B (4). As a result, the now activated NF-
B,
which is comprised of dimers made up of various combinations of
Rel homology-sharing subunits, can translocate to the nucleus
and act to regulate expression of genes coding for various
proinflammatory factors (5). The phosphorylated I
Bs are, in
parallel, degraded by proteasome.
B activation has been reported to occur
during the very early stages of caerulein-induced pancreatitis (6, 7)
and evidence has been presented that suggests that prevention of
caerulein-induced NF-
B activation in the pancreas can ameliorate the
severity of caerulein-induced pancreatitis (6). Supramaximal
stimulation of acini in vitro with caerulein also causes
activation of NF-
B by a mechanism that has been shown to involve a
rise in cytoplasmic free calcium levels ([Ca2+]i)
and activation of protein kinase C (8-9).
B. We show that prior water immersion stress prevents
caerulein-induced in vivo NF-
B activation in the rat pancreas. Similarly, NF-
B activation in vitro is not
observed when acini prepared from prior water-immersed animals are
exposed to a supramaximally stimulating dose of caerulein. In contrast, NF-
B activation, both in vivo and in vitro, in
response to TNF-
is not altered by prior water immersion. Finally,
we show that prior water immersion stress markedly attenuates the
acinar cell [Ca2+]i changes that follow exposure
to a supramaximally stimulating dose of caerulein, and in acini
prepared from animals not stressed by prior water immersion, chelation
of cytoplasmic Ca2+ with BAPTA/AM can prevent
caerulein-induced NF-
B activation in those acini. Taken together,
these observations lead us to conclude that water immersion stress
prevents caerulein-induced NF-
B activation by interfering with
caerulein-induced changes in [Ca2+]i. This
phenomenon may contribute to the protective effects of water immersion
on secretagogue-induced pancreatitis.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
was from
BIOSOURCE, collagenase (CLS-4) was from
Worthington, and both Fura-2/AM and BAPTA/AM were from Molecular Probes
(Ridgefield, CT). All other reagents and chemicals were purchased from
Sigma. All experimental protocols were approved by the Institutional
Animal Use Committee of the Beth Israel Deaconess Medical Center
(Boston, MA).
(10 µg/kg) were dissolved in saline and administered as a single
intraperitoneal dose to normal and water-immersed animals. Control
animals received no injection. Animals were sacrificed in a
CO2 chamber at various times after the injections, and
pancreatic tissue samples were collected immediately.
(200 ng/ml). Other acini were preincubated with BAPTA/AM (50 µM) for 30 min at 37 °C before adding caerulein or
TNF-
. After an additional 30 or 90 min of incubation, the acini were
washed in ice-cold HEPES-Ringer buffer, and nuclear and cytoplasmic
protein extracts were prepared. Viability of acini after washing, as
assessed by trypan blue exclusion, was >95%.
B activation and I
B-
degradation, nuclear and cytoplasmic protein extracts were prepared as
described by Dyer and Herzog (13). Protein concentrations were
determined by the method of Bradford (14). For
[Ca2+]i measurements, pancreatic acini from
normal and water-immersed rats were loaded with 2 µM
Fura-2/AM for 30 min at 37 °C and then washed extensively.
[Ca2+]i was quantitated using a Spex dual
excitation spectrofluorometer as described previously (11, 15).
Excitation was at 340 and 380 nm, and emission was measured at 505 nm.
The results are expressed as the fluorescence ratio.
B binding
motif was end-labeled with [
-32P]ATP using
T4 polynucleotide kinase and purified over two successive 1-ml G-50 columns (Amersham Pharmacia Biotech). 1 × 106 cpm of the probe was added to the reaction mixture, and
the binding reaction was allowed to proceed for 20 min at room
temperature. For supershift assays, 2 µl of specific antibody against
NF-
B subunits p50, p52, p65, or c-Rel (Santa Cruz Biotechnology,
Inc., Santa Cruz, CA) was added to the reaction mixture and the
resulting solution was allowed to incubate for 30 min at 4 °C before
adding the oligonucleotide probe. DNA-protein complexes were resolved in a 6% nondenaturing polyacrylamide gel in a TBE buffer (22.5 mM Tris, 22.5 mM boric acid, and 0.5 mM EDTA, pH 8.3) at 140 V for 2-3 h. Gels were
dried and exposed to Kodak Bio Max MR films at
70 °C. NF-
B
bands from films were quantitated by using an HP Scanjet 4100 scanner
and a Scion image analysis program.
B-
antibody (sc-371, Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) at 1:1000 (v/v) dilution in PBST-milk at 4 °C overnight.
The membranes then were washed in PBST and incubated with horseradish
peroxidase-conjugated anti-rabbit IgG at 1:5000 (v/v) dilution in
PBST-milk for 1 h. After washing, I
B-
protein bands in the
membranes were visualized by enhanced chemiluminescence (PerkinElmer
Life Sciences).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B Activation in
Vivo--
In preliminary experiments, rats were exposed to a
supramaximally stimulating dose of caerulein (50 µg/kg) and
sacrificed at various times thereafter. Activation of pancreatic
NF-
B and degradation of I
B-
were observed to occur in a
biphasic manner with an initial peak of 30 min (11.1 ± 1.8-fold
control) after caerulein administration, a subsequent decline, and then
a second phase of increase that reached a maximal level between 90 min (9.4 ± 1.5-fold control) and 180 min (8.7 ± 1.7-fold
control) after caerulein administration. These observations are in
accord with those reported recently by Gukovsky et al. (6).
In our subsequent experiments, NF-
B activation and the effects of
water immersion stress on this process were evaluated at the selected times of 30 and 90 min after caerulein administration.
B activation and I
B-
degradation
observed 30 as well as 90 min after caerulein administration. The time
course of the water immersion-induced blockade of caerulein-induced
NF-
B activation is shown in Fig. 2.
Half-maximal inhibition is observed after ~3 h of water immersion,
and at that time water immersion-induced HSP60 expression is also
approximately half-maximal (Fig. 2B). In contrast to these
effects on caerulein-induced NF-
B activation, TNF-
-induced
NF-
B activation and I
B-
degradation are not altered by prior
water immersion (Fig. 3).
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Fig. 1.
Effect of water immersion stress on
caerulein-induced NF- B activation.
A, NF-
B DNA binding activity and I
B-
protein
levels. Nuclear and cytoplasmic protein extracts were prepared from
water-immersed and non-water-immersed animals either before (0 time) or
30 and 90 min after supramaximal stimulation with caerulein. The
upper panel represents EMSA, and the lower
panel represents the corresponding I
B-
Western blot.
B, densitometric quantitation of NF-
B binding activity.
Normal (i.e. non-water-immersed) and water-immersed animals
were evaluated either before (black bars), 30 min after
(white bars), or 90 min after (shadowed bars)
supramaximal caerulein stimulation. The values shown represent the fold
increase over non-caerulein stimulation. *, p < 0.05 when the water-immersed group was compared with the corresponding
non-water-immersed group.
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Fig. 2.
Time-dependent effects of water
immersion on caerulein-induced NF- B
activation. A, NF-
B DNA binding activity as measured by
EMSA. Nuclear protein extracts were prepared from pancreatic tissue
samples collected after 30 min of supramaximal caerulein stimulation.
Caerulein was administered after varying times of water immersion.
B, time-dependent effects of water immersion on
the inhibition of caerulein-induced NF-
B activation and the
expression of HSP60. Black circles represent the relative
inhibition of NF-
B DNA binding activity, shown in the right
vertical axis, as a function of water immersion duration. NF-
B
activation after 30 min of supramaximal stimulation in
non-water-immersed animals represents 100%. The bars,
defined in the left vertical axis, represent HSP60
expression as a function of water immersion duration (3).
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Fig. 3.
Effect of water immersion on
TNF- -induced NF-
B
activation. A, NF-
B DNA binding activity and
I
B-
protein levels. Nuclear and cytoplasmic protein extracts were
prepared from water-immersed and non-water-immersed animals 90 min
after supramaximal stimulation with caerulein (C) or after
stimulation with TNF-
(T, 10 µg/kg). Nonstimulated
animals served as controls (Co). The upper panel
represents EMSA, and the lower panel represents the
corresponding I
B-
Western blot. B, densitometric
quantitation of NF-
B binding activity. Water-immersed and
non-water-immersed animals before (black bars) and after 90 min of either supramaximal caerulein stimulation (white
bars) or TNF-
(10 µg/kg) stimulation (gray bars)
were evaluated. The values represent fold increase over nonstimulated
control animals. *, p < 0.05 when the water-immersed
group was compared with the corresponding non-water-immersed
group.
B activation, but not
TNF-
-induced NF-
B activation, is prevented by prior water immersion suggested the possibility that caerulein and TNF-
might activate different species of NF-
B. To evaluate this possibility, supershift assays were performed using antibodies to the p50, p52, p65,
and c-Rel subunits of the NF-
B. As shown in Fig.
4 and in accord with results reported by
others (6, 16), the caerulein- or cholecystokinin-induced activation of
NF-
B involves p50/p50 and p50/p65 dimers. p50/p50 and p50/p65 dimers
are involved also in the TNF-
-induced activation of NF-
B. These
observations indicate that caerulein and TNF-
activate the same
species of NF-
B in rat pancreas.
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Fig. 4.
The subunits involved in caerulein- and
TNF- -induced activation of
NF-
B. Nuclear protein extracts were
prepared from non-water-immersed animals subjected to supramaximal
stimulation with caerulein for 90 min or with TNF-
(10 µg/kg) for
90 min. Nuclear extracts were incubated for 30 min at 4 °C in the
presence of 2 µl of either anti-p50, anti-p52, anti-p65, or
anti-c-Rel antibodies before adding the labeled oligonucleotide
probe.
B Activation in
Vitro--
Acini were prepared from control rats and from rats
immediately after 6 h of water immersion stress. Those acini then
were exposed to a supramaximally stimulating concentration of caerulein or to TNF-
in vitro, and NF-
B activation was evaluated
30 or 90 min later. As shown in Fig. 5,
caerulein-induced NF-
B activation and I
B-
degradation are not
observed in acini prepared from prior water-immersed animals, but water
immersion does not interfere with either TNF-
-induced degradation of
I
B-
or activation of NF-
B.
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Fig. 5.
Effects of water immersion stress on
NF- B activation by caerulein and
TNF-
in pancreatic acini. A,
NF-
B DNA binding activity and I
B-
protein levels. Pancreatic
acini from water-immersed and non-water-immersed animals were freshly
prepared. After incubating the acini for 30 or 90 min in either buffer
alone, buffer containing a supramaximally stimulating concentration of
caerulein (0.1 µM), or buffer containing TNF-
(200 µg/ml), nuclear and cytoplasmic protein extracts were prepared. The
upper panel represents EMSA and the lower panel
shows I
B-
Western blot. B, densitometric quantitation
of NF-
B binding activity. Pancreatic acini were prepared from
water-immersed and non-water-immersed animals before (black
bars) and after 30 min of either supramaximal caerulein
stimulation (white bars) or exposure to TNF-
(gray
bars, 200 µg/ml). The values represent fold increase over that
noted for nonstimulated control acini. *, p = <0.05
when the water-immersed group was compared with the corresponding
non-water-immersed group.
-induced NF-
B Activation--
Freshly
prepared acini obtained from control (i.e. not
water-immersed) animals were incubated with the Ca2+
chelator BAPTA/AM and then exposed to either caerulein or TNF-
. As
shown in Fig. 6, chelation of
intracellular Ca2+ with BAPTA/AM, which prevents the
caerulein-induced rise in [Ca2+]i (11), prevents
caerulein- but not TNF-
-induced NF-
B activation.
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Fig. 6.
Effects of chelating intracellular
Ca2+ on caerulein- and
TNF- -induced activation of
NF-
B in pancreatic acini. A,
NF-
B binding activity and I
B-
protein levels. Pancreatic acini
were freshly prepared from nonmanipulated animals. The acini were
preincubated for 30 min with or without BAPTA/AM (50 µM)
and then incubated for an additional 30 min with either a
supramaximally stimulating concentration of caerulein (0.1 µM) or with TNF-
(200 µg/ml). Nuclear and
cytoplasmic protein extracts then were prepared. The upper
panel represents EMSA, and the lower panel represents
the corresponding I
B-
Western blot. B, densitometric
quantitation of NF-
B binding activity. Pancreatic acini were
prepared from nonmanipulated animals and preincubated with or without
BAPTA/AM (50 µM) for 30 min. They then were stimulated
with either a supramaximally stimulating concentration of caerulein
(white bars) or with TNF-
(gray bars, 200 µg/ml). The values shown represent fold increases over nonstimulated
controls (black bars). *, p = <0.05 when
the group exposed to BAPTA/AM was compared with the corresponding group
not exposed to BAPTA/AM.
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Fig. 7.
Effects of prior water immersion on
[Ca2+]i changes induced by supramaximal
stimulation with caerulein. Freshly prepared pancreatic acini from
water-immersed (open circles) and non-water-immersed
(closed circles) rats were loaded with Fura-2/AM for 30 min,
washed, and then exposed to a supramaximally stimulating dose of
caerulein (0.1 µM), and fluorescence was monitored as
described in the text. Results shown are representative of three
separate experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
B is a widely expressed transcription factor that in many systems
has been shown to play a critical role in regulating inflammatory
processes by modulating the expression of genes coding for inflammatory
mediators including cytokines, chemokines, and adhesion molecules (5,
18). Studies from several groups have indicated that activation of
pancreatic acinar cell NF-
B is an early event in
secretagogue-induced pancreatitis. In a recent study we showed that
although caerulein-induced trypsinogen activation and NF-
B
activation are closely related temporally, they are independent events
in pancreatic acinar cells (19). Although still somewhat controversial
(6, 7), the preponderance of evidence suggests that NF-
B activation
is a proinflammatory event in this model of pancreatitis and that
interventions that interfere with acinar cell NF-
B activation reduce
the severity of secretagogue-induced pancreatitis (6, 16, 20).
B activation by supramaximally
stimulating doses of caerulein or cholecystokinin has been examined recently by several groups, and the results of their studies have shown
that (a) activation depends on a rise in acinar cell
[Ca2+]i and activation of protein kinase C (8,
9), (b) activation is accompanied by degradation of
I
B-
(8, 9), and (c) the NF-
B dimers activated in
this process are composed of p50 and p65 subunits (6, 16). Pancreatic
acinar cell NF-
B also can be activated by TNF-
(21), but this
process has not been studied as extensively. Our own results (Figs.
3-5) indicate that TNF-
also promotes I
B-
degradation and
translocation of p50/p50 and p50/p65 NF-
B dimers into the nucleus.
These observations suggest that caerulein and TNF-
activate the same
species of NF-
B and that the final steps in activation
(i.e. activation of I
B kinase and phosphorylation
of I
B-
leading to dissociation of I
B-
from NF-
B and
degradation of I
B-
) are similar for caerulein- and
TNF-
-induced activation of NF-
B.
B by supramaximally stimulating doses of
caerulein both in vivo (Figs. 1-3) and in vitro
(Fig. 5) is inhibited by prior water immersion. Under these conditions, HSP60 expression is up-regulated and the time course of HSP60 expression after water immersion stress roughly correlates with that of
prevention of NF-
B activation (Fig. 2). It is tempting, therefore,
to speculate and simplify these observations by concluding that HSP60
mediates the process by which NF-
B activation is inhibited, but this
conclusion can only be tentative because water immersion stress also
might set in motion other as yet unidentified events that prevent
NF-
B activation beside HSP60 expression. Further studies will be
needed before the relationship between HSP60 expression and prevention
of caerulein-induced NF-
B activation can be defined more clearly and unambiguously.
B activation. Our studies indicate that
water immersion stress interferes with caerulein-induced NF-
B
activation, but it does not alter TNF-
-induced NF-
B activation either in vivo (Figs. 1 and 3) or in vitro (Fig.
5). This finding suggests that water immersion stress affects the
activation process at a step that occurs before I
B kinase activation
and I
B-
phosphorylation because, as noted above, these steps seem
to be shared by both the TNF-
- and caerulein-induced NF-
B
activation process. The finding that caerulein-induced NF-
B
activation but not TNF-
-induced NF-
B activation can be blocked by
chelation of cytoplasmic Ca2+ with BAPTA/AM (Fig. 6)
indicates that the two pathways for NF-
B activation differ in their
requirement for a rise in [Ca2+]i, and this
observation suggested to us that prior water immersion stress might
interfere with caerulein-induced NF-
B activation by interfering with
caerulein-induced [Ca2+]i changes in pancreatic
acinar cells. To examine this possibility,
[Ca2+]i changes in acini prepared from control
and water-immersed animals were evaluated (Fig. 7). Prior water
immersion was found to reduce the resting [Ca2+]i
level in acini and to attenuate markedly the caerulein-induced rise in
[Ca2+]i. This observation leads us to conclude
that prior water immersion prevents caerulein-induced NF-
B
activation by interfering with caerulein-induced
[Ca2+]i changes and to suggest that this may at
least in part explain the protection against caerulein-induced
pancreatitis that is afforded by prior water immersion.
activate the same species of NF-
B in
pancreatic acinar cells but that they do so by different mechanisms.
Caerulein-induced activation is a
Ca2+-dependent process, whereas TNF-
-induced
activation is independent of a rise in [Ca2+]i.
Prior water immersion stress induces HSP60 expression and also prevents
caerulein-induced NF-
B activation. We suggest that prior water
immersion stress prevents caerulein-induced NF-
B activation by
interfering with the caerulein-induced rise in
[Ca2+]i, which is critical to that event.
Chelation of cytoplasmic Ca2+ with BAPTA/AM can bring about
the same effect. The reduction in caerulein-induced
[Ca2+]i rise and the activation of NF-
B that
follows water immersion stress is correlated with the rise in HSP60
expression that also follows water immersion stress, but whether HSP60
actually mediates the effects of water immersion stress on
Ca2+ dynamics and NF-
B activation will require further studies.
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FOOTNOTES |
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* This work supported in part by National Institutes of Health Grants DK-58694 and DK-31396.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.
Financially supported by the Finnish Academy of Sciences, Sigrid
Jusélius Foundation, Finnish Cultural Foundation, Maud Kuistila Foundation, Finnish Medical Association Duodecim, and Finnish Foundation for Alcohol Studies.
§ To whom correspondence should be addressed: Dept. of Surgery, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-5369; Fax: 617-667-8679; E-mail: asaluja@bidmc.harvard.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M009721200
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ABBREVIATIONS |
---|
The abbreviations used are:
HSP60, heat shock
protein 60;
NF-B, nuclear factor-
B;
I
B, inhibitory-
B;
TNF-
, tumor necrosis factor-
;
BAPTA/AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate/acetoxymethyl
ester;
EMSA, electrophoretic mobility shift assay;
PBS, phosphate-buffered saline.
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