From the Department of Physiology II, University of Saarland, D-66421 Homburg/Saar, Germany
Received for publication, February 6, 2001, and in revised form, February 26, 2001
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ABSTRACT |
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In pancreatic acinar cells analysis
of the propagation speed of secretagogue-evoked Ca2+
waves can be used to examine coupling of hormone receptors to intracellular signal cascades that cause activation of protein kinase C
or production of arachidonic acid (AA). In the present study we have
investigated the role of cytosolic phospholipase A2
(cPLA2) and AA in acetylcholine (ACh)- and
bombesin-induced Ca2+ signaling. Inhibition of
cPLA2 caused acceleration of ACh-induced Ca2+
waves, whereas bombesin-evoked Ca2+ waves were unaffected.
When enzymatic metabolization of AA was prevented with the
cyclooxygenase inhibitor indomethacin or the lipoxygenase inhibitor
nordihydroguaiaretic acid, ACh-induced Ca2+ waves
were slowed down. Agonist-induced activation of cPLA2
involves mitogen-activated protein kinase (MAPK) activation. An
increase in phosphorylation of p38MAPK and
p42/44MAPK within 10 s after stimulation could be
demonstrated for ACh but was absent for bombesin. Rapid phosphorylation
of p38MAPK and p42/44MAPK could also be
observed in the presence of cholecystokinin (CCK), which also causes
activation of cPLA2. ACh-and CCK-induced Ca2+
waves were slowed down when p38MAPK was inhibited with
SB 203580, whereas inhibition of p42/44MAPK with PD
98059 caused acceleration of ACh- and CCK-induced Ca2+
waves. The spreading of bombesin-evoked Ca2+ waves was
affected neither by PD 98059 nor by SB 203580. Our data indicate that
in mouse pancreatic acinar cells both ACh and CCK receptors couple to
the cPLA2 pathway. cPLA2 activation occurs within 1-2 s after hormone application and is promoted by
p42/44MAPK and inhibited by p38MAPK.
Furthermore, the data demonstrate that secondary
(Ca2+-induced) Ca2+ release, which supports
Ca2+ wave spreading, is inhibited by AA itself and not by a
metabolite of AA.
Stimulation of pancreatic acinar cells with the secretagogues
acetylcholine (ACh),1
cholecystokinin (CCK), or bombesin evokes cytosolic Ca2+
waves that initiate within the secretory cell pole and subsequently spread toward the basal cell membrane (1-3). Ca2+ wave
propagation has been explained by sequential Ca2+ release
from stores in series involving Ca2+-induced
Ca2+ release (CICR) (4, 5). The speed of Ca2+
waves depends on the type of agonist used for stimulation and to some
extent also on the agonist concentration (6). The propagation rate can
be modified by application of arachidonic acid (AA) or by activation of
protein kinase C (PKC) with phorbol esters (6, 7). Both application of
AA and activation of PKC leads to inhibition of CICR and thereby slows
down spreading of Ca2+ signals. Analysis of the propagation
rate of secretagogue-evoked Ca2+ waves in pancreatic acinar
cells can therefore be used to investigate coupling of hormone
receptors to intracellular signal cascades that lead to endogenous
production of AA and/or to activation of PKC. In previous studies we
could demonstrate that in mouse pancreatic acinar cells bombesin
receptors couple to phospholipase D (7), which produces diacylglycerol
and thereby activates PKC, whereas high affinity CCK receptors couple
to cytosolic phospholipase A2 (cPLA2) (6), an
enzyme that leads to release of AA from membrane phospholipids.
Bombesin-induced activation of PKC via phospholipase
D-dependent diacylglycerol production, as well as CCK-induced formation of AA by cPLA2, takes place within
the very first seconds after hormone application, before or during
development of the initial inositol 1,4,5-trisphosphate-induced
Ca2+ signal in the luminal cell pole.
In the present study we investigated the role of cPLA2 in
ACh- and bombesin-evoked Ca2+ signaling. Our data indicate
that in pancreatic acinar cells activation of cPLA2 is an
early event in ACh- but not in bombesin-evoked intracellular signaling.
ACh- and CCK-induced activation of cPLA2 involves MAP
kinase activation. Furthermore, we demonstrate that secretagogue-induced Ca2+ signaling in pancreatic acinar
cells is modified by AA itself and not by metabolites of AA.
Cell Preparation--
Adult male CD-1 mice (35-40 g) were
sacrificed by cervical dislocation. The pancreas was removed and
transferred into a "preparation buffer" consisting of (in
mM): 130 NaCl, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 1.2 KH2PO4, 10 glucose,
0.2% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 10 HEPES, pH 7.4. Single acinar cells were isolated by enzymatic digestion with
collagenase type V (30 units/ml, 10 min, 37 °C; Sigma) as
described previously (8). Enzymatic digestion was followed by
mechanical dissociation of cells by gentle pipetting. The resulting
cell suspension was centrifuged, and the cells were washed twice in
preparation buffer without collagenase.
Confocal Microscopy--
Freshly prepared acinar cells were
loaded with 4 µM fluo-3/AM for 30 min at room
temperature and then stored at 4 °C. Experiments were performed
within 4 h after cell isolation. For measurement of cytosolic
Ca2+ signals, cells were transferred to a perfusion chamber
and were allowed to adhere to the glass coverslip for several minutes. Then, the cells were continuously superfused with a standard bath solution containing (in mM): 140 NaCl, 4.7 KCl, 1.3 CaCl2, 1 MgCl2, 10 HEPES, 10 glucose, pH 7.4. Fluorescence images (excitation, 488 nm; emission, >515 nm) of
128 × 128 pixels (0.463 µm/pixel) were taken with a confocal
laser-scanning microscope (MRC-600; Bio-Rad) every 0.28 s. To
monitor local changes in [Ca2+]cyt small
rectangular areas were selected in the luminal and basal cell region.
The time when [Ca2+]cyt increased in the
respective area was determined, and the speed of agonist-induced
Ca2+ waves was calculated from the distance between these
areas and the time lag between the increase in
[Ca2+]cyt at the luminal and basal cell
region. Signals from single cells, as well as from individual cells in
small cell clusters, were analyzed. All experiments were performed at
room temperature. Where appropriate data are presented as mean ± S.E. Statistical analysis was performed with student's t test.
Phosphorylation of p42/44MAPK and
p38MAPK--
Aliquots of freshly prepared cells were
stimulated by addition of the respective agonist, and the reactions
were stopped after the appropriate time by rapid freezing in liquid
nitrogen. Cells were sonicated in lysis buffer (in mM: 150 NaCl, 4 EGTA, 4 dithiothreitol, 20 HEPES, 0.2 phenylmethylsulfonyl
fluoride, 2 sodium orthovanadate, 80 µg/ml trypsin inhibitor, 0.1 mg/ml leupeptin), and the resulting suspension was centrifuged at
1000 × g. The supernatant was diluted with SDS loading
buffer (10 mM Tris, 1% mercaptoethanol, 1% SDS, 10%
glycerol, 0.015% bromphenol blue) and heated for 5 min at 95 °C
prior to SDS polyacrylamide gel electrophoresis (8.75% acrylamide). Proteins were transferred onto nitrocellulose membranes and blocked with 1% bovine serum albumin. Protein phosphorylation was detected with antibodies against the phosphorylated form of the respective MAP
kinases (Santa Cruz Biotechnology). Optical density of the chemoluminescence was scanned and quantified with a Bioprofil imaging
and scanning system (Vilber-Lourmat). The optical density measured under the control condition was set to 100%.
Quantitative Measurement of PHAS-I
Phosphorylation--
MAP kinase activity in crude cell lysates was
determined using PHAS-I as substrate (9). Cell lysates (25 µg of
protein) were mixed with 3 µl of 10-fold "reaction buffer" (250 mM HEPES, 100 mM magnesium acetate, 500 µM ATP, pH 7.5) and 2 µl of [ Measurement of p42/44MAPK-dependent
PHAS-I Phosphorylation--
p42/44MAPK were purified by
standard immunoprecipitation. Briefly, 400 µg of protein from cell
lysates were dissolved in Buffer A (in mM: 10 Tris, 5 EDTA,
50 NaCl, 30 sodium pyrophosphate, 50 NaF, 0.2 phenylmethylsulfonyl
fluoride, 2 orthovanadate, 0.1 mg/ml leupeptin) with 1% SDS and 2 µl
of polyclonal antibody against extracellular signal-regulated kinase
(Santa Cruz Biotechnology). The mixture was agitated for 90 min at
4 °C and then 18 µl of protein G plus/A agarose was added. After a
1-h agitation at 4 °C the agarose beads were washed twice with
Buffer A and sedimented. The pellets were used for measurement of MAP
kinase activity as described above.
It has been shown that stimulation of pancreatic acinar cells with
low doses of CCK causes rapid activation of cPLA2 and
thereby formation of AA, which slows down spreading of cytosolic
Ca2+ signals (6). To test whether ACh and bombesin
receptors also couple to the signal cascade involving cPLA2
activation we analyzed the propagation rate of ACh- and bombesin-evoked
Ca2+ waves in the presence of AA, the product of
cPLA2 activity, or the cPLA2 inhibitor
arachidonyltrifluoromethyl ketone (AACOCF3) (Fig.
1A). Preincubation of acinar
cells with 5 µM AA for 5 min caused slow-down of both
ACh- and bombesin-evoked Ca2+ waves. This observation is
consistent with the idea that AA inhibits secondary CICR from
intracellular stores and therefore causes slower spreading of cytosolic
Ca2+ signals (4). The two concentrations of ACh were chosen
as a typical physiological and a supramaximal agonist concentration. The concentration dependence of the propagation rate of ACh-evoked Ca2+ waves is presented in Fig. 1B. For a
reproducible initiation of Ca2+ waves at least 20 nM ACh were necessary. ACh concentrations above 500 nM did not significantly further increase the spreading
speed of Ca2+ waves. Fitting of the data with a Hill
equation yielded a half-maximal propagation rate at 108 nM
ACh. On the other hand, bombesin-evoked Ca2+ waves had more
or less the same propagation rate (~ 5 µm/s) over a wide
concentration range (100 pM to 5 nM) (Fig.
1C). Only at very low bombesin concentration (30 pM) a slightly decreased propagation rate (3.7 µm/s) and
at supramaximally high concentration (10 nM) a slightly
increased propagation rate (6.4 µm/s) could be measured. For all
further experiments we used bombesin at a concentration of 1 nM.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP
(1 µCi/µl), and the volume of the reaction mixture was adjusted with H20 to 25 µl. In control experiments 1 µl of
activated MAP kinase (0.05 µg/µl) (Stratagene, Heidelberg, Germany)
was used instead of cell lysate. The phosphorylation reaction was
started by adding 5 µl of PHAS-I (1.0 µg/µl) (Stratagene,
Heidelberg, Germany). After a 20-min incubation at 30 °C, reactions
were stopped by the addition of 10 µl of 3× SDS polyacrylamide gel
electrophoresis sample buffer and heating of the samples for 5 min to
95 °C. Proteins were separated in a 15% denaturing acrylamide gel
by gel electrophoresis. Finally, gels were washed and exposed to x-ray
film for 2-4 days at
70 °C. The autoradiographies showed the
phosphorylated substrate protein PHAS-I as a predominant signal at
~21 kDa.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
A, effect of arachidonic acid and of the
cPLA2 inhibitor AACOCF3 on the propagation rate
of ACh- and bombesin-evoked Ca2+ waves. Cells were
incubated with either 5 µM AA or 10 µM
AACOCF3 for 5 min before Ca2+ waves were
elicited with the respective agonist. Application of AA reduces the
spreading speed of ACh- and bombesin-evoked Ca2+ waves,
whereas inhibition of cPLA2 only affected ACh-induced
Ca2+ waves. B, Concentration dependence of the
propagation rate of ACh-evoked Ca2+ waves. The half-maximal
value of the Hill function was 108 nM ACh. The
concentration dependence of Ca2+ wave propagation rates for
bombesin is given in C. n.s., not significant;
**, p < 0.01; ***, p < 0.001.
When cells were preincubated with the cPLA2 inhibitor AACOCF3 (10 µM) for 5 min and then stimulated with ACh, a faster propagation of Ca2+ waves could be observed at low (50 nM), as well as high (10 µM), ACh concentrations. In contrast, when bombesin was used for stimulation, AACOCF3 had no effect on Ca2+ wave spreading. The experiments with AACOCF3 indicate a rapid activation of cPLA2 in the presence of ACh but not in the presence of bombesin. Because AACOCF3 did not affect bombesin-evoked Ca2+ waves an unspecific effect of AACOCF3 on cytosolic Ca2+ signaling is unlikely.
The enzymatic metabolization of AA produces a large number of
regulatory active metabolites. To investigate whether AA itself or one
of its metabolites exerts the inhibitory effect on sequential Ca2+ release we stimulated pancreatic acinar cells in the
presence of either the cyclooxygenase inhibitor indomethacin (10 µM, 5 min preincubation) or the lipoxygenase inhibitor
nordihydroguaiaretic acid (NDGA; 30 µM, 5 min
preincubation). Both indomethacin and NDGA slowed down spreading of
Ca2+ waves elicited at low or high ACh concentrations (Fig.
2A). This demonstrates that AA
itself and not a metabolite of AA slows down spreading of cytosolic
Ca2+ waves by down-regulation of secondary Ca2+
release. Ca2+ waves elicited by bombesin were affected
neither by indomethacin nor by NDGA indicating that these substances do
not nonspecifically change Ca2+ signaling (Fig.
2A).
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It is known that in pancreatic acinar cells stimulation of high affinity CCK receptors leads to strong activation of cPLA2 (10) and rapid formation of AA, which causes slow spreading of cytosolic Ca2+ waves (6). The propagation rate of Ca2+ waves at low CCK concentration (20 pM) could not further be reduced by addition of exogenous AA (6). This was explained by production of already saturating endogenous AA concentrations in the presence of low CCK concentrations. Therefore, it is not surprising that now, in a new series of experiments, indomethacin and NDGA had no or only a small effect, respectively, on the propagation rate of Ca2+ waves elicited by activation of high affinity CCK receptors (Fig. 2B). However, when low affinity CCK receptors were stimulated by application of CCK at supramaximal concentration (10 nM), indomethacin and NDGA significantly reduced the propagation rate of Ca2+ waves (Fig. 2B). The differences in the effect of indomethacin and NDGA on Ca2+ waves elicited by low and high concentrations of CCK, respectively, might be because of different delays between hormone application and the initial rise in [Ca2+]cyt. The delay in the Ca2+ response to 50 pM CCK was 8.7 s longer than in the response to 10 nM CCK. The short latency in the presence of high CCK concentrations allows only very little production of AA, which is reflected by a fast spreading of Ca2+ waves. In this case, inhibition of AA metabolization by indomethacin and NDGA increases the amount of AA and therefore slows down the propagation of Ca2+ waves. On the other hand, the long latency between application of 50 pM CCK and the initial Ca2+ signal in the cytosol gives time for production of high AA concentrations. Spreading of Ca2+ waves in the presence of low CCK concentrations is therefore slow and, if AA reaches saturating concentrations, cannot be further slowed down by inhibition of AA metabolization.
There is growing evidence that the MAP kinases p42/44MAPK
and p38MAPK can regulate the activity of cPLA2
(11-15). To investigate whether in mouse pancreatic acinar cells
p42/44MAPK and p38MAPK are also involved in
agonist-induced activation of cPLA2 we analyzed the
propagation rate of cytosolic Ca2+ waves in the presence
and absence of the specific MAP kinase inhibitors PD 98059 and SB
203580. Inhibition of p42/44MAPK by preincubation of cells
with 10 µM PD 98059 for 5 min significantly accelerated
the speed of CCK-induced Ca2+ waves at low and high agonist
concentration, whereas a slower spreading of CCK-induced
Ca2+ waves could be observed when cells were preincubated
with the p38MAPK inhibitor SB 203580 (10 µM)
for 5 min (Fig. 3B). In
contrast, neither PD 98509 nor SB 203580 had any significant effect on
Ca2+ waves elicited with bombesin (Fig. 3A).
When ACh was used for stimulation, preincubation of the cells with the
p38MAPK inhibitor SB 203580 (10 µM) caused a
significant slow-down of Ca2+ waves at both low and high
ACh concentrations. On the other hand, inhibition of
p42/44MAPK with PD 98059 produced a faster spreading of
Ca2+ waves only when 50 nM ACh was used for
initiation of Ca2+ waves but had no significant effect on
Ca2+ waves elicited by 10 µM ACh. A possible
explanation for this observation might be that in pancreatic acinar
cells, similar as in other cell types, several parallel pathways exist
that activate cPLA2 (16, 17). In the experiments with high
ACh the inhibitory effect of p38MAPK could prevail over the
stimulatory effect of p42/44MAPK. Nevertheless, activation
of cPLA2 could be accomplished by a parallel pathway
bypassing the p42/44 MAP kinase pathway. In consequence, we would have
little effect of PD 98059 that might be overlooked in our experiments,
whereas inhibition of p38MAPK with SB 203580 (Fig.
3A) or inhibition of cPLA2 with
AACOCF3 (Fig. 1A) would produce clear effects.
The data suggest that in mouse pancreatic acinar cells activation of
p42/44MAPK and p38MAPK is an early event in
stimulus-secretion coupling that already occurs before cytosolic
Ca2+ signals are generated. Furthermore, the data suggest
that there is a regulatory cross-talk between p42/44MAPK
and p38MAPK. cPLA2 activation is promoted by
p42/44MAPK whereas p38MAPK has an inhibitory
effect.
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Agonist-induced phosphorylation of p42/44MAPK and
p38MAPK could also be demonstrated by Western blot analysis
with antibodies recognizing the activated form of
p42/44MAPK and p38MAPK. Stimulation of
pancreatic acinar cells with either CCK or ACh caused a significant
increase in p42/44MAPK phosphorylation within 10 s
after hormone application (Fig.
4A). When bombesin was used
instead, after 10 s there was no significant increase in
p42/44MAPK phosphorylation compared with control
experiments without agonist (Fig. 4A). Stimulation of acinar
cells with ACh and CCK also caused an increase in phosphorylation of
p38MAPK, whereas no increase in p38MAPK
phosphorylation could be found 10 s after addition of bombesin (Fig. 4B). Agonist-dependent activation of MAP
kinases could be demonstrated in in vitro experiments using
exogenously added PHAS-I as MAP kinase phosphorylation substrate (Fig.
4C) (9). A significantly higher amount of phosphorylated
PHAS-I could be detected when cells were stimulated with ACh or CCK for
10 s, whereas there was no significant increase in PHAS-I
phosphorylation in experiments with bombesin. MAP kinase activation in
the presence of bombesin could only be observed when cells were
stimulated for more than 1 min (Fig. 4C). However, this late
activation of MAP kinases in the presence of bombesin occurs when the
agonist-evoked cytosolic Ca2+ wave had already traveled
through the cell, and it is therefore not surprising that MAP kinase
inhibitors could not affect the propagation rate of bombesin-elicited
Ca2+ waves.
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To confirm a regulatory cross-talk between p38MAPK and
p42/44MAPK we stimulated pancreatic acinar cells with ACh
in the presence and absence of MAP kinase inhibitors and measured
p42/44MAPK-dependent PHAS-I phosphorylation
after cell lysis and immunoprecipitation of p42/44MAPK.
Stimulation of acinar cells with 1 µM ACh for 30 s
produced an ~2-fold increase in
p42/44MAPK-dependent PHAS-I phosphorylation
compared with control experiments without ACh (Fig. 4D).
More or less the same p42/44MAPK-dependent
PHAS-I phosphorylation as under control conditions could be found when
cells were treated with either 10 µM PD 98059 or 10 µM SB 203580 for 5 min prior to cell lysis. Activity of p42/44MAPK was measured after immunoprecipitation of
p42/44MAPK and removal of PD 98059 and SB 203580, respectively. When cells were preincubated with PD 98059 and then
stimulated with ACh in the presence of PD 98059 (Fig. 4D,
4th column), about the same increase in
p42/44MAPK activity could be detected as in stimulation
experiments without PD 98059 (Fig. 4D, 2nd
column). This is not astonishing, because the presence of PD 98059 during stimulation of the cells with ACh inhibits
p42/44MAPK activity but not ACh-dependent
p42/44MAPK activation. For the measurement of
p42/44MAPK activity PD 98059 was removed by
immunoprecipitation of p42/44MAPK. On the other hand when
cells were stimulated with ACh in the presence of the
p38MAPK inhibitor SB 203580 a much higher
p42/44MAPK activity could be observed after
immunoprecipitation of p42/44MAPK. These experiments
indicate that stimulation of pancreatic acinar cells with ACh causes
rapid activation of both p42/44MAPK and
p38MAPK. Activation of p42/44MAPK is negatively
controlled by simultaneous activation of p38MAPK.
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DISCUSSION |
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The propagation rate of agonist-evoked Ca2+ waves in mouse pancreatic acinar cells is modulated by activation of PKC- and cPLA2-dependent formation of AA (7). Measurement of the propagation speed of Ca2+ waves can therefore be used to demonstrate agonist-dependent activation of PKC and cPLA2 in single cells within the first seconds after hormone application. By measurements of Ca2+ waves we could show in a previous study that in pancreatic acinar cells high affinity CCK receptors couple to a signal cascade that leads to activation of cPLA2 (6). Activation of cPLA2 and formation of AA occurred before the CCK-evoked Ca2+ wave spread from the luminal to the basal cell pole. In the present study we show that preincubation of cells with the cPLA2 inhibitor AACOCF3 also accelerates ACh-evoked Ca2+ waves whereas bombesin-evoked Ca2+ waves were unaffected. We therefore conclude that ACh receptors activate the same signal cascade as high affinity CCK receptors, whereas in bombesin-induced Ca2+ signaling the cPLA2 pathway is not involved. The fact that Ca2+ waves evoked by 10 µM ACh are traveling much faster than Ca2+ waves elicited by low ACh concentrations might be because of the different delays between agonist binding and initiation of cytosolic Ca2+ waves. Ca2+ waves elicited by 10 µM ACh started 1.8 ± 0.2 s (n = 94) after hormone application, whereas Ca2+ signals elicited by 50 nM ACh had a delay of 4.7 ± 0.5 s (n = 86). Therefore, the amount of AA produced before initiation of Ca2+ waves might be higher in the presence of 50 nM ACh. This means stronger inhibition of secondary Ca2+ release and slower spreading of Ca2+ waves in the presence of 50 nM ACh compared with experiments with 10 µM ACh.
In a previous study we showed that the inhibitory effects of activated PKC and AA on Ca2+ wave spreading are not additive, suggesting that both signal pathways converge to the same Ca2+ release mechanism (6). However, the molecular mechanism by which AA inhibits Ca2+ release is still unclear. Because inhibition of cyclooxygenase and lipoxygenase with indomethacin and NDGA slowed down CCK- and ACh-evoked Ca2+ waves, we can conclude that AA itself and not a metabolite exerts the inhibitory effect on Ca2+ wave spreading. With electrophysiological methods an inhibitory effect of AA has also been shown on inositol polyphosphate-evoked long term Ca2+ signaling in mouse (18) and rat (19) pancreatic acinar cells. As in our experiments this long term effect of AA could not be abolished by inhibition of AA metabolization with indomethacin and NDGA (19).
Stimulation of pancreatic acinar cells with CCK causes activation of p42/44MAPK and p38MAPK (20-22) and initiates protein synthesis of pancreatic digestive enzymes by MAP kinase-dependent phosphorylation of the pancreatic translation factor eIF4E and its binding protein PHAS-I (20, 22). Furthermore, it has been shown that in pancreatic acinar cells due to the tissue isolation procedure cytokine expression can be up-regulated by p38MAPK (23). Our experiments indicate that besides this long term effect on protein synthesis MAP kinases are also part of feedback mechanisms that control agonist-evoked Ca2+ signaling. With biochemical methods we could demonstrate that there is a significant phosphorylation of both p42/44MAPK and p38MAPK within 10 s after ACh and CCK application. Furthermore, the analysis of the propagation rate of Ca2+ waves in the presence and absence of MAP kinase inhibitors gives evidence that MAP kinase activation already occurs within the delay between agonist application and initiation of cytosolic Ca2+ signals. This means that there is already significant MAP kinase activation within less than 1.8 s after ACh (10 µM) or CCK (10 nM) application. p42/44MAPK and p38MAPK exert antagonizing effects on Ca2+ wave spreading; p42/44MAPK activation causes slower propagation of Ca2+ waves whereas activation of p38MAPK causes faster Ca2+ wave spreading. This might be explained by a regulatory cross-talk between p38MAPK and p42/44MAPK. Inhibition of the p42/44MAPK signaling cascade by p38MAPK has been shown in a study on the human hepatoma cell line HepG2 (24). The rapid activation of p38MAPK might also be involved in reorganization of actin cytoskeleton (21) and regulation of enzyme secretion.
Our data indicate that analysis of the propagation rate of
agonist-evoked Ca2+ waves is a powerful tool to study
coupling of different hormone receptors to intracellular signal
cascades. In the present study we demonstrate that in pancreatic acinar
cells not only high affinity CCK but also ACh receptors couple to the
cPLA2 pathway. cPLA2-dependent formation of AA is an early event in intracellular signaling and exerts
a negative control on Ca2+ wave spreading.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (Schm 876/2-1 and SFB 530).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.
Present address: Dept. of Physiology, University of Extremadura,
Faculty of Veterinary Sciences, Avenida Universidad, P. O. Box 643, 10071 Cáceres, Spain.
§ To whom correspondence should be addressed. Tel.: 49-6841-166454; Fax: 49-6841-166655; E-mail: andreas.schmid@med-rz.uni-saarland.de.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M101136200
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ABBREVIATIONS |
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The abbreviations used are: ACh, acetylcholine; AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; bom, bombesin; NDGA, nordihydroguaiaretic acid; MAP, mitogen-activated protein; MAPK, MAP kinase; CCK, cholecystokinin; CICR, Ca2+-induced Ca2+ release; PKC, protein kinase C; AACOCF3, arachidonyltrifluoromethyl ketone.
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