Arachidonic Acid Modulates the Spatiotemporal Characteristics of Agonist-evoked Ca2+ Waves in Mouse Pancreatic Acinar Cells*

Gregor Siegel, Lutz Sternfeld, Antonio GonzálezDagger, Irene Schulz, and Andreas Schmid§

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 [gamma -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.

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


View larger version (16K):
[in this window]
[in a new window]
 
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).


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 2.   The effects of the cyclooxygenase inhibitor indomethacin (indo.) and of the lipoxygenase inhibitor NDGA on the propagation rate of ACh-, bombesin- and CCK-evoked Ca2+ waves are shown. Indomethacin (10 µM) and NDGA (30 µM) were added to the bath solution 5 min before the respective agonist was added. n.s., not significant; *, p < 0.05; ***, p < 0.001.

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.


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 3.   The effect of MAP kinase inhibitors PD 98059 and SB 203580 on the propagation rate of ACh-, bombesin- and CCK-evoked Ca2+ waves is shown. PD 98059 (10 µM) or SB 203580 (10 µM) was added to the bath solution 5 min before the respective agonist was added. n.s., not significant; ***, p < 0.001.

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.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 4.   Stimulation of pancreatic acinar cells with ACh or CCK for 10 s causes a significant increase in the phosphorylation of p42/44MAPK (A) and p38MAPK (B). No increase in MAP kinase phosphorylation could be found when bombesin was applied for 10 s. MAP kinase phosphorylation was detected with antibodies against the phosphorylated form of p42/44MAPK and p38MAPK (middle panels). Antibodies raised against the non-phosphorylated form were used as control (upper panels). MAP kinase activation is demonstrated using PHAS-I as substrate (C). In a 10-s period after ACh and CCK application MAP kinase activity increased about 40% compared with control experiments. Short term stimulation with bombesin had no effect on MAP kinase activity. However, when bombesin was present for more than 1 min this agonist caused MAP kinase activation. Negative regulation of p42/44MAPK by p38MAPK could be demonstrated when p42/44MAPK-dependent PHAS-I phosphorylation was measured after cell lysis and immunoprecipitation of p42/44MAPK (D). Inhibition of p38MAPK activity with SB 203580 during cell stimulation with ACh caused a significantly higher p42/44MAPK activity than in the absence of SB 203580. n.s., not significant; *, p < 0.05; **, p < 0.01.

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.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* 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.

Dagger 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

    ABBREVIATIONS

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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Kasai, H., and Augustine, G. J. (1990) Nature 348, 735-738[CrossRef][Medline] [Order article via Infotrieve]
2. Kasai, H., Li, Y. X., and Miyashita, Y. (1993) Cell 74, 669-677[Medline] [Order article via Infotrieve]
3. Thorn, P., Lawrie, A. M., Smith, P. M., Gallacher, D. V., and Petersen, O. H. (1993) Cell 74, 661-668[Medline] [Order article via Infotrieve]
4. Schulz, I., Krause, E., González, A., Göbel, A., Sternfeld, L., and Schmid, A. (1999) Biol. Chem. Hoppe-Seyler 380, 903-908
5. Straub, S. V., Giovannucci, D. R., and Yule, D. I. (2000) J. Gen. Physiol. 116, 547-560[Abstract/Free Full Text]
6. González, A., Schmid, A., Sternfeld, L., Krause, E., Salido, G. M., and Schulz, I. (1999) Biochem. Biophys. Res. Commun. 261, 726-733[CrossRef][Medline] [Order article via Infotrieve]
7. Pfeiffer, F., Sternfeld, L., Schmid, A., and Schulz, I. (1998) Am. J. Physiol. 274, C663-C672[Medline] [Order article via Infotrieve]
8. González, A., Schulz, I., and Schmid, A. (2000) J. Biol. Chem. 275, 38680-38686[Abstract/Free Full Text]
9. Haystead, T. A., Haystead, C. M., Hu, C., Lin, T. A., and Lawrence, J. C., Jr. (1994) J. Biol. Chem. 269, 23185-23191[Abstract/Free Full Text]
10. Tsunoda, Y., and Owyang, C. (1995) Am. J. Physiol. 269, G435-G444[Abstract/Free Full Text]
11. Kramer, R. M., Roberts, E. F., Um, S. L., Borsch-Haubold, A. G., Watson, S. P., Fisher, M. J., and Jakubowski, J. A. (1996) J. Biol. Chem. 271, 27723-27739[Abstract/Free Full Text]
12. Wheeler-Jones, C., Abu-Ghazaleh, R., Cospedal, R., Houliston, R. A., Martin, J., and Zachary, I. (1997) FEBS Lett. 420, 28-32[CrossRef][Medline] [Order article via Infotrieve]
13. Hiller, G., and Sundler, R. (1999) Cell. Signal. 11, 863-869[CrossRef][Medline] [Order article via Infotrieve]
14. Syrbu, S. I., Waterman, W. H., Molski, T. F., Nagarkatti, D., Hajjar, J. J., and Sha'afi, R. I. (1999) J. Immunol. 162, 2334-2340[Abstract/Free Full Text]
15. Fonteh, A. N., Atsumi, G., LaPorte, T., and Chilton, F. H. (2000) J. Immunol. 165, 2773-2782[Abstract/Free Full Text]
16. Lin, W. W., and Chen, B. C. (1998) Br. J. Pharmacol. 125, 1601-1609[Abstract]
17. Balsinde, J., Balboa, M. A., Li, W. H., Llopis, J., and Dennis, E. A. (2000) J. Immunol. 164, 5398-5402[Abstract/Free Full Text]
18. Rowles, S. J., and Gallacher, D. V. (1996) Biochem. J. 319, 913-918[Medline] [Order article via Infotrieve]
19. Maruyama, Y. (1993) J. Physiol. (Lond.) 463, 729-746[Abstract]
20. Bragado, M. J., Groblewski, G. E., and Williams, J. A. (1998) Gastroenterology 115, 733-742[Medline] [Order article via Infotrieve]
21. Schäfer, C., Ross, S. E., Bragado, M. J., Groblewski, G. E., Ernst, S. A., and Williams, J. A. (1998) J. Biol. Chem. 273, 24173-24180[Abstract/Free Full Text]
22. Bragado, M. J., Tashiro, M., and Williams, J. A. (2000) Gastroenterology 119, 1731-1739[Medline] [Order article via Infotrieve]
23. Blinman, T. A., Gukovsky, I., Mouria, M., Zaninovic, V., Livingston, E., Pandol, S. J., and Gukovskaya, A. S. (2000) Am. J. Physiol. 279, C1993-C2003[Abstract/Free Full Text]
24. Singh, R. P., Dhawan, P., Golden, C., Kapoor, G. S., and Mehta, K. D. (1999) J. Biol. Chem. 274, 19593-19600[Abstract/Free Full Text]


Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.