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Cholecystokinin-Evoked Ca2+ Waves in Isolated Mouse Pancreatic Acinar Cells Are Modulated by Activation of Cytosolic Phospholipase A2, Phospholipase D, and Protein Kinase C
Biochemical and Biophysical Research Communications 261, 726 –733 (1999) Article ID bbrc.1999.1106, available online at http://www.idealibrary.com on
Cholecystokinin-Evoked Ca 21 Waves in Isolated Mouse Pancreatic Acinar Cells Are Modulated by Activation of Cytosolic Phospholipase A 2, Phospholipase D, and Protein Kinase C Antonio Gonza´lez,* Andreas Schmid,* Lutz Sternfeld,* Elmar Krause,* Gine´s M. Salido,† and Irene Schulz* *Department of Physiology II, Faculty of Medicine, University of Saarland, D-66421, Homburg/Saar, Germany; and †Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, 10079, Ca´ceres, Spain
Received May 17, 1999
We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca 21 signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8induced Ca 21 signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca 21 waves elicited by stimulation of highaffinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a.CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca 21 release was the same at both CCK-8 concentrations, suggesting that the secondary Ca 21 release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca 21 waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a.CCK-R are linked to phospholipase C (PLC) and phospholipase A 2 (PLA 2) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA 2 and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca 21 wave propagation by yet unknown mechanisms. © 1999 Academic Press Key Words: pancreas; calcium wave; CCK-8; PKC, PLA 2; PLD.
Abbreviations used: AA, arachidonic acid; AACOCF 3, arachidonyl trifluoromethyl-ketone; BEL, bromoenol lactone; CICR, calcium-induced calcium release; CCK-8, cholecystokinin octapeptide; h.a.CCK-R, high-affinity cholecystokinin-receptor; l.a.CCK-R, low-affinity cholecystokinin-receptor; [Ca 21] i, intracellular free calcium concentrationl DAG, diacylglycerol; MAFP, methyl-arachidonyl-fluorophosphonate; MAPkinase, mitogen-activated protein-kinase; PMA, phorbol-12-myristate13-acetate; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PA, phosphatidic acid; PC, phosphatidylcholine; cPLA 2, cytosolic phospholipase A 2; iPLA 2, calcium-independent phospholipase A 2; sPLA 2, secretory phospholipase A 2. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
In the exocrine pancreas high- and low-affinity cholecystokinin (CCK) receptors exist. Therefore, depending on the concentration, CCK binds either only to the high or to a variable extent to high and low affinity binding sites (1). This may result in generation of different second messengers in the signal cascades leading to amylase secretion. It has been shown that activation of high affinity CCK-receptors (h.a.CCK-R) by low CCK concentrations (,100 pM) is coupled to phospholipase A 2 (PLA 2), and probably also involves phospholipase C (PLC)-mediated inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) production to mediate Ca 21 oscillations, protein kinase C (PKC) activation and amylase secretion. In contrast, lowaffinity CCK receptors (l.a.CCK-R), which are activated at high CCK concentrations (.100 pM) are coupled to the PLC pathway without implication of PLA 2 (1– 6). Activation of PLC causes breakdown of phosphatidylinositol 4,5-bisphosphate (PIP 2) to IP 3 and DAG. IP 3 induces Ca 21 release from intracellular non-mitochondrial stores with a consequent rise in cytosolic free Ca 21 concentration ([Ca 21] i), whereas DAG activates PKC (7, 8). Another source for DAG is phosphatidylcholine (PC) which can be cleaved by phospholipase D (PLD) to choline and phosphatidic acid (PA). In a second step PA is dephosphorylated by a phosphatidic acid phosphohydrolase to DAG (9). In addition to these two well characterized second messengers, IP 3 and DAG, also arachidonic acid (AA) has been recognized as an important factor in hormonal stimulation of different tissues and cell types (10). AA can be liberated via PLA 2 from PC as well as from DAG by a DAG-lipase (11). Furthermore, DAG is generated by PLC or by sequential activation of PLD and phosphatidate phosphohydrolase (12). AA is the precursor of several bioactive molecules, termed eico-
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sanoids, including prostaglandins, leukotrienes and thromboxanes (10). It has been reported that in isolated pancreatic acini AA released in response to hormonal stimulation induces amylase secretion (11, 13–15), and that it releases Ca 21 from the endoplasmic reticulum (6). Other studies have shown an inhibitory effect of AA in pancreatic secretion (16) as well as in IP 3-dependent Ca 21 mobilization (17, 18). Furthermore, inhibition of PLA 2 potentiates IP 3-induced Ca 21-activated currents in mouse pancreatic acinar cells (19). The role of PLD in stimulation of exocrine secretion is not clear at present. It has been suggested that inhibition of PLD has no consequence for the secretory response in the exocrine pancreas (20). However, in studies carried out on rat submandibular acinar cells (21) PLD has been shown to play a role in signal transduction. In the present study, carried out in isolated mouse pancreatic acinar cells, we have tested if modulation of PKC-, PLA 2- and PLD-activities has any effects on the propagation of Ca 21 signals. In pancreatic acinar cells hormone-evoked Ca 21 signals start in the luminal cell pole and then spread towards the basolateral side of the cell (22). Ca 21 wave propagation has been explained by sequential Ca 21 release from Ca 21 stores in series involving calcium-induced calcium release (CICR) (23–25). In a previous study we have demonstrated that activation of PKC slows down ACh-evoked Ca 21 wave propagation, probably due to inhibition of CICR (24). In the present study we have monitored the propagation of Ca 21 signals in response to CCK-8. We have employed confocal laser scanning microscopy to follow local changes of [Ca 21] i in individual mouse pancreatic acinar cells loaded with the fluorescent probe fluo-3. The results show that the propagation of CCK-evoked Ca 21 waves in mouse pancreatic acinar cells can be modulated by activation of cPLA 2, PLD and PKC. Concentration-dependent differences in the pattern of Ca 21 mobilization could be explained by differential coupling of high and low affinity CCK-R to intracellular phospholipase-mediated signaling pathways. MATERIALS AND METHODS Materials Fluo-3/AM was obtained from Molecular Probes Inc. (Europe), Ro31-8220, phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA) from Calbiochem-Novabiochem Corp. (Germany), arachidonyl trifluoromethyl-ketone (AACOCF 3) from Biomol Res. Lab. (Germany), methyl-arachidonyl-fluorophosphonate (MAFP) and bromoenol lactone (BEL) from Cayman Chemical Company (Germany) and n-butanol from Merck-Schuchardt (Germany). All other materials used were obtained from Sigma Chemicals Co. (Germany).
Methods Preparation of isolated acinar cells. Adult male CD-1 mice (35– 40 g) were used for this study. They had free access to water and
food. Animals were sacrificed by cervical dislocation, the pancreas was rapidly removed and the acinar cells were isolated as described previously by a collagenase digestion method (24). With this isolation procedure single cells as well as small clusters consisting of 2 up to 5 cells were obtained. Fluorescence measurements. Freshly isolated mouse pancreatic acinar cells were loaded with 4 mM fluo-3/AM for 30 min at room temperature (22°C) following previously established methods (26). After dye loading, the cells were kept at 4°C and the experiments were performed within the next 4 h. For monitoring Ca 21-dependent fluorescence signals, aliquots of the cell suspension were placed onto polylysine coated glass coverslips attached to the bottom of a perfusion chamber. The cells were continuously superfused with a NaHepes buffer containing in mM: 140 NaCl, 4.7 KCl, 1.3 CaCl 2, 1 MgCl 2, 10 Hepes, 10 glucose, pH adjusted to 7.4 with NaOH. Employing a confocal laser-scanning system (Bio-Rad, MRC-1024) fluorescence images (excitation 488 nm/emission . 515 nm) of 128 3 128 pixels with a resolution of 0.526 mm/pixel were recorded every 0.25 s. Small rectangular areas were selected in the luminal and the basolateral pole of the cells. The difference in time between the increase in [Ca 21] i at both cell regions was determined and the speed (mm/s) of the CCK-induced Ca 21 signal was calculated. All experiments were performed at room temperature. Determination of intracellular free calcium concentration. Absolute values of intracellular free calcium concentration ([Ca 21 ] i ) were calculated from the self-ratio method described by Cheng et al. (27) for the non-ratiometric dye fluo-3, assuming a dissociation constant of 400 nM for Ca 21 binding of fluo-3 and a resting [Ca 21 ] i of 100 nM (2). Analysis of data. Data show the mean [Ca 21] i expressed in nM 6 SEM and the mean propagation rate of Ca 21 waves expressed as mm/s 6 SEM. Statistical analysis was performed by Student9s t test and only P values less than 0.05 were considered as significant.
RESULTS Cholecystokinin-Evoked Ca 21 Signals As shown in Fig. 1 stimulation of pancreatic acinar cells with 20 pM or 10 nM CCK-8 resulted in an initial increase in [Ca 21] i at the luminal cell pole and subsequent spreading of the Ca 21 signal towards the basolateral cell membrane. From the distance between both areas selected at each side of the cell and the difference in time between the increase in the fluorescence in these regions, we could determine the speed of the Ca 21 wave. The speed of Ca 21 wave in response to CCK-8 was dependent on the concentration used, being faster at higher concentrations of CCK-8. The mean values for the propagation rate of Ca 21 signals in response to the CCK-8 concentrations are summarized in Fig. 2A. In addition to a slower propagation rate of Ca 21 signals, a larger delay between hormone application and the initial Ca 21 release was observed at the lower concentrations of CCK-8 (Fig. 2B). It has been described that CCK-8 at low concentrations (,100 pM) stimulates amylase secretion via activation of h.a.CCK-R, whereas at high CCK-8 concentrations (.100 pM) amylase release is reduced when l.a.CCK-R are activated (1). To investigate the role of high and low affinity CCK-R activation on cytosolic Ca 21 signals we stimulated cells with either 20 pM or
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1 mM of the phorbol ester PMA to activate PKC, spreading of Ca 21 waves in response to both CCK-8 concentrations was significantly slower compared to the controls (CCK-8 20 pM: 5.28 6 0.27 mm/s, 43 experiments/89 cells; CCK-8 20 pM 1 1 mM PMA: 4.05 6 0.53 mm/s, 20 experiments/44 cells, P , 0.05; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 1 mM PMA: 12.22 6 0.53 mm/s, 34 experiments/83 cells, P , 0.001). Preincubation with 5 mM of the PKC-inhibitor Ro31-8220 produced a faster propagation of Ca 21 waves at 10 nM CCK-8. The mean speed of Ca 21 waves in response to 20 pM CCK-8 was unchanged by this treatment (Fig. 3A) (CCK-8 20 pM: 5.28 6 0.27 mm/s, 43 experiments/89 cells; CCK-8 20 pM 1 5 mM Ro31-8220: 5.21 6 0.44 mm/s, 22 experiments/42 cells; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 5 mM Ro318220: 22.10 6 1.89 mm/s, 31 experiments/65 cells, P , 0.001). Effect of AA on CCK-Evoked Ca 21 Waves
FIG. 1. Time-course of Ca 21 wave propagation in mouse pancreatic acinar. (A) Small areas located at the luminal (lu) and basolateral (ba) pole of the cells were selected and the changes in fluorescence were monitored employing a confocal laser-scanning microscope. Following stimulation, the fluorescence first increased at the luminal cell pole and then spread as a Ca 21 wave towards the basolateral pole of the cells. The time between the increase in fluorescence at both sides of the cell was monitored and the speed of the Ca 21 wave was determined. Following stimulation of the cells with 20 pM CCK-8, which only activates the h.a.CCK-R, the Ca 21 wave traversed the cell within 1.69 6 0.10 s, corresponding to a speed of the Ca 21 wave of 7.78 6 0.52 mm/s (n 5 12 experiments/24 cells). (B) When cells were stimulated with a higher concentration, 10 nM CCK-8, that activates both h.a.CCK-R and l.a.CCK-R, the Ca 21 wave spread from the luminal to the basolateral pole within 0.63 6 0.03 s corresponding to a speed of 21.84 6 1.44 mm/s (n 5 23 experiments/52 cells).
10 nM CCK-8. At 20 pM CCK-8 the speed of Ca 21 wave was 7.78 6 0.51 mm/s (n 5 12 experiments/24 cells) and at 10 nM CCK-8 it was 21.84 6 1.43 mm/s (n 5 23 experiments/52 cells). The initial rise in [Ca 21] i in the luminal cell pole required about 13 more seconds with 20 pM CCK-8 than with 10 nM CCK-8. However, the magnitude of the Ca 21 signal in the luminal cell pole was not significantly different at both CCK-8 concentrations. Table 1 summarizes the values for [Ca 21] i in the luminal cell pole at 0.5 s and 1 s after beginning of the Ca 21 signal as well as at its maximum. Effect of PKC on CCK-Evoked Ca 21 Waves When pancreatic acinar cells were stimulated with CCK-8 following 5 min preincubation in the presence of
When pancreatic acinar cells were stimulated with 10 nM CCK-8 after 5 min preincubation with 5 mM of AA, spreading of the CCK-8-induced Ca 21 wave was much slower compared to the controls. On the other hand, AA did not change the propagation rate of Ca 21 waves elicited by stimulation of the cells with 20 pM CCK-8 (Fig. 3B) (CCK-8 20 pM: 4.76 6 0.40 mm/s, 24 experiments/51 cells; CCK-8 20 pM 1 5 mM AA: 5.38 6 0.66 mm/s, 9 experiments/26 cells; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 5 mM AA: 10.22 6 0.82 mm/s, 22 experiments/54 cells, P , 0.001). AA, in the concentration we used in our experiments, had no effect on the resting [Ca 21] i since changes in the fluorescence baseline were not observed during pretreatment of cells with AA. To decide if the effect of both AA and PKC-activation on CCK-induced Ca 21 waves are additive or not, we performed a set of experiments in which pancreatic acinar cells were pretreated with both AA and PMA for 5 min prior to stimulation with 10 nM CCK-8. Under these conditions we did not observe any higher reduction in the speed of Ca 21 waves compared to the effect of these agents applied separately (data not shown). Effect of PLA 2 Inhibition on CCK-Evoked Ca 21 Waves Three groups of PLA 2 have been described in mammalian cells: Ca 21-dependent cytosolic PLA 2 (cPLA 2), Ca 21-independent PLA 2 (iPLA 2) and Ca 21-dependent secretory PLA 2 (sPLA 2) (28 –30). To investigate the role of cPLA 2 we examined the effect of the cPLA 2-specific inhibitor AACOCF 3 (31). In the presence of 10 mM AACOCF 3 we could observed a faster propagation of Ca 21 signals at 20 pM CCK-8 compared to the response in the presence of the same concentration of CCK-8 alone. The mean speed of Ca 21 waves in response to 10
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FIG. 2. CCK-8-induced Ca 21 wave propagation in mouse pancreatic acinar cells. (A) Cells were stimulated with increasing concentrations of CCK-8. As a consequence, a faster spreading of the Ca 21 wave could be observed in a dose-dependent manner. Data show the mean speed of propagation of Ca 21 waves in mm/s 6 SEM. (B) Delay in the appearance of luminal Ca 21 response after switching from Na-Hepes buffer to solutions containing the respective concentration of CCK-8. Data show the mean time delay in s 6 SEM. The dashed line indicates the mean lag time produced by our perfusion system.
nM CCK-8 was unchanged by treatment of the cells with AACOCF 3 (Fig. 3B) (CCK-8 20 pM: 4.76 6 0.40 mm/s, 24 experiments/51 cells; CCK-8 20 pM 1 10 mM AACOCF 3: 9.89 6 0.90 mm/s, 14 experiments/35 cells, P , 0.001; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72
experiments/171 cells; CCK-8 10 nM 1 10 mM AACOCF 3: 16.07 6 1.28 mm/s, 16 experiments/34 cells). To study if a Ca 21-independent PLA 2 (iPLA 2) could be involved in production of AA with a consequent effect on the propagation of Ca 21 signals, we preincu-
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Calculated Luminal [Ca 21] i (in nM) after 0.5 s, 1 s, and at Its Maximum after Stimulation of Mouse Pancreatic Acinar Cells with 20 pM and 10 nM CCK-8
CCK-8 20 pM
CCK-8 10 nM
Point/Time
[Ca 21] i, nM
n, expt/cells
Luminal/0.5 s Luminal/1 s Luminal/peak Luminal/0.5 s Luminal/1 s Lumnal/peak
172.70 6 13.06 239.30 6 21.96 376.10 6 57.42 156.50 6 13.39 208.60 6 19.53 305.70 6 33.77
38/83 38/83 38/65 25/56 25/56 25/55
6.40 6 0.45 mm/s, 11 experiments/24 cells; CCK-8 10 nM: 15.13 6 1.53 mm/s, 9 experiments/22 cells; CCK-8 10 nM 1 0.3% 2-butanol: 14.60 6 1.53 mm/s, 11 experiments/28 cells). DISCUSSION
Note. Values show the mean 6 SEM of [Ca 21] i at the luminal cell pole in nM. Absolute values of [Ca 21] i were calculated from the self-ratio method for the nonratiometric dye fluo-3, assuming a dissociation constant of 400 nM for Ca 21 binding of fluo-3 and a resting [Ca 21] i of 100 nM (27). Values obtained after stimulation of cells with 10 nM CCK-8 were compared to those obtained in the same area after stimulation of cells with 20 pM CCK-8 (0.5 s 5 0.5 seconds after the initial rise in Ca 21; 1 s 5 1 second after the initial rise in Ca 21; peak 5 maximal response obtained after the initial rise in Ca 21).
bated pancreatic acinar cells with two known inhibitors of this enzyme, MAPF and BEL (32, 33). Both inhibitors at maximal effective concentrations (25 mM and 50 mM, respectively) did not modify the speed of the Ca 21 waves at both concentrations of CCK-8 tested (data not shown) indicating that iPLA 2 has no implications in the regulation of Ca 21 signals induced by CCK-8. Effect of PLD on CCK-Evoked Ca 21 Waves DAG can be generated by activation of PLC and PLD. DAG activates PKC and therefore slows down the propagation rate of bombesin-evoked Ca 21 signals (24). To investigate whether PLD activation is also involved in the control of CCK-evoked Ca 21 signals, we have preincubated pancreatic acinar cells with 0.3% n-butanol. Activation of PLD in the presence of n-butanol leads to production of the ineffective phosphatidylbutanol instead of PA (34) and therefore should cause a reduction in the DAG-dependent activation of PKC. In the presence of 0.3% n-butanol, activation of l.a.CCK-R by 10 nM CCK-8 led to faster propagation rates of Ca 21 signals (Fig. 3C). However, no change in the mean speed of Ca 21 waves in response to 20 pM CCK-8 was observed (CCK-8 20 pM: 5.24 6 0.37 mm/s, 11 experiments/22 cells; CCK-8 20 pM 1 0.3% n-butanol: 6.50 6 0.54 mm/s, 11 experiments/ 30 cells; CCK-8 10 nM: 15.13 6 1.53 mm/s, 9 experiments/22 cells; CCK-8 10 nM 1 0.3% n-butanol: 21.8 6 2.56 mm/s, 12 experiments/36 cells; P , 0.05). When the ineffective secondary alcohol 2-butanol was used instead of n-butanol, no changes in the propagation speed of Ca 21 waves were observed at both CCK-8 concentrations (CCK-8 20 pM: 5.24 6 0.37 mm/s, 11 experiments/22 cells; CCK-8 20 pM 1 0.3% 2-butanol:
In the exocrine pancreas activation of h.a.CCK-R and l.a.CCK-R leads to generation of different second messengers and therefore produces different biological responses (5). The generation of Ca 21 waves may represent an important early step for the coordination of cellular functions in pancreatic acinar cells (22). Ca 21 wave propagation has been explained by sequential Ca 21 release from Ca 21 stores in series and it has been suggested that the mechanism underlying Ca 21 wave propagation involves CICR (3, 23, 24). Secretagogue-dependent differences in the propagation of cytosolic Ca 21 signals have been shown in pancreatic and parotid acinar cells (24, 35–37). In a previous study on mouse pancreatic acinar cells we could demonstrate that bombesin-induced Ca 21 waves spread slower than ACh-evoked Ca 21 waves. We suggested that bombesin induces, via activation of PLD, a higher production of DAG compared to ACh. This leads to a PKC-dependent inhibition of CICR and consequently to a slower propagation of bombesin-evoked Ca 21 waves (24). In the present study we have investigated the influence of intracellular signal cascades on CCK-evoked Ca 21 waves. Our data show that Ca 21 waves elicited by activation of h.a.CCK-R spread with a slower propagation rate than Ca 21 waves elicited by activation of l.a.CCK-R. The time between application of CCK-8 and initial release of Ca 21 at the luminal cell pole increased with decreasing concentrations of CCK-8 (Fig. 2). The longer lag time at low CCK-8 concentrations could be explained by a lower production rate of IP 3, which of course increases the time until the threshold concentration for IP 3-evoked Ca 21 release is reached. After the initial release of Ca 21 at the luminal cell pole, spreading of the Ca 21 signal is supported by CICR. As shown in Table 1, the increase in [Ca 21] i at the luminal cell pole after stimulation with 20 pM and 10 nM CCK-8, respectively, was in the same order of magnitude. Therefore, we can conclude that the differences in the propagation rate of Ca 21 waves at 20 pM and 10 nM CCK-8 must be due to modulation of secondary Ca 21 release from stores that promote spreading of the Ca 21 signal throughout the cytosol. Facilitated Ca 21 release from these secondary Ca 21 pools leads to a faster spreading of CCK-8-induced Ca 21 signals, whereas inhibition of secondary Ca 21 release leads to a slower propagation rate of Ca 21 waves (24, 25).
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Regulation of CCK-Evoked Ca 21 Waves by PKC In a previous study we have shown that in mouse pancreatic acinar cells the propagation rate of AChand bombesin-evoked Ca 21 waves can be modulated by PKC activity (24). Here we show that PKC also modulates cytosolic Ca 21 signals evoked by high and low affinity .CCK-R activation. In our experiments activation of PKC with PMA slowed down the propagation rate of Ca 21 waves at both concentrations of CCK-8 tested. On the other hand, inhibition of PKC led to faster propagation of Ca 21 waves only at the higher concentration of CCK-8 (Fig. 3A). These results suggest that, within the period between hormone application and initiation of the cytosolic Ca 21 signal stimulation of l.a.CCK-R but not of h.a.CCK-R results in a sufficient production of DAG to activate PKC. The coupling of l.a.CCK-R to the PLC/PKC pathway has been reported in other studies (1, 2, 4, 6). There is also evidence that cytosolic Ca 21 signals evoked by stimulation of h.a.CCK-R involve formation of IP 3, probably due to activation of PLC (3). In principle, PLCmediated breakdown of PIP 2 should also lead to production of DAG and activation of PKC. However, our data indicate that PLC-mediated formation of DAG by stimulation of h.a.CCK-R seems not to play a significant role in activation of PKC in the time between h.a.CCK-R activation and start of the Ca 21 signal. Regulation of CCK-Evoked Ca 21 Waves by AA and cPLA 2 The role of AA in Ca 21 mobilization is still a matter of debate (6, 17, 18). In our experiments, AA led to slower propagation of Ca 21 waves when l.a.CCK-R were activated, whereas Ca 21 waves following h.a.CCK-R
FIG. 3. Involvement of protein kinase C (PKC), phospholipase A 2 (cPLA 2) and phospholipase D (PLD) in propagation of CCK-8induced Ca 21 waves in mouse pancreatic acinar cells. (A) Inhibition of PKC by 5 mM Ro31-8220 (5 min preincubation) did not change the propagation rate of Ca 21 waves elicited with 20 pM CCK-8, which only activates the h.a.CCK-R. However, in the presence of 10 nM CCK-8, which activates both h.a.CCK-R and l.a.CCK-R, a faster propagation rate of Ca 21 waves was observed. Activation of PKC by preincubation of the cells with 1 mM PMA (5 min) led to a slower Ca 21 wave propagation at both CCK-8 concentrations. (B) Preincubation of the cells with 5 mM AA for 5 min reduced the propagation rate of Ca 21 waves induced with 10 nM CCK-8, whereas the speed of Ca 21 waves in the presence of 20 pM CCK-8 was not affected. Inhibition of cPLA 2 with 10 mM AACOCF 3 led to an acceleration in the Ca 21 wave propagation induced by 20 pM CCK-8, but had no effect at 10 nM CCK-8. (C) Inhibition of PLD-mediated production of PA/DAG by preincubation of the cells with 0.3% n-butanol for 5 min led to a faster propagation rate of Ca 21 waves when the cells were stimulated with 10 nM CCK-8, whereas the speed of propagation of Ca 21 waves in response to 20 pM CCK-8 was not changed. When the cells were stimulated with CCK-8 after 5 min preincubation with the ineffective secondary alcohol 2-butanol, no changes in the propagation of CCK-8-induced Ca 21 waves were observed. 731
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activation were not affected (Fig. 3B). This could be explained by endogenous production of AA after h.a.CCK-R stimulation via activation of PLA 2. Additional application of AA in this case would not further slow down propagation of Ca 21 signals. PLA 2 is a group of enzymes which leads to liberation of AA from the sn-2 position of membrane phospholipids. Three types of PLA 2 had been described in mammalian cells: Ca 21-dependent cPLA 2, Ca 21-independent iPLA 2 and secretory sPLA 2 (28 –30). In pancreatic acinar cells inactive sPLA 2 (type I) is stored in granules (38) from which it can be released into the acinar lumen by exocytosis after hormonal stimulation of the cell. In the extracellular space sPLA 2 becomes activated by high Ca 21 concentrations. Involvement of sPLA 2 in Ca 21 signaling of pancreatic acinar cells is unlikely since secretion and activation of sPLA 2 is at the end in the secretagogue-evoked signal cascade, and therefore can hardly influence intracellular Ca 21 release which is an early event in signal transduction. Furthermore, in our experiments all secreted enzymes were removed from the bath medium by continuous perfusion of the cells. The experiments with inhibitors of cPLA 2 and iPLA 2 show that a role of iPLA 2 in control of Ca 21 wave propagation can also be excluded. Only cPLA 2 seems to be involved in cytosolic Ca 21 signaling of pancreatic acinar cells. An interesting question is how cPLA 2 is activated during signal transduction. It is known that cPLA 2 can be activated by Ca 21-dependent translocation to membranes where the enzyme can cleave membrane lipids. A Ca 21-dependent translocation of cPLA 2 to intracellular membranes, including endoplasmic reticulum and nucleus, has been demonstrated for several cell types (39, 40). Therefore, it could be possible that cPLA 2 is located near cytosolic Ca 21 stores and that Ca 21-dependent activation of cPLA 2 in direct neighborhood to the Ca 21 releasing sites can produce a negative feedback for secondary Ca 21 release. However, since cPLA 2 plays only a role in h.a.CCK-Rmediated Ca 21 signaling a simple activation of cPLA 2 by cytosolic Ca 21 ions alone can not explain our experimental results. It has been reported that besides Ca 21dependent translocation cPLA 2 can also be activated by agonist-induced MAP-kinase-dependent phosphorylation, resulting in stimulation of the intrinsic enzymatic activity of cPLA 2 (41). Further investigations should be carried out to investigate the role of the MAP-kinase cascade in the regulation of Ca 21 waves in response to secretagogues. Regulation of CCK-Evoked Ca 21 Waves by PLD In the presence of n-butanol, which prevents formation of DAG following activation of PLD (42), stimulation of l.a.CCK-R led to a faster propagation of cytosolic Ca 21 signals (Fig. 3C), whereas the propagation of Ca 21
waves in response to stimulation of h.a.CCK-R was unchanged. These results suggest that in mouse pancreatic acinar cells l.a.CCK-R is coupled to PLD, and that PLD-mediated formation of DAG modulate Ca 21 wave propagation in response to high CCK-8 concentrations. Taken together our results show that high and lowaffinity CCK-R are coupled to different intracellular signaling pathways, which regulate Ca 21 release and spreading of cytosolic Ca 21 waves. We have shown that CCK-evoked Ca 21 signals can be modulated by activation of cPLA 2, PLD and PKC. Stimulation of h.a.CCK-R mainly activate the cPLA 2 cascades, whereas l.a.CCK-R are coupled to the PLC and PLD cascades. The products in these pathways, AA and DAG, respectively, inhibit the propagation of CCKinduced Ca 21 waves. Since the effects of AA and PMA were not additive it is suggested that both signal cascades act on the same Ca 21 releasing mechanism. The molecular mechanism of this inhibition is not yet known. ACKNOWLEDGMENTS The authors wish to thank the Deutsche Forschungsgemeinschaft (Schu 429/11-1; Schm 876/2-1), the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (01 VM 9310) and Consejerı´a de Educacio´n y Juventud-Junta de Extremadura (PRI96100010) for support of this study.
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17. Maruyama, Y. (1990) J. Physiol. (London) 430, 471– 482. 18. Striggow, F., and Ehrlich, B. E. (1997) Biochem. Biophys. Res. Commun. 237, 413– 418. 19. Rowles, S. J., and Gallacher, D. V. (1996) Biochem. J. 319, 913–918. 20. Hou, W., Arita, Y., and Morisset, J. (1996) Cell. Signal. 8, 487– 496. 21. Chung, H. C., and Fleming, N. (1995) Pflu¨gers Arch. 431, 161–168. 22. Kasai, H., and Augustine, G. J. (1990) Nature 348, 735–738. 23. Nathanson, M. H., Padfield, P. J., O9Sullivan, A. J., Burgstahler, A. D., and Jamieson, J. D. (1992) J. Biol. Chem. 267, 18118 – 18121. 24. Pfeiffer, F., Sternfeld, L., Schmid, A., and Schulz, I. (1998) Am. J. Physiol. 274, C663–C672. 25. Gonza´lez, A., Pfeiffer, F., Schmid, A., and Schulz, I. (1998) Am. J. Physiol. 275, C810 –C817. 26. Pfeiffer, F., Schmid, A., and Schulz, I. (1995) Pflu¨gers Arch. 430, 916 –922. 27. Cheng, H., Lederer, W. J., and Cannell, M. B. (1993) Science 262, 740 –744. 28. Kudo, I., Murakami, M., Hara, S., and Inoue, K. (1993) Biochim. Biophys. Acta 1170, 217–231. 29. Dennis, E. A. (1994) J. Biol. Chem. 269, 13057–13060. 30. Mukherjee, A. B., Miele, L., and Pattabiraman, N. (1994) Biochem. Pharmacol. 48, 1–10.
31. Riendeau, D., Guay, J., Weech, P. K., Laliberte, F., Yergey, J., Li, C., Desmarais, S., Perrier, H., Liu, S., Nicoll Griffith, D., and Street, I. P. (1994) J. Biol. Chem. 269, 15619 –15624. 32. Balsinde, J., and Dennis, E. A. (1996) J. Biol. Chem. 271, 6758 – 6765. 33. Hazen, S. L., Zupan, L. A., Weiss, R. H., Getmann, D. P., and Gross, R. W. (1991) J. Biol. Chem. 266, 7227–7232. 34. El Bawab, S., Macovschi, O., Lagarde, M., and Prigent, A. F. (1995) Biochem. J. 308, 113–118. 35. Xu, X., Zeng, W., Diaz, J., and Muallem, S. (1996) J. Biol. Chem. 271, 24684 –24690. 36. Tojyo, Y., Tanimura, A., and Matsumoto, Y. (1997) Cell Calcium 22, 455– 462. 37. Sjo¨din, L., Ito, K., Miyashita, Y., and Kasai, H. (1997) Pflu¨gers Arch. 433, 397– 402. 38. Kortesuo, P. T., Hietaranta, A. J., Jamia, M., Hirsimaki, P., and Nevalainen T. J. (1993) Int. J. Pancreatol. 13, 111–118. 39. Channon, J. Y., and Leslie, C. C. (1990) J. Biol. Chem. 265, 5409 –5413. 40. Schievella, A. R., Regier, M. K., Smith, W. L., and Lin, L. L. (1995) J. Biol. Chem. 270, 30749 –30754. 41. Lin, L. L., Wartmann, M., Lin, A. Y., Knopf, J. L., Seth, A., and Davis, R. J. (1993) Cell 72, 269 –278. 42. Xie, M. S., Jacobs, L. S., and Dubyak, G. R. (1991) J. Clin. Invest. 88, 45–54.
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Cholecystokinin-Evoked Ca 21 Waves in Isolated Mouse Pancreatic Acinar Cells Are Modulated by Activation of Cytosolic Phospholipase A 2, Phospholipase D, and Protein Kinase C Antonio Gonza´lez,* Andreas Schmid,* Lutz Sternfeld,* Elmar Krause,* Gine´s M. Salido,† and Irene Schulz* *Department of Physiology II, Faculty of Medicine, University of Saarland, D-66421, Homburg/Saar, Germany; and †Department of Physiology, Faculty of Veterinary Medicine, University of Extremadura, 10079, Ca´ceres, Spain
Received May 17, 1999
We employed confocal laser-scanning microscopy to monitor cholecystokinin (CCK)-evoked Ca 21 signals in fluo-3-loaded mouse pancreatic acinar cells. CCK-8induced Ca 21 signals start at the luminal cell pole and subsequently spread toward the basolateral membrane. Ca 21 waves elicited by stimulation of highaffinity CCK receptors (h.a.CCK-R) with 20 pM CCK-8 spread with a slower rate than those induced by activation of low-affinity CCK receptors (l.a.CCK-R) with 10 nM CCK-8. However, the magnitude of the initial Ca 21 release was the same at both CCK-8 concentrations, suggesting that the secondary Ca 21 release from intracellular stores is modulated by activation of different intracellular pathways in response to low and high CCK-8 concentrations. Our experiments suggest that the propagation of Ca 21 waves is modulated by protein kinase C (PKC) and arachidonic acid (AA). The data indicate that h.a.CCK-R are linked to phospholipase C (PLC) and phospholipase A 2 (PLA 2) cascades, whereas l.a.CCK-R are coupled to PLC and phospholipase D (PLD) cascades. The products of PLA 2 and PLD activation, AA and diacylglycerol (DAG), cause inhibition of Ca 21 wave propagation by yet unknown mechanisms. © 1999 Academic Press Key Words: pancreas; calcium wave; CCK-8; PKC, PLA 2; PLD.
Abbreviations used: AA, arachidonic acid; AACOCF 3, arachidonyl trifluoromethyl-ketone; BEL, bromoenol lactone; CICR, calcium-induced calcium release; CCK-8, cholecystokinin octapeptide; h.a.CCK-R, high-affinity cholecystokinin-receptor; l.a.CCK-R, low-affinity cholecystokinin-receptor; [Ca 21] i, intracellular free calcium concentrationl DAG, diacylglycerol; MAFP, methyl-arachidonyl-fluorophosphonate; MAPkinase, mitogen-activated protein-kinase; PMA, phorbol-12-myristate13-acetate; PLC, phospholipase C; PLD, phospholipase D; PKC, protein kinase C; PA, phosphatidic acid; PC, phosphatidylcholine; cPLA 2, cytosolic phospholipase A 2; iPLA 2, calcium-independent phospholipase A 2; sPLA 2, secretory phospholipase A 2. 0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
In the exocrine pancreas high- and low-affinity cholecystokinin (CCK) receptors exist. Therefore, depending on the concentration, CCK binds either only to the high or to a variable extent to high and low affinity binding sites (1). This may result in generation of different second messengers in the signal cascades leading to amylase secretion. It has been shown that activation of high affinity CCK-receptors (h.a.CCK-R) by low CCK concentrations (,100 pM) is coupled to phospholipase A 2 (PLA 2), and probably also involves phospholipase C (PLC)-mediated inositol 1,4,5-trisphosphate (IP 3) and diacylglycerol (DAG) production to mediate Ca 21 oscillations, protein kinase C (PKC) activation and amylase secretion. In contrast, lowaffinity CCK receptors (l.a.CCK-R), which are activated at high CCK concentrations (.100 pM) are coupled to the PLC pathway without implication of PLA 2 (1– 6). Activation of PLC causes breakdown of phosphatidylinositol 4,5-bisphosphate (PIP 2) to IP 3 and DAG. IP 3 induces Ca 21 release from intracellular non-mitochondrial stores with a consequent rise in cytosolic free Ca 21 concentration ([Ca 21] i), whereas DAG activates PKC (7, 8). Another source for DAG is phosphatidylcholine (PC) which can be cleaved by phospholipase D (PLD) to choline and phosphatidic acid (PA). In a second step PA is dephosphorylated by a phosphatidic acid phosphohydrolase to DAG (9). In addition to these two well characterized second messengers, IP 3 and DAG, also arachidonic acid (AA) has been recognized as an important factor in hormonal stimulation of different tissues and cell types (10). AA can be liberated via PLA 2 from PC as well as from DAG by a DAG-lipase (11). Furthermore, DAG is generated by PLC or by sequential activation of PLD and phosphatidate phosphohydrolase (12). AA is the precursor of several bioactive molecules, termed eico-
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sanoids, including prostaglandins, leukotrienes and thromboxanes (10). It has been reported that in isolated pancreatic acini AA released in response to hormonal stimulation induces amylase secretion (11, 13–15), and that it releases Ca 21 from the endoplasmic reticulum (6). Other studies have shown an inhibitory effect of AA in pancreatic secretion (16) as well as in IP 3-dependent Ca 21 mobilization (17, 18). Furthermore, inhibition of PLA 2 potentiates IP 3-induced Ca 21-activated currents in mouse pancreatic acinar cells (19). The role of PLD in stimulation of exocrine secretion is not clear at present. It has been suggested that inhibition of PLD has no consequence for the secretory response in the exocrine pancreas (20). However, in studies carried out on rat submandibular acinar cells (21) PLD has been shown to play a role in signal transduction. In the present study, carried out in isolated mouse pancreatic acinar cells, we have tested if modulation of PKC-, PLA 2- and PLD-activities has any effects on the propagation of Ca 21 signals. In pancreatic acinar cells hormone-evoked Ca 21 signals start in the luminal cell pole and then spread towards the basolateral side of the cell (22). Ca 21 wave propagation has been explained by sequential Ca 21 release from Ca 21 stores in series involving calcium-induced calcium release (CICR) (23–25). In a previous study we have demonstrated that activation of PKC slows down ACh-evoked Ca 21 wave propagation, probably due to inhibition of CICR (24). In the present study we have monitored the propagation of Ca 21 signals in response to CCK-8. We have employed confocal laser scanning microscopy to follow local changes of [Ca 21] i in individual mouse pancreatic acinar cells loaded with the fluorescent probe fluo-3. The results show that the propagation of CCK-evoked Ca 21 waves in mouse pancreatic acinar cells can be modulated by activation of cPLA 2, PLD and PKC. Concentration-dependent differences in the pattern of Ca 21 mobilization could be explained by differential coupling of high and low affinity CCK-R to intracellular phospholipase-mediated signaling pathways. MATERIALS AND METHODS Materials Fluo-3/AM was obtained from Molecular Probes Inc. (Europe), Ro31-8220, phorbol 12-myristate 13-acetate (PMA) and arachidonic acid (AA) from Calbiochem-Novabiochem Corp. (Germany), arachidonyl trifluoromethyl-ketone (AACOCF 3) from Biomol Res. Lab. (Germany), methyl-arachidonyl-fluorophosphonate (MAFP) and bromoenol lactone (BEL) from Cayman Chemical Company (Germany) and n-butanol from Merck-Schuchardt (Germany). All other materials used were obtained from Sigma Chemicals Co. (Germany).
Methods Preparation of isolated acinar cells. Adult male CD-1 mice (35– 40 g) were used for this study. They had free access to water and
food. Animals were sacrificed by cervical dislocation, the pancreas was rapidly removed and the acinar cells were isolated as described previously by a collagenase digestion method (24). With this isolation procedure single cells as well as small clusters consisting of 2 up to 5 cells were obtained. Fluorescence measurements. Freshly isolated mouse pancreatic acinar cells were loaded with 4 mM fluo-3/AM for 30 min at room temperature (22°C) following previously established methods (26). After dye loading, the cells were kept at 4°C and the experiments were performed within the next 4 h. For monitoring Ca 21-dependent fluorescence signals, aliquots of the cell suspension were placed onto polylysine coated glass coverslips attached to the bottom of a perfusion chamber. The cells were continuously superfused with a NaHepes buffer containing in mM: 140 NaCl, 4.7 KCl, 1.3 CaCl 2, 1 MgCl 2, 10 Hepes, 10 glucose, pH adjusted to 7.4 with NaOH. Employing a confocal laser-scanning system (Bio-Rad, MRC-1024) fluorescence images (excitation 488 nm/emission . 515 nm) of 128 3 128 pixels with a resolution of 0.526 mm/pixel were recorded every 0.25 s. Small rectangular areas were selected in the luminal and the basolateral pole of the cells. The difference in time between the increase in [Ca 21] i at both cell regions was determined and the speed (mm/s) of the CCK-induced Ca 21 signal was calculated. All experiments were performed at room temperature. Determination of intracellular free calcium concentration. Absolute values of intracellular free calcium concentration ([Ca 21 ] i ) were calculated from the self-ratio method described by Cheng et al. (27) for the non-ratiometric dye fluo-3, assuming a dissociation constant of 400 nM for Ca 21 binding of fluo-3 and a resting [Ca 21 ] i of 100 nM (2). Analysis of data. Data show the mean [Ca 21] i expressed in nM 6 SEM and the mean propagation rate of Ca 21 waves expressed as mm/s 6 SEM. Statistical analysis was performed by Student9s t test and only P values less than 0.05 were considered as significant.
RESULTS Cholecystokinin-Evoked Ca 21 Signals As shown in Fig. 1 stimulation of pancreatic acinar cells with 20 pM or 10 nM CCK-8 resulted in an initial increase in [Ca 21] i at the luminal cell pole and subsequent spreading of the Ca 21 signal towards the basolateral cell membrane. From the distance between both areas selected at each side of the cell and the difference in time between the increase in the fluorescence in these regions, we could determine the speed of the Ca 21 wave. The speed of Ca 21 wave in response to CCK-8 was dependent on the concentration used, being faster at higher concentrations of CCK-8. The mean values for the propagation rate of Ca 21 signals in response to the CCK-8 concentrations are summarized in Fig. 2A. In addition to a slower propagation rate of Ca 21 signals, a larger delay between hormone application and the initial Ca 21 release was observed at the lower concentrations of CCK-8 (Fig. 2B). It has been described that CCK-8 at low concentrations (,100 pM) stimulates amylase secretion via activation of h.a.CCK-R, whereas at high CCK-8 concentrations (.100 pM) amylase release is reduced when l.a.CCK-R are activated (1). To investigate the role of high and low affinity CCK-R activation on cytosolic Ca 21 signals we stimulated cells with either 20 pM or
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1 mM of the phorbol ester PMA to activate PKC, spreading of Ca 21 waves in response to both CCK-8 concentrations was significantly slower compared to the controls (CCK-8 20 pM: 5.28 6 0.27 mm/s, 43 experiments/89 cells; CCK-8 20 pM 1 1 mM PMA: 4.05 6 0.53 mm/s, 20 experiments/44 cells, P , 0.05; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 1 mM PMA: 12.22 6 0.53 mm/s, 34 experiments/83 cells, P , 0.001). Preincubation with 5 mM of the PKC-inhibitor Ro31-8220 produced a faster propagation of Ca 21 waves at 10 nM CCK-8. The mean speed of Ca 21 waves in response to 20 pM CCK-8 was unchanged by this treatment (Fig. 3A) (CCK-8 20 pM: 5.28 6 0.27 mm/s, 43 experiments/89 cells; CCK-8 20 pM 1 5 mM Ro31-8220: 5.21 6 0.44 mm/s, 22 experiments/42 cells; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 5 mM Ro318220: 22.10 6 1.89 mm/s, 31 experiments/65 cells, P , 0.001). Effect of AA on CCK-Evoked Ca 21 Waves
FIG. 1. Time-course of Ca 21 wave propagation in mouse pancreatic acinar. (A) Small areas located at the luminal (lu) and basolateral (ba) pole of the cells were selected and the changes in fluorescence were monitored employing a confocal laser-scanning microscope. Following stimulation, the fluorescence first increased at the luminal cell pole and then spread as a Ca 21 wave towards the basolateral pole of the cells. The time between the increase in fluorescence at both sides of the cell was monitored and the speed of the Ca 21 wave was determined. Following stimulation of the cells with 20 pM CCK-8, which only activates the h.a.CCK-R, the Ca 21 wave traversed the cell within 1.69 6 0.10 s, corresponding to a speed of the Ca 21 wave of 7.78 6 0.52 mm/s (n 5 12 experiments/24 cells). (B) When cells were stimulated with a higher concentration, 10 nM CCK-8, that activates both h.a.CCK-R and l.a.CCK-R, the Ca 21 wave spread from the luminal to the basolateral pole within 0.63 6 0.03 s corresponding to a speed of 21.84 6 1.44 mm/s (n 5 23 experiments/52 cells).
10 nM CCK-8. At 20 pM CCK-8 the speed of Ca 21 wave was 7.78 6 0.51 mm/s (n 5 12 experiments/24 cells) and at 10 nM CCK-8 it was 21.84 6 1.43 mm/s (n 5 23 experiments/52 cells). The initial rise in [Ca 21] i in the luminal cell pole required about 13 more seconds with 20 pM CCK-8 than with 10 nM CCK-8. However, the magnitude of the Ca 21 signal in the luminal cell pole was not significantly different at both CCK-8 concentrations. Table 1 summarizes the values for [Ca 21] i in the luminal cell pole at 0.5 s and 1 s after beginning of the Ca 21 signal as well as at its maximum. Effect of PKC on CCK-Evoked Ca 21 Waves When pancreatic acinar cells were stimulated with CCK-8 following 5 min preincubation in the presence of
When pancreatic acinar cells were stimulated with 10 nM CCK-8 after 5 min preincubation with 5 mM of AA, spreading of the CCK-8-induced Ca 21 wave was much slower compared to the controls. On the other hand, AA did not change the propagation rate of Ca 21 waves elicited by stimulation of the cells with 20 pM CCK-8 (Fig. 3B) (CCK-8 20 pM: 4.76 6 0.40 mm/s, 24 experiments/51 cells; CCK-8 20 pM 1 5 mM AA: 5.38 6 0.66 mm/s, 9 experiments/26 cells; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72 experiments/171 cells; CCK-8 10 nM 1 5 mM AA: 10.22 6 0.82 mm/s, 22 experiments/54 cells, P , 0.001). AA, in the concentration we used in our experiments, had no effect on the resting [Ca 21] i since changes in the fluorescence baseline were not observed during pretreatment of cells with AA. To decide if the effect of both AA and PKC-activation on CCK-induced Ca 21 waves are additive or not, we performed a set of experiments in which pancreatic acinar cells were pretreated with both AA and PMA for 5 min prior to stimulation with 10 nM CCK-8. Under these conditions we did not observe any higher reduction in the speed of Ca 21 waves compared to the effect of these agents applied separately (data not shown). Effect of PLA 2 Inhibition on CCK-Evoked Ca 21 Waves Three groups of PLA 2 have been described in mammalian cells: Ca 21-dependent cytosolic PLA 2 (cPLA 2), Ca 21-independent PLA 2 (iPLA 2) and Ca 21-dependent secretory PLA 2 (sPLA 2) (28 –30). To investigate the role of cPLA 2 we examined the effect of the cPLA 2-specific inhibitor AACOCF 3 (31). In the presence of 10 mM AACOCF 3 we could observed a faster propagation of Ca 21 signals at 20 pM CCK-8 compared to the response in the presence of the same concentration of CCK-8 alone. The mean speed of Ca 21 waves in response to 10
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FIG. 2. CCK-8-induced Ca 21 wave propagation in mouse pancreatic acinar cells. (A) Cells were stimulated with increasing concentrations of CCK-8. As a consequence, a faster spreading of the Ca 21 wave could be observed in a dose-dependent manner. Data show the mean speed of propagation of Ca 21 waves in mm/s 6 SEM. (B) Delay in the appearance of luminal Ca 21 response after switching from Na-Hepes buffer to solutions containing the respective concentration of CCK-8. Data show the mean time delay in s 6 SEM. The dashed line indicates the mean lag time produced by our perfusion system.
nM CCK-8 was unchanged by treatment of the cells with AACOCF 3 (Fig. 3B) (CCK-8 20 pM: 4.76 6 0.40 mm/s, 24 experiments/51 cells; CCK-8 20 pM 1 10 mM AACOCF 3: 9.89 6 0.90 mm/s, 14 experiments/35 cells, P , 0.001; CCK-8 10 nM: 17.02 6 0.79 mm/s, 72
experiments/171 cells; CCK-8 10 nM 1 10 mM AACOCF 3: 16.07 6 1.28 mm/s, 16 experiments/34 cells). To study if a Ca 21-independent PLA 2 (iPLA 2) could be involved in production of AA with a consequent effect on the propagation of Ca 21 signals, we preincu-
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Calculated Luminal [Ca 21] i (in nM) after 0.5 s, 1 s, and at Its Maximum after Stimulation of Mouse Pancreatic Acinar Cells with 20 pM and 10 nM CCK-8
CCK-8 20 pM
CCK-8 10 nM
Point/Time
[Ca 21] i, nM
n, expt/cells
Luminal/0.5 s Luminal/1 s Luminal/peak Luminal/0.5 s Luminal/1 s Lumnal/peak
172.70 6 13.06 239.30 6 21.96 376.10 6 57.42 156.50 6 13.39 208.60 6 19.53 305.70 6 33.77
38/83 38/83 38/65 25/56 25/56 25/55
6.40 6 0.45 mm/s, 11 experiments/24 cells; CCK-8 10 nM: 15.13 6 1.53 mm/s, 9 experiments/22 cells; CCK-8 10 nM 1 0.3% 2-butanol: 14.60 6 1.53 mm/s, 11 experiments/28 cells). DISCUSSION
Note. Values show the mean 6 SEM of [Ca 21] i at the luminal cell pole in nM. Absolute values of [Ca 21] i were calculated from the self-ratio method for the nonratiometric dye fluo-3, assuming a dissociation constant of 400 nM for Ca 21 binding of fluo-3 and a resting [Ca 21] i of 100 nM (27). Values obtained after stimulation of cells with 10 nM CCK-8 were compared to those obtained in the same area after stimulation of cells with 20 pM CCK-8 (0.5 s 5 0.5 seconds after the initial rise in Ca 21; 1 s 5 1 second after the initial rise in Ca 21; peak 5 maximal response obtained after the initial rise in Ca 21).
bated pancreatic acinar cells with two known inhibitors of this enzyme, MAPF and BEL (32, 33). Both inhibitors at maximal effective concentrations (25 mM and 50 mM, respectively) did not modify the speed of the Ca 21 waves at both concentrations of CCK-8 tested (data not shown) indicating that iPLA 2 has no implications in the regulation of Ca 21 signals induced by CCK-8. Effect of PLD on CCK-Evoked Ca 21 Waves DAG can be generated by activation of PLC and PLD. DAG activates PKC and therefore slows down the propagation rate of bombesin-evoked Ca 21 signals (24). To investigate whether PLD activation is also involved in the control of CCK-evoked Ca 21 signals, we have preincubated pancreatic acinar cells with 0.3% n-butanol. Activation of PLD in the presence of n-butanol leads to production of the ineffective phosphatidylbutanol instead of PA (34) and therefore should cause a reduction in the DAG-dependent activation of PKC. In the presence of 0.3% n-butanol, activation of l.a.CCK-R by 10 nM CCK-8 led to faster propagation rates of Ca 21 signals (Fig. 3C). However, no change in the mean speed of Ca 21 waves in response to 20 pM CCK-8 was observed (CCK-8 20 pM: 5.24 6 0.37 mm/s, 11 experiments/22 cells; CCK-8 20 pM 1 0.3% n-butanol: 6.50 6 0.54 mm/s, 11 experiments/ 30 cells; CCK-8 10 nM: 15.13 6 1.53 mm/s, 9 experiments/22 cells; CCK-8 10 nM 1 0.3% n-butanol: 21.8 6 2.56 mm/s, 12 experiments/36 cells; P , 0.05). When the ineffective secondary alcohol 2-butanol was used instead of n-butanol, no changes in the propagation speed of Ca 21 waves were observed at both CCK-8 concentrations (CCK-8 20 pM: 5.24 6 0.37 mm/s, 11 experiments/22 cells; CCK-8 20 pM 1 0.3% 2-butanol:
In the exocrine pancreas activation of h.a.CCK-R and l.a.CCK-R leads to generation of different second messengers and therefore produces different biological responses (5). The generation of Ca 21 waves may represent an important early step for the coordination of cellular functions in pancreatic acinar cells (22). Ca 21 wave propagation has been explained by sequential Ca 21 release from Ca 21 stores in series and it has been suggested that the mechanism underlying Ca 21 wave propagation involves CICR (3, 23, 24). Secretagogue-dependent differences in the propagation of cytosolic Ca 21 signals have been shown in pancreatic and parotid acinar cells (24, 35–37). In a previous study on mouse pancreatic acinar cells we could demonstrate that bombesin-induced Ca 21 waves spread slower than ACh-evoked Ca 21 waves. We suggested that bombesin induces, via activation of PLD, a higher production of DAG compared to ACh. This leads to a PKC-dependent inhibition of CICR and consequently to a slower propagation of bombesin-evoked Ca 21 waves (24). In the present study we have investigated the influence of intracellular signal cascades on CCK-evoked Ca 21 waves. Our data show that Ca 21 waves elicited by activation of h.a.CCK-R spread with a slower propagation rate than Ca 21 waves elicited by activation of l.a.CCK-R. The time between application of CCK-8 and initial release of Ca 21 at the luminal cell pole increased with decreasing concentrations of CCK-8 (Fig. 2). The longer lag time at low CCK-8 concentrations could be explained by a lower production rate of IP 3, which of course increases the time until the threshold concentration for IP 3-evoked Ca 21 release is reached. After the initial release of Ca 21 at the luminal cell pole, spreading of the Ca 21 signal is supported by CICR. As shown in Table 1, the increase in [Ca 21] i at the luminal cell pole after stimulation with 20 pM and 10 nM CCK-8, respectively, was in the same order of magnitude. Therefore, we can conclude that the differences in the propagation rate of Ca 21 waves at 20 pM and 10 nM CCK-8 must be due to modulation of secondary Ca 21 release from stores that promote spreading of the Ca 21 signal throughout the cytosol. Facilitated Ca 21 release from these secondary Ca 21 pools leads to a faster spreading of CCK-8-induced Ca 21 signals, whereas inhibition of secondary Ca 21 release leads to a slower propagation rate of Ca 21 waves (24, 25).
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Regulation of CCK-Evoked Ca 21 Waves by PKC In a previous study we have shown that in mouse pancreatic acinar cells the propagation rate of AChand bombesin-evoked Ca 21 waves can be modulated by PKC activity (24). Here we show that PKC also modulates cytosolic Ca 21 signals evoked by high and low affinity .CCK-R activation. In our experiments activation of PKC with PMA slowed down the propagation rate of Ca 21 waves at both concentrations of CCK-8 tested. On the other hand, inhibition of PKC led to faster propagation of Ca 21 waves only at the higher concentration of CCK-8 (Fig. 3A). These results suggest that, within the period between hormone application and initiation of the cytosolic Ca 21 signal stimulation of l.a.CCK-R but not of h.a.CCK-R results in a sufficient production of DAG to activate PKC. The coupling of l.a.CCK-R to the PLC/PKC pathway has been reported in other studies (1, 2, 4, 6). There is also evidence that cytosolic Ca 21 signals evoked by stimulation of h.a.CCK-R involve formation of IP 3, probably due to activation of PLC (3). In principle, PLCmediated breakdown of PIP 2 should also lead to production of DAG and activation of PKC. However, our data indicate that PLC-mediated formation of DAG by stimulation of h.a.CCK-R seems not to play a significant role in activation of PKC in the time between h.a.CCK-R activation and start of the Ca 21 signal. Regulation of CCK-Evoked Ca 21 Waves by AA and cPLA 2 The role of AA in Ca 21 mobilization is still a matter of debate (6, 17, 18). In our experiments, AA led to slower propagation of Ca 21 waves when l.a.CCK-R were activated, whereas Ca 21 waves following h.a.CCK-R
FIG. 3. Involvement of protein kinase C (PKC), phospholipase A 2 (cPLA 2) and phospholipase D (PLD) in propagation of CCK-8induced Ca 21 waves in mouse pancreatic acinar cells. (A) Inhibition of PKC by 5 mM Ro31-8220 (5 min preincubation) did not change the propagation rate of Ca 21 waves elicited with 20 pM CCK-8, which only activates the h.a.CCK-R. However, in the presence of 10 nM CCK-8, which activates both h.a.CCK-R and l.a.CCK-R, a faster propagation rate of Ca 21 waves was observed. Activation of PKC by preincubation of the cells with 1 mM PMA (5 min) led to a slower Ca 21 wave propagation at both CCK-8 concentrations. (B) Preincubation of the cells with 5 mM AA for 5 min reduced the propagation rate of Ca 21 waves induced with 10 nM CCK-8, whereas the speed of Ca 21 waves in the presence of 20 pM CCK-8 was not affected. Inhibition of cPLA 2 with 10 mM AACOCF 3 led to an acceleration in the Ca 21 wave propagation induced by 20 pM CCK-8, but had no effect at 10 nM CCK-8. (C) Inhibition of PLD-mediated production of PA/DAG by preincubation of the cells with 0.3% n-butanol for 5 min led to a faster propagation rate of Ca 21 waves when the cells were stimulated with 10 nM CCK-8, whereas the speed of propagation of Ca 21 waves in response to 20 pM CCK-8 was not changed. When the cells were stimulated with CCK-8 after 5 min preincubation with the ineffective secondary alcohol 2-butanol, no changes in the propagation of CCK-8-induced Ca 21 waves were observed. 731
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activation were not affected (Fig. 3B). This could be explained by endogenous production of AA after h.a.CCK-R stimulation via activation of PLA 2. Additional application of AA in this case would not further slow down propagation of Ca 21 signals. PLA 2 is a group of enzymes which leads to liberation of AA from the sn-2 position of membrane phospholipids. Three types of PLA 2 had been described in mammalian cells: Ca 21-dependent cPLA 2, Ca 21-independent iPLA 2 and secretory sPLA 2 (28 –30). In pancreatic acinar cells inactive sPLA 2 (type I) is stored in granules (38) from which it can be released into the acinar lumen by exocytosis after hormonal stimulation of the cell. In the extracellular space sPLA 2 becomes activated by high Ca 21 concentrations. Involvement of sPLA 2 in Ca 21 signaling of pancreatic acinar cells is unlikely since secretion and activation of sPLA 2 is at the end in the secretagogue-evoked signal cascade, and therefore can hardly influence intracellular Ca 21 release which is an early event in signal transduction. Furthermore, in our experiments all secreted enzymes were removed from the bath medium by continuous perfusion of the cells. The experiments with inhibitors of cPLA 2 and iPLA 2 show that a role of iPLA 2 in control of Ca 21 wave propagation can also be excluded. Only cPLA 2 seems to be involved in cytosolic Ca 21 signaling of pancreatic acinar cells. An interesting question is how cPLA 2 is activated during signal transduction. It is known that cPLA 2 can be activated by Ca 21-dependent translocation to membranes where the enzyme can cleave membrane lipids. A Ca 21-dependent translocation of cPLA 2 to intracellular membranes, including endoplasmic reticulum and nucleus, has been demonstrated for several cell types (39, 40). Therefore, it could be possible that cPLA 2 is located near cytosolic Ca 21 stores and that Ca 21-dependent activation of cPLA 2 in direct neighborhood to the Ca 21 releasing sites can produce a negative feedback for secondary Ca 21 release. However, since cPLA 2 plays only a role in h.a.CCK-Rmediated Ca 21 signaling a simple activation of cPLA 2 by cytosolic Ca 21 ions alone can not explain our experimental results. It has been reported that besides Ca 21dependent translocation cPLA 2 can also be activated by agonist-induced MAP-kinase-dependent phosphorylation, resulting in stimulation of the intrinsic enzymatic activity of cPLA 2 (41). Further investigations should be carried out to investigate the role of the MAP-kinase cascade in the regulation of Ca 21 waves in response to secretagogues. Regulation of CCK-Evoked Ca 21 Waves by PLD In the presence of n-butanol, which prevents formation of DAG following activation of PLD (42), stimulation of l.a.CCK-R led to a faster propagation of cytosolic Ca 21 signals (Fig. 3C), whereas the propagation of Ca 21
waves in response to stimulation of h.a.CCK-R was unchanged. These results suggest that in mouse pancreatic acinar cells l.a.CCK-R is coupled to PLD, and that PLD-mediated formation of DAG modulate Ca 21 wave propagation in response to high CCK-8 concentrations. Taken together our results show that high and lowaffinity CCK-R are coupled to different intracellular signaling pathways, which regulate Ca 21 release and spreading of cytosolic Ca 21 waves. We have shown that CCK-evoked Ca 21 signals can be modulated by activation of cPLA 2, PLD and PKC. Stimulation of h.a.CCK-R mainly activate the cPLA 2 cascades, whereas l.a.CCK-R are coupled to the PLC and PLD cascades. The products in these pathways, AA and DAG, respectively, inhibit the propagation of CCKinduced Ca 21 waves. Since the effects of AA and PMA were not additive it is suggested that both signal cascades act on the same Ca 21 releasing mechanism. The molecular mechanism of this inhibition is not yet known. ACKNOWLEDGMENTS The authors wish to thank the Deutsche Forschungsgemeinschaft (Schu 429/11-1; Schm 876/2-1), the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie (01 VM 9310) and Consejerı´a de Educacio´n y Juventud-Junta de Extremadura (PRI96100010) for support of this study.
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