Epidermal growth factor inhibits hormone- and fibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini

Peptides, Vol. 16, No. 1, pp. 123-128, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in the USA. All rights reserved 0196-9781/95 $9.50 + .OO

Pergamon 0196-9781(94)00164-2

Epidermal Growth Factor Inhibits Hormone- and Fibroblast Growth Factor-Induced Activation of Phospholipase C in Rat Pancreatic Acini D. STRYJEK-KAMINSKA,

A. PIIPER, W. F. CASPARY

II. Medical Department,

University

Received

of Frankfurt

AND

S. ZEUZEM’

a. M., Germany

2 May 1994

STRYJEK-KAMINSKA, D., A. PIIPER, W. F. CASPARY AND S. ZEUZEM. Epidennal growth factor inhibits hormone- and jibroblast growth factor-induced activation of phospholipase C in rat pancreatic acini. PEPTIDES 16(l) 123-128, 1995.Epidermal growth factor (EGF) inhibits cholecystokinin-octapeptide-stimulated amylase release and inositol 1,4,5trisphosphate (1,4,5-IP,) production in isolated rat pancreatic acini. In the present study, pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP3 production induced by secretagogues that activate either phospholipase C-/3 (carbachol, bombesin) or phospholipase C-y [basic fibroblast growth factor (bFGF)]. The results show that EGF (100 @ml) inhibited bombesin (0.1 nM- 1 CLM)-induced amylase release almost completely. Similarly, the effect of EGF on carbachol-stimulated amylase release was substantial at submaximal (0.1 fl: 44% inhibition), maximal (1 fl: 75% inhibition), and supramaximal (100 @f: 33% inhibition) carbachol concentrations. EGF reduced amylase release at submaximal bFGF concentrations (0.1 nA4: 40% inhibition), but not at supramaximal bFGF concentrations (1 and 10 nM). EGF decreased the peak increase of 1,4,5-IP3 in response to bombesin and carbachol (5 s after beginning of the incubation) and bFGF (15 s after beginning of the incubation) by 81 & 19%. 65 2 15%, and fi6 -C 18%. respectively. Receptor binding characteristics for secretagogues that activate phospholipase C were not influenced by coincubation with EGF excluding heterologous transmembrane receptor modulation. These results suggest that EGF inhibits the action of phospholipase C-p- and y-isoenzyme-activating secretagogues in the exocrine pancreas by a

postreceptor mechanism. Basic fibroblast growth factor Pancreatic Phospholipase C

Carbachol acini

Bombesin

Cholecystokinin

IT is well established that the action of calcium-mobilizing secretagogues is initiated by bnnding of secretagogues to specific receptors, activation of phosphoinositide-specific phospholipase C, and subsequent hydrolysis of phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5trisphosphate ( 1,4,5-IP,) and 1,2-diacylglycerol (25). In reconstituted systems, occupation of heptahelical receptors leads to activation of pertussis- and cholera toxin-insensitive GTP binding proteins (G-proteins) of the G,,, I type (5,21) that activate P-i,soenzymes of phospholipase C. In contrast, growth factors bind to receptors possessing a cytoplasmic tyrosine kinase domain that is activated upon receptor occupation and directly stimulates phospholipase C-y. Numerous studies have shown that the calcium-mobilizing secretagogues CCK, bombesin, and carbachol stimulate enzyme secretion in the exocrine pancreas (25). In contrast, there are only a few studies examining regulation of secretion by growth factors. The fibroblast growth factor (FGF) family currently consists of seven different heparin-binding polypeptides that have mitogenic activity towards a wide variety of cells and have

Inositol 1,4,5-trisphosphate

EGF

roles in cell differentiation and angiogenesis (2). Among the different FGFs, acidic FGF (aFGF) and basic FGF (bFGF) are best characterized. bFGF is a potent secretagogue eliciting amylase release, increase in the intracellular 1,4,5-IP3 level, and release of intracellular calcium. Thus, bFGF appears to act via similar signal transducing pathways as cholecystokinin-octapeptide (CCK-8) (4). EGF is a single-chain, 53-amino acid polypeptide that has diverse physiological functions such as mitogenic effects and inhibition of gastric acid secretion (7). EGF has dual effects on pancreatic amylase secretion. It is a weak stimulator of amylase release and mobilizes calcium from intracellular stores without significant increase in 1,4,5-IP3 production (4,18,24). In the presence of CCK-8, however, EGF inhibits CCK-%induced 1,4,5-IP3 production, release of intracellular calcium, and activation of the Cl- conductance in zymogen granules (10,16,18). Moreover, EGF shifts the dose-response curve for CCK-S-induced amylase release to higher concentrations of the hormone (24), indicating that EGF is a negative modulator of pancreatic exocrine function.

’ Requests for reprints should be addressed to Stefan Zeuzem, M.D., University D-60590 Frankfurt a. M., Germany.

123

of Frankfurt

a. M., II. Medical Department,

Theodor-Stem-Kai

7,

STRYJEK-KAMINSKA

124

In the present study, rat pancreatic acini were used to investigate the effect of EGF on amylase release and 1,4,5-IP1 production in response to various calcium-mobilizing secretagogues that have been shown to activate different isoenzymes of phospholipase C. METHOD

Chemicals Collagenase type III, soybean trypsin inhibitor, digitonin, bovine serum albumin, bombesin, CCK-8, bovine bFGF, reagents for the detection of amylase, human recombinant epidermal growth factor (EGF), and carbachol were from Sigma (Deisenhofen, Germany). 1,4,5-IP3 assay kit, [‘251]bombesin, BoltonHunter [“‘I]CCK-8, [‘251]bFGF, and [3H]N-methylscopolamine bromide were purchased from New England Nuclear (Dreieich, Germany). Standard chemicals (p. a. quality) were obtained from Merck (Darmstadt, Germany). Preparation

of Pancreatic Acini

Acini were isolated from the pancreas of male Wistar rats by collagenase digestion as described (15,26). Acini from two rats were suspended in 30 ml Na+-Krebs-Ringer HEPES buffer of the following composition (in mM): 140 NaCl; 4.7 KCl; 2 MgC&; 1.2 KH2P04; 1.2 CaCl,; 10 glucose; 0.2% (w/v) bovine serum albumin; 0.01% (w/v) soybean trypsin inhibitor; 10 HEPES/ NaOH, pH 7.4. Viability of the cells after the experiments exceeded 90%, as estimated by the trypan blue dye exclusion method. Measurement

of Amylase Release

The detection of amylase released into the medium from isolated rat pancreatic acini was performed essentially as described previously (17,24). Briefly, 5-ml aliquots of acinar suspension were incubated with appropriate secretagogues for 30 min at 37°C under continuous supply of oxygen in a shaking water bath. Aliquots (300 ~1) were removed at the beginning and at the end of the incubation. Cells were subsequently pelleted by brief (2 min) centrifugation (1000 X g). The supematant (200 PI) was removed and assayed for amylase content measuring the hydrolysis rate of nitrophenyl-maltoheptaosid. Amylase release was expressed as a percentage of total amylase content of the samples at the beginning of the incubation. 1,4,5-IP, Determination 1,4,5-IP, was measured using a radioreceptor assay specific for the 1,4,5-isomer of inositol trisphosphate as described previously (17,24). Acini from two rats were suspended in 20 ml Na+-Krebs-Ringer HEPES buffer, and 2-ml aliquots were incubated with appropriate secretagogues in a continuously stirred cuvette at 37°C. After the indicated time, 200~~1 aliquots were removed and mixed with an equal volume of chilled 20% (w/v) trichloroacetic acid. After centrifugation at 10,000 X g (4”C), 300 ~1 of the supematant was removed and extracted with 800 ~1 1,1,2-trichloro-l,2,2-trifluoroethane/trioctylamine (3: 1 by vol.); 100 ~1 of the upper aqueous phase was assayed for 1,4,5-IP, content according to the protocol of the manufacturer. Protein was determined according to the method of Bradford (1) using bovine serum albumin as a standard. Receptor Binding Assay Receptor binding studies in isolated pancreatic acini were performed in 40 ml Na+-Krebs-Ringer HEPES buffer, using 70,000

ET AL.

cpm/ml of radiolabeled ligand label ([‘251]bombesin, BoltonHunter [?]CCK-8, [“‘I]bFGF, or [‘H]N-methylscopolamine bromide), and increasing amounts of unlabeled reagents (bombesin, CCK-8, bFGF, or carbachol). After incubation for 1 h at 37°C and continuous supply of 100% oxygen, the acini were separated from the medium by centrifugation. Cell-associated radioactivity was measured after the pellets were washed three times with ice-cold saline solution. Nonsaturable binding of the radioligands was determined as the amount of radioactivity associated with the acini in the presence of excess unlabeled ligand and was < 30% of total binding except for [‘2’I]bFGF. Data Analysis All experiments were repeated at least three times with acini from different preparations. The data presented are means 2 SEM. Statistical significance was calculated using an one-tailed Student‘s t-test for paired values. RESULTS

Effect of EGF on Amylase Release From Pancreatic Acini In isolated pancreatic acini, EGF (100 ng/ml) had no significant influence on basal amylase release of 3.4 & 2.1% of total amylase content present at the beginning of the experiment [Fig. l(A-D)]. Increasing concentrations of bombesin led to a monophasic increase in amylase release from isolated rat pancreatic acini [Fig. l(A)]. EGF (100 @ml) strongly decreased amylase release at all bombesin concentrations used. At a bombesin concentration of 100 nM, EGF reduced hormone-stimulated amylase secretion from 10.3 t 1.7% to 0.8 5 1.6% of total above basal (n = 3, p < 0.001). A lower concentration of EGF (50 ng/ml) reduced the bombesin (100 m-induced amylase release from 10.5 + 1.5% to 6.3 2 1.8% of total amylase content (n = 3), whereas 10 ng/ml EGF had no effect. Carbachol elicits similar responses in rat pancreatic acini as bombesin, but binds to a different receptor and has no mitogenic effects (25). As shown in Fig. l(B), the dose-response curve for carbachol-induced amylase release was biphasic, with an increase at low (< 1 p&f) and a decrease at supramaximal (> 1 @4) carbachol concentrations. Maximum amylase release of 11.0 2 1.8% of total above basal was observed at 1 @f carbachol. In contrast to the effect of EGF on CCK-8-stimulated amylase release [Fig. 2(C)], EGF (100 ng/ml) reduced carbachol-induced amylase release at both submaximal and supramaximal concentrations of the secretagogue. At a carbachol concentration of 1 PM, EGF (100 @ml) reduced amylase secretion from 11 .O t 1.8% to 2.6 ? 1.7% of total above basal (n = 3, p < 0.01). EGF (100 @ml) inhibited amylase release at submaximal, but not at maximal or supramaximal, CCK-8 concentrations. At the supramaximal CCK-8 concentration of 100 nM, EGF increased the CCK-8-induced amylase secretion [Fig. l(C)]. These data show that EGF inhibits amylase release in response to agonists acting on heptahelical receptors, which activate phospholipase C-/l isoenzymes. It has recently been shown that bFGF is a potent secretagogue in the exocrine pancreas, and that this response is likely due to the ability of bFGF to stimulate phosphoinositide-specific phospholipase C (4). In contrast to CCK-8, bombesin, and carbachol, bFGF binds to a receptor with an intrinsic tyrosine kinase activity (2,3,5,21,25). As illustrated in Fig. l(D), bFGF increased amylase release with an E&, of 5 i 3 pM. EGF (100 ng/ml) shifted the dose-response curve for bFGFinduced amylase release by at least one order of magnitude to higher bFGF concentrations [Fig. l(D)].

EGF INHIBITS ACTIVATION

OF PHOSPHOLIPASE

Bombesln

CCK-8

125

C

(M)

Carbachol

(M)

bFGF

(M)

(M)

HG. 1. Effect of EGF on (A) bombesin-, (B) carbachol-, (C) CCK-8-, and (D) bFGFstimulated amylase secretion. Rat pancreatic acini were incubated for 30 min without (open symbols) or with (filled symbols) EGF (100 @ml). The amount of amylase released into the medium was measured as described in the Method section. Results are mean values 2 SEM of three independent experiments. Amylase release of 10% corresponds to an amylase activity of 6.7 IJ/mg protein. *Indicates significant differences between points on the two different curves. *p < 0.05; **p < 0.01; ***p < 0.001.

Effect of EGF on Secretagogue-Induced 1,4,5-IP, Production All secretagogues investigated in this study are known to stimulate secretion by activation of phospholipase C. To investigate whether the inhibitory effect of EGF on secretagogue-stimulated amylase release correlates with inhibition of phosphoinositide-specific phospholipase C, we examined the effect of EGF (100 q/ml) on bombesin (100 nM)-, carbachol (100 @4)-, CCK-8 (100 r&f)-, and bFGF (1 w-induced 1,4,5-IPs production in isolated pancreatic acini. All four secretagogues caused a rapid increase in the 1,4,5IP, level with a maximum at 5 s (bombesin, carbachol, and CCK8) or 15 s (bFGF) after beginning of the incubation (n = 3) [Fig. 2(A-D)]. Then, the 1,4,5-IPr content declined gradually towards basal values, but remained elevated during the whole observation period of 5 min. In the absence of secretagogue, the 1,4,5-IP, level did not change significantly (data not shown). Whereas EGF (100 nglml) alone had no effect on the basal 1,4,5-IP3 content of pancreatic acini (data not shown), EGF inhibited 1,4,5IP3 accumulation in response to all four secretagogues. EGF reduced the peak increase of 1,4,5-IP, concentration at 5 s after beginning of the incubation by 81 + 19% (bombesin), 65 2 15% (carbachol), and 60 + 12% (CCK-8) (n = 3). The maximum of bFGF-induced 1,4,5-IPJ production (15 s after addition of the growth factor) was decreased by 56 + 18% (n = 3). EGF also inhibited 1,4,5-IP3 production at lower concentrations of the secretagogues (e.g., at CCK-8 concentrations of 1 and 10 nkf and

a bFGF concentration of 0.1 n&f) (data not shown). These results show that EGF inhibits 1,4,5-IP, accumulation induced by heptahelical receptor activating agonists as well as by tyrosine kinase receptor agonists.

at

Effect of EGF on the Receptor Binding of the Secretagogues to Isolated Acini Competition studies were performed by incubating isolated pancreatic acini with [‘251]bombesin, [3H]N-methylscopolamine bromide, Bolton-Hunter [‘251]CCK-8, and [‘*‘I]bFGF, and increasing concentrations of the respective unlabeled ligand. The displacement curves for bombesin were similar in the presence and absence of EGF (100 &ml) (Fig. 3). Half-maximal displacement of tracer occurred at 4 nM bombesin in the absence of EGF and at 2 nM in the presence of 100 ng/ml EGF. Scatchard analysis of the binding data revealed linear plots with no differences in the respective slope. The abscissa1 intercepts yielded similar numbers of receptor sites, indicating that bombesin binds to a similar number of receptors in the absence and presence of EGF (data not shown). Similarly, EGF (100 ng/ml) had no significant effect on the displacement curves for [3H]N-methylscopolamine and Bolton-Hunter [“‘I]CCK-8 (data not shown). Half-maximal displacement of [3H]N-methylscopolamine by carbachol and [‘*‘I]CCK-8 by unlabeled CCK-8 was observed at 0.08 and 2 nM in the absence and at 0.11 m&f and 1 n&f in the presence of EGF, respectively (n = 3). The high nonspecific binding of [‘*‘I]bFGF

STRYJEK-KAMINSKA

126

ET AL.

(> 90% of the cell-bound fraction at tracer concentration) did not allow determination of specific binding parameters for bFGF. DISCUSSION

In the present study, we show inhibitory effects of EGF on both amylase secretion and 1,4,5-IP, production in response to bombesin, carbachol, CCK-8, and bFGF. Coincubation of the acini with EGF and either bombesin, carbachol, or CCK-8 revealed no heterologous transmembrane modulation of the bombesin, cholinergic, and CCK receptor binding. However, due to high nonspecific binding, we were unable to investigate the effect of EGF on [‘*‘I]bFGF receptor binding. All four substances are potent secretagogues in the exocrine pancreas. Bombesin, carbachol, and CCK-8 activate receptors of the seven-transmembrane o-helix type that activate phospholipase C-e by a G-protein-dependent mechanism (25). In contrast, the receptors for EGF and bFGF belong to another class of receptors that activate phospholipase C-y in a G-protein-independent fashion (2). Thus, our data suggest that EGF inhibits activation of these two isoenzymes of phospholipase C in pancreatic acini, presumably by postreceptor mechanisms. The inhibitory effect of EGF on 1,4,5IP, production was paralleled by inhibition of amylase release in response to carbachol, bombesin, and submaximal concentrations of CCK-8 or bFGF. However, at supramaximal CCK-8 concentration (100 nM), EGF increased hormone-stimulated amylase release (24). As discussed elsewhere (14,16,23), several lines of evidence indicate that the

60

0 010-'2

10-10 Bombesfn

[‘2SI]bombesin to pancreatic acini. Acini were incubated for 60 min at 37°C with 70,000 cpm/ml of [“‘I]bombesin with and without increasing concentrations of unlabeled bombesin. The data shown are representative for three independent experiments.

downstroke of the dose-response curve for CCK-&stimulated amylase release is due to an overstimulation of phospholipase C and/or activation of protein kinase C (6). Therefore, EGF may abolish overstimulation of phospholipase C at supramaximal CCK-8 concentrations and thus stimulate amylase. Similar to CCK-8, the dose-response curve for carbacholstimulated amylase release in acini is also biphasic, with an in-

3o(A) 0 \ 15-

Bombesln

O\

q \

q

10.

Yl--o

5 '%

Bombesln+EGF .I n +k-m

07 0

50

100

150

300 Time (WC)

Ttme (WC)

0

50

100

150

10-s

(M)

FIG. 3. Effect of EGF (100 ngknl) on specific receptor binding of

25-O 20. \

10-e

300

Time (set)

FIG. 2. Time course of (A) bombesin (100 n&f)-, (B) carbachol(O.1 nnW)-, (C) CCK8 (100 nM)-, and (D) bFGF (1 m-induced 1,4,5-IP, production in the presence (closed symbols) and absence (open symbols) of EGF (100 ng/ml). Acini were incubated with the indicated substances at 37°C in a stirred cuvette. The 1,4,5-IP, content of the acini was subsequently measured as described in the Method section. The results shown are representative for at least three different experiments performed on different days using separate acini preparations.

EGF INHIBITS ACTIVATION

OF PHOSPHOLIPASE

127

C

crease at low and a decrease at supramaximal concentrations of the secretagogue. Furthermore, carbachol is a potent stimulator of 1,4,5-IP, production in Ipancreatic acini (14,25). Thus, it is feasible to assume that the mechanisms of inhibition of amylase release at supramaximal concentrations of the two secretagogues are similar (i.e., due to overstimulation of phospholipase C). However, in the present study we found that EGF decreased amylase release also at supramaximal carbachol concentrations. Presently, the different effects of EGF on amylase release in response to CCK-8 and carbachol cannot be explained conclusively. Carbachol is not as effective as CCK-8 in stimulating phosphoinositide-specific phospholipase C (14). It is possible that, in the presence of EGF, even very high concentrations of carbachol are not sufficient to cause maximum amylase release [see Fig. l(B)]. Alternatively, different mechanisms may account for high dose inhibition of amylase release by CCK-8 and carbachol. Signal transductions of both secretagogues are at variance in several regards: 1. Both secretagogues bind to different receptors. 2. CCK-8- and bombesin-induced, but not carbachol-induced, signal transduction involves activation of pertussis toxinsensitive G-proteins (20,22). High concentrations of CCK8, but not carbachol, cause activation of adenylate cyclase (25). 3. In parotid acini, different proportions of cyclic phosphoinositides generated upon stimulation with carbachol and bombesin suggest that they activate different isoenzymes of phospholipase C (23). EGF is produced and secreted by both pancreatic acinar and duct cells (10). Although physiological levels of EGF in the interstitial fluid near the acini are unknown, the present results suggest that EGF is an autocrine/paracrine regulator of acinar amylase secretion. The finding that one growth factor inhibits the action of another is novel. EGF and bFGF bind to. different receptors. Five different FGF receptors have been cloned (8). These receptors have a particular structure possessing immunoglobulin-like domains at the extracellular site and an interrupted tyrosine kinase domain in the cytoplasmic part of the molecule. The opposing effects of EGF and bFGF suggest that they activate different signaling pathways in pancreatic acini, one causing stimulation (bFGF), the other inhibition (EGF) of phospholipase C and amylase release. In contrast, both growth

factors stimulate DNA synthesis in the exocrine pancreas and are overexpressed in pancreatic carcinomas (9,10), suggesting that both growth factors may act as autocrine/paracrine stimulators of pancreatic growth. The mechanism of EGF inhibition of amylase secretion in response to calcium-mobilizing hormones is not known. EGF did not affect receptor binding of carbachol, bombesin, and CCK-8 to the acini. A high degree of nonspecific binding did not allow determination of specific binding parameters for bFGF. Because EGF did not affect the binding of three out of four ligands and the effect of EGF on secretagogue-induced 1,4,5-IP, production could be detected within seconds, it appears unlikely that the effect of EGF can be explained by heterologous receptor downregulation. It has been shown that the EGF receptor interacts with cholera toxin- and pertussis toxin-sensitive G-proteins in rat pancreatic acini (19). However, the same G-proteins also interact with CCK and bombesin receptors, which stimulate phospholipase C activity (20,22). Thus, evidence for the involvement of pertussis toxinsensitive G-proteins in EGF-mediated inhibition of secretagogueinduced 1,4,5-IP, production in pancreatic acini is weak. In a different secretory system, the parietal cell of the stomach, EGF inhibits histamine-induced acid secretion, apparently by activation of pertussis toxin-sensitive G-proteins (11). In these cells EGF also inhibits acid secretion in response to carbachol, which activates phosphoinositide-specific phospholipase C and subsequently mobilizes intracellular calcium, but does not activate adenylate cyclase. However, EGF does not inhibit carbachol-induced calcium mobilization in parietal cells (1 l), indicating that it is not an inhibitor of phospholipase C in these cells. Thus, the mechanism of inhibition of carbachol-induced acid secretion by EGF differs from the action of EGF in response to calcium-mobilizing secretagogues in pancreatic acinar cells. In conclusion, we showed in the present study that EGF inhibits amylase secretion and 1,4,5-IP, production in response to various calcium-mobilizing secretagogues. This suggests that EGF decreases amylase release in response to calcium-mobilizing secretagogues by inhibition of phosphoinositide-specific phospholipase C. Moreover, these data raise the possibility that EGF acts as an autocrine/paracrine regulator of amylase secretion in vivo. ACKNOWLEDGEMENT

This study

was supported

in part by the

Deutsche Forschungsge-

meinschaft (Ze 237/4-l).

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