Regulatory Peptides, 30 (1990) 239-253

239

Elsevier REGPEP 00959

Stimulation of pancreatic amylase release is associated with a parallel sustained increase of cytoplasmic calcium L. SjOdin l, H . G . D a h l t n 1, P . E . L u n d 2 and E. Gylfe 2 l Department of Drugs, Pharmacological Division. National Board of Health and Welfare and 2Department of Cell Biology, Uppsala University, Uppsala (Sweden)

(Received 21 March 1989; revised version received and accepted 6 June 1990) K e y words: Acinar cell; Bombesin; Calcium; Caerulein; Carbachol;

Cholecystokinin; Eledoisin; Pancreas; Substance P

Summary The kinetics of the changes in the cytoplasmic Ca 2÷ concentration (Cai/ +) and amylase release were measured in fura-2-1oaded pancreatic acinar cells and perifused pancreatic acini, respectively. Cholecystokinin octapeptide (CCK-8) and its amphibian analogue caerulein induced similar dose-related increases of Caiz + and amylase secretion with threshold concentrations of 2 - 6 . 1 0 - 1 2 M, and maximal effects at 2 . 1 0 - l o M. The action of CCK/caerulein on Ca~2+ was complex and similar to that of carbachol and bombesin with a prompt several-fold increase within seconds followed by a gradual decline over more than 5 min to a new sustained suprabasal level. The kinetics of amylase release in response to CCK and carbachol correlated with the changes in Cai2 +. Additions of the antagonists N2,OZ-dibutyrylguanosine 3' : 5'-cyclic monophosphate and atropine after 30 min of CCK-8 and carbachol stimulation, respectively, were associated with prompt lowerings of Ca~z ÷ and inhibitions of amylase secretion. The patterns observed with substance P (SP) and eledoisin were different with high concentrations ( 1 0 - s - 1 0 - 7 M) giving monophasic increases of Caiz+ and amylase release. An initial stimulation of cells with a high dose of CCK eliminated the Cai2 + response to further stimulation with CCK, carbachol, bombesin and SP, whereas cells subjected to initial stimulation with SP responded to subsequent exposure to CCK with prolonged elevation of Ca~2 ÷. The data indicate that stimulation with CCK, carbachol and bombesin may be associated with intracellular mobilization of calcium

Correspondence: L. SjOdin, Socialstyrelsens LSkemedelsavdelning,P.O. Box 607, S-751 25 Uppsala,

Sweden. 0167-0115/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

240 from more than one pool, and that an increase of Cai2 ÷ is involved even in threshold stimulation of amylase release.

Introduction

Amylase secretion from guinea pig pancreatic acinar cells is stimulated by a number of agents such as acetylcholine, bombesin, cholecystokinin (CCK), secretin, substance P (SP) and vasoactive intestinal polypeptide (VIP). Whereas the effects of secretin and VIP are mediated by cyclic AMP [1,2], agents such as acetylcholine, bombesin, CCK/caerulein, and SP are thought to act by lysing polyphosphoinositides with formarion of CaZ+-mobilizing inositol 1,4,5-trisphosphate (IP3) and protein kinase C (PKC)-activating diacylglycerol [3-8]. However, a major role for Ca 2+ has been questioned, since caerulein and carbachol stimulated amylase release [8,9] and PKC translocation [9] at concentrations without detectable effects on the cytoplasmic Ca 2 + concentration (Cai2 + ). In the present study, the Ca 2 + indicator fura-2 has been used together with sensitive dual-wavelength fluorometry to study Ca~2 + of dispersed pancreatic acinar cells exposed to CCK-8/caerulein, bombesin, carbachol and SP. This approach enabled the demonstration of Ca~2 + elevations at all CCK/caerulein and SP concentrations stimulating amylase release from perifused acini. Furthermore, the kinetics of the changes of Cai2 + and amylase release were correlated throughout the response to these secretagogues.

Materials and Methods

Male guinea-pigs (150-200 g) were obtained from AB Sahlins fOrsOksdjursfarm, Malm6, (Sweden). Crude and purified collagenase, hyaluronidase, purified soybean trypsin inhibitor, N-methyl-D-glucamine and N2,O2-dibutyryl-guanosine 3': 5'-cyclic monophosphate (Bt2cGMP) were from Sigma Chemical Co., St Louis, MO. Bovine serum albumin was obtained from ICN Life Sciences, Cleveland, OH, and Hepes (2-hydroxy-ethyl-piperazine-N-sulfonic acid) from Kebo Lab AB, Stockholm, Sweden. Biogel P4 was from Bio-Rad, Richmond, CA. Phadebas amylase test tablets were purchased from Pharmacia Diagnostics AB, Uppsala, Sweden. Modified Eagle's medium amino acid solution (50-times concentrated) and vitamin solution (100-times concentrated) were from Gibco Bin-Cult, Paisley, U.K. Molecular Probes Inc., Eugene, OR supplied fura-2 tetracetoxymethylester. Synthetic bombesin, cholecystokinin octapeptide (CCK-8), eledoisin, somatostatin and substance P were obtained from Peninsula Lab, Belmont, CA whereas caerulein was from Farmitalia, Milan, Italy. Porcine secretin was a gift from Professor Viktor Mutt, Karolinska Institutet, Stockholm. All other chemicals were of the highest grade commercially available.

241

Preparation of dispersed pancreatic acinar cells and acini Guinea pigs were starved overnight and killed by a blow to the head. Pancreas was dissected free from fat and mesentery. Dispersed acinar cells were prepared using the technique of Amsterdam and Jamieson [10], and pancreatic acini were isolated according to the method of Peikin et al. [ 11 ]. Cells and acini were normally suspended in an incubation solution containing 24.5 mM Hepes (pH 7.4), 98 mM NaC1, 6 mM KC1, 2.5 mM NaH2PO 4, 0.5 mM or 2 mM CaCI 2, 1.0 mM MgCI 2, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 1% (v/v) albumin, 0.01% trypsin inhibitor, 2% (v/v) amino acid and 1% (v/v) vitamin mixture. The solution was equilibrated with 100% 02. Fura-2 loading and measurement of C ~ + For each experiment, 1 - 3 . 1 0 6 cells, or in three experiments acini, were suspended in 5 ml medium and 5 #1 1 mM fura-2 acetoxymethylester in dimethylsulphoxide added. After incubation for 40 min, the cells were spun down and washed with medium lacking the indicator. The cells were then suspended in 1 ml medium and transferred to a 1 cm quartz cuvette placed in the thermostatically controlled (37 °C) cuvette holder of a time-sharing multichannel spectrophoto-fluorometer [12]. The suspension was continuously stirred from the top by a rotating plastic spiral not interfering with the light path. A 75 W xenon arc lamp combined with 5-nm half-bandwidth interference filters in the rotating air-turbine fdter changer provided excitation light flashes of 1 ms duration at 340 and 380 nm every 10 ms. Emission was measured with a photomultiplier at 510 nm using a 30 nm half-bandwidth filter. The electronically separated signals excited at 340 and 380 nm were fed into an analog ratio meter. The ratio as well as the 380 nm excitation fluorescence were recorded on a strip chart recorder. The initial contribution of extracellular fura-2 was determined by addition of excess E G T A followed by an identical concentration of Ca 2 +. Leakage of fura-2 into the medium was estimated from the initial slope of the ratio recording and compensated for [13]. At the end of each experiment, the cells were disrupted by addition of 0.05~ Triton X-100. The 340/380 nm fluorescence excitation ratio and the 380 nm excitation fluorescence were then obtained both at saturation Ca 2 + concentrations and at less than 1 nM Ca 2 +. The latter was accomplished by addition of excess E G T A as well as Tris to raise pH above 8.3. Cai2÷ could now be calculated as previously described [ 13]. Measurement of amylase release The kinetics of amylase release were studied by perifusing acini mixed with Biogel P4 polyacrylamide beads in a 0.5 ml column kept at 37 °C in an incubator. A 30-50 #1 sample of acini from a 50 g pellet was transferred to a column after mixing with 0.5 ml suspension of beads in incubation solution containing 0.5 or 2 mM Ca 2 +. A flow of medium at a rate of 0.5 ml/min was delivered to the column by a peristaltic pump. The perifusate was collected in 1 min samples by a fraction collector and analyzed for amylase using the Phadebas reagent as substrate [2]. Similar perifusion techniques have also been described by Frandsen [14] and Singh et al. [15].

242 Results

T h e Ca~2 + r e s p o n s e s o f one p r e p a r a t i o n o f d i s p e r s e d acinar cells to different concentrations o f C C K - 8 are shown in Fig. 1. Caerulein induced similar d o s e - r e l a t e d effects (not shown). T h e stimulation a p p e a r e d to result in a c o m p l e x increase o f Cai2 +. There was an initial severalfold rise from a b a s a l level o f about 200 n M within seconds. This p e a k was followed by a shoulder declining during several minutes with stabilization o f Ca~2 + at a new s u p r a b a s a l level. In a series o f experiments, the threshold concentration was 2 - 6 . 1 0 - 12 M, a n d m a x i m a l effects required 2 - 6 . 1 0 - lo M (Fig. 2). Similar concentrations were required for threshold and m a x i m a l release o f amylase from perifused pancreatic acini. In a m e d i u m with an axcess o f E G T A a d d e d 1-3 min before stimulation, the threshold concentration for C C K - 8 - i n d u c e d increase o f Cai2 + was 2 . 1 0 - 11 M a n d the m a x i m a l effect was smaller than in presence o f extracellular C a 2 + (Fig. 2). A d d i t i o n o f 1 m M o f the receptor antagonist B t 2 c G M P about 30 min after C C K stimulation resulted in a r a p i d return o f C a i2 + to the b a s a l level (Fig. 3). A small lowering

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243

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Fig. 10. Effects of different concentrations of SP on amylase release (leR panel) and on Ca~2+ (right panel). Isolated pancreatic acini were perifused with a medium containing 0.5-2 mM Ca~+. Amylase release is expressed as maximal increase above basal secretion (~o). Fura-2-1oaded acinar cells were stimulated with 2.10-11-6.10 -s M SP in the presence of 0.5 mM ( 0 ) or about 10 -s M Ca2+ (O; with EGTA added 1-3 min before stimulation). Each addition was made to previously unstimulated cells. Results are peak increases ofCa~2+ above basal level. Means of 2 (amylase) and 2-5 (Cai2+ ) separate experiments. Vertical bars denote S.E. (Cai2 + ) or range (amylase release).

250 However, a slight stimulatory effect of a high concentration of secretin on Cai2 ÷ in rat pancreatic acini has been reported [16]. The present data demonstrate great similarities in the actions of C C K and carbachol on Ca~2 + of pancreatic acinar cells and on amylase secretion from isolated acini. Initial peaks of Caiz ÷ were thus followed by slow declines to new levels, which at least in the presence of extracellular Ca 2 ÷ were suprabasal and sustained. In the parotid gland, the corresponding prolonged phase of the Ca~2 + response to carbachol has been found to depend on external Ca 2+ [17]. Also, bombesin may have a similar effect whereas it became clear that high doses of SP and eledoisin induced only an initial Ca~2 + peak. The more sustained effect at lower doses of SP, may be due to a continued action on an undepleted intracellular pool of calcium. The current results from kinetic experiments indicate that SP evokes similar maximal increases of Ca~2 + and amylase release as CCK. Such findings may appear to be at variance with our earlier results showing that SP stimulates 45Ca efflux and amylase release from isolated acinar cells less effectively than caerulein [18]. However, those results were based on amylase release measured during incubations for 30 min and 4 5 C a effiux during 5 min. The smaller responses to SP may consequently at least in part be due to less sustained effects of SP stimulation. The Ca~2 + peaks can to an important degree be attributed to intracellular mobilization of Ca 2 +, since they occur also in the absence of external Ca 2 +. This effect is probably at least partly mediated by IP 3. However, in analogy with previous suggestions [ 19] the data indicate that more than one intracellular pool may be involved in the actions of CCK, carbachol and bombesin. The C C K induced Ca~2+ peaks thus exhibited shoulders even after maximal stimulation in the absence of external Ca 2 ÷. Since the Ca~2+ response patterns were similar when external N a ÷ was entirely replaced with choline or N-methyl-D-glucamine, a stimulated influx of N a + with mobilization of intracellular Ca 2 + does not seem to be implicated. Maximal stimulation with C C K was the only stimulus which could abolish subsequent responses to all other agonists providing additional evidence for the involvement of different pools. However, the failure of a maximal stimulation with, e.g., bombesin or SP to prevent responses to C C K may also reflect the lower number of receptors for bombesin [20] and SP [21] than for C C K [22]. It is likely that the sustained Cai2 + responses include effects on the plasma membrane permeability to Ca 2 +. One alternative is inhibition of the outward transport of Ca z + and another is opening of Ca z + channels. The modest effects of N a + replacement with choline or N-methyl-D-glucamine on basal Ca~2 + or on the actions of agonists make the involvement of the N a +/Ca 2 + countertransport mechanisms unlikely. Moreover, the lack of acute effect of K + depolarization on Ca~2 + seems to exclude voltage-dependent Ca z + channels. If a plasma membrane permeability is involved in the sustained effect on Ca~2 + the C a 2 + concentration in the submembrane space can be expected to be considerably higher than in the rest of the cytoplasm. The present technique which measures average fluorescence cannot reveal a Cai2+ heterogeneity. Moreover, even average Ca~2+ may be underestimated under such conditions due to the nonlinear Ca 2 + -binding characteristics of fura-2 [ 13]. It was previously believed that the rise of Ca~2 + is the factor triggering pancreatic

251 enzyme secretion in response to acetylcholine, bombesin, CCK and SP [3,7,18,23,24]. However, this view was later challenged by fmdings of a striking dissociation between Caiz+ and secretion. Amylase release was thus stimulated by much lower agonist concentrations than those required for a rise of Ca~z ÷ [8]. Since observations indicated that even these very low concentrations of carbachol or caerulein induce PKC translocation [ 9], interest was focussed on the involvement of diacylglycerol in signal transduction. Although the present data by no means exclude a role for diacylglycerol, they undoubtedly question that amylase secretion, stimulated by physiological stimuli, occurs at basal Cai2 + levels. The reason why changes in C a i2 + have now been detected at much lower CCK concentrations than previously [8,9,25] is not entirely clear. The sensitive low-noise dual-wavelength real-time fluorescence system may be a contributing element. Another important factor is probably that suspensions of single ceils were utilized. When the more common approach with suspended acini was tested, the Cai2+ responses were considerably diminished (data not shown). The acinar approach which is superior for studies of enzyme secretion [ 11 ] may be disadvantageous in fluorescence analysis, possibly due to tissue quenching of the fluorescence. The current results indicate that threshold concentrations of CCK and SP for increase of Ca~2 + are similar to those required for stimulation of amylase release from perifused acini and to those previously reported for secretion from suspended isolated acinar cells [18] and acini [8,19,25,26]. It has not been possible to study the kinetics for amylase release from isolated acinar cells due to the limited amylase responses from the isolated cells [ 18]. The arguments for an involvement of Cai2 + in sustained amylase secretion are fortified by the lowering obtained with addition of Bt2cGMP 30 min after CCK or atropine 30 min after carbachol stimulation. Atropine has previously been reported to lack effect on Cai2 ÷ when added only 5 min after a carbachol challenge [8], and amylase release stimulated with carbachol, CCK, and bombesin has been reported to occur at basal Cai2+ 2 - 5 min after stimulants were added [3,27]. The arguments for calcium involvement are further strengthened, by the finding that SP at high concentrations only transiently increases Cai2 ÷ and amylase release. Even ifPKC activation should be found to be more important for amylase secretion than activation of Ca 2 ÷ -calmodulin dependent protein kinases it is noteworthy that PKC depends on both Ca 2÷ and phospholipid. The physiological activation of PKC should consequently be expected to involve a rise of Ca~2 +. The argument that PKC activation with phorbol esters can stimulate secretion at basal Cai2 + [3,25] is irrelevant in this context, since these drugs activate the kinase independently of a rise of Cai2 + [28,29]. Since this article was initially submitted, the prolonged Ca~2+ responses of pancreatic acinar cells to stimulation with carbachol or CCK and the low threshold concentrations for such stimulation have been confirmed [30].

Acknowledgements Excellent technical and secretarial assistance was given by Christina Karlsson, Elisabeth Lindberg, Birgitta Persson, Katarina Ryckenberg, Eila Viitanen and Linn6a

252 W a l l s t e n . T h i s study w a s s u p p o r t e d (12x-6240).

by the S w e d i s h M e d i c a l R e s e a r c h

Council

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