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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2009 Aug;90(4):387–399. doi: 10.1111/j.1365-2613.2009.00638.x

Calpain-mediated breakdown of cytoskeletal proteins contributes to cholecystokinin-induced damage of rat pancreatic acini

Heike Weber *, Saskia Hühns *, Frank Lüthen †, Ludwig Jonas ‡
PMCID: PMC2741149  PMID: 19659897

Abstract

The cytosolic cysteine protease calpain is implicated in a multitude of cellular functions but also plays a role in cell damage. Our previous results suggest that an activation of calpain accompanied by a decrease in its endogenous inhibitor calpastatin may contribute to pancreatic damage during cerulein-induced acute pancreatitis. The present study aimed at the time course of secretagogue-induced calpain activation and cellular substrates of the protease. Isolated rat pancreatic acini were incubated with a supramaximal concentration of cholecystokinin (0.1 μM CCK) for 30 min in the presence or absence of the calpain inhibitor Z-Val-Phe methyl ester (100 μM ZVP). The activation of calpain and the expression of calpastatin and the actin cytoskeleton-associated proteins αII-spectrin, E-cadherin and vinculin were studied by immunoblotting. The cell damage was assessed by lactate dehydrogenase release and ultrastructural analysis including fluorescence-labelled actin filaments. Immediately after administration, CCK led to activation of both calpain isoforms, μ- and m-calpain. The protease activation was accompanied by a decrease in the E-cadherin level and formation of calpain-specific breakdown products of αII-spectrin. A calpain-specific cleavage product of vinculin appeared concomitantly with changes in the actin filament organization. No effect of CCK on calpastatin was found. Inhibition of calpain by ZVP reduced CCK-induced damage of the actin-associated proteins and the cellular ultrastructure including the actin cytoskeleton. The results suggest that CCK-induced acinar cell damage requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Keywords: acute pancreatitis, calpain, calpastatin, cytoskeletal proteins, protease activation

Calpains are cytoplasmic neutral cysteine proteases of the papain family. In addition to several tissue-specific calpains (n-calpains), two ubiquitous isoforms, termed μ-calpain (calpain 1) and m-calpain (calpain 2), have been studied extensively. Both the activation and the catalytical activity of calpain are strictly regulated by cytosolic calcium (Ca2+) and its endogenous inhibitor, named calpastatin (Croall & DeMartino 1991). Unlike digestive proteases such as trypsin or chymotrypsin, calpain modifies its substrates by limited proteolytical degradation, producing distinct peptide fragments without further degradation. Regulated proteolysis by calpain is required for the control of fundamental cellular processes including cytoskeletal remodelling, membrane fusion, cell proliferation and differentiation, and activation of proteolytical cascades leading to apoptosis (Saido et al. 1994). Situations under which the proteolytical activity cannot be regulated within physiological ranges may result in cellular damage. In this regard, calpain has been reported to play a role in a variety of diseases including neurodegenerative diseases, muscular dystrophies and cataract development (Carafoli & Molinari 1998). Our previous results show for the first time a role of calpain in acute pancreatitis. Indeed, we observed that both the ubiquitous calpain isoforms are activated in the pancreatic tissue of rats suffering from cerulein-induced acute pancreatitis. Inhibition of calpain activation by prophylactic administration of the specific calpain inhibitor Z-Val-Phe methyl ester (ZVP) reduces cerulein-induced pancreatic damage (Weber et al. 2004). Support of our data is provided by a study demonstrating a protective effect of calpain inhibition in cerulein-induced acute pancreatitis of the mouse (Virlos et al. 2004).

One of the initial and critical events in cerulein-induced acinar cell damage appears to be the breakdown of the actin cytoskeleton resulting in inhibition of enzyme secretion (O’Konski & Pandol 1990; Jungermann et al. 1995). In this study, we investigated whether calpain may be at least in part responsible for this. We used freshly isolated rat pancreatic acini stimulated with supramaximal secretory concentrations of cholecystokinin (CCK), a well-established cellular system to study secretagogue effects on acinar cell integrity (Adler et al. 1984; Gorelick et al. 1993).

Our findings suggest that acinar cell damage following CCK hyperstimulation requires activation of calpain and that the actin cytoskeleton belongs to the cellular targets of the protease.

Materials and methods

Antibodies and reagents

For immunoblotting, rabbit polyclonal antibodies against μ-calpain (domain I; dilution 1:5000) and m-calpain (domain III; dilution 1:5000) were purchased from Sigma-Aldrich (Deisenhofen, Germany) and Calbiochem-Novabiochem (San Diego, CA, USA) respectively. A mouse monoclonal anti-calpastatin antibody (clone 1F7E3D10; domain IV; dilution 1:1000) was obtained from Sigma. A rabbit polyclonal anti E-cadherin antibody (H-108; dilution 1: 200) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Mouse monoclonal antibodies to αII-spectrin (1:1000) and vinculin (clone V284, 1:500) were from BIOMOL (Hamburg, Germany).

All cell culture material was purchased from Invitrogen (Paisley, UK). Collagenase was obtained from Serva (Heidelberg, Germany) and Bodipy FL phallacidin from Molecular Probes (Eugene, OR, USA). SDS and PVDF membranes were purchased from Bio-Rad (Munich, Germany). Tyr (SO3H) 27-cholecystokinin fragment*2 (CCK), Z-Val-Phe methyl ester (ZVP), and most other chemicals used were obtained from Sigma.

Preparation of pancreatic acini

Acini were prepared by collagenase digestion from pancreata of female rats (150–180 g body weight) starved for 18 h as described previously (Siegmund et al. 2004). Finally, the cells were suspended in Krebs-Ringer’s-HEPES buffer (pH 7.4, 37 °C). Cell viability was tested with the trypan blue exclusion assay immediately after preparation using a Neubauer chamber for blood cell counting. Preparations were accepted for the experiments if more than 95% of the cells excluded the dye.

Experimental design

All investigations were carried out between 8 and 12 am to avoid any potential circadian effects. Aliquots of acini were incubated for 30 min with 100 μM ZVP dissolved in DMSO or with vehicle only (final DMSO concentration, 1%). Thereafter, the cells were stimulated with 0.1 μM CCK dissolved in physiological saline. Control cells were incubated with DMSO and saline in the same manner. Samples were taken immediately after starting the experiments by CCK application (labelled 0 min) and after 15 and 30 min.

ZVP is a hydrophobic cell-permeable dipeptidyl methyl ester that has only minimal inhibitory effects on other cysteine proteases such as cathepsin B and several caspases such as caspase-1, -2,-3, or -7 (Edelstein et al. 1995; Kohli et al. 1999). In own preliminary in vitro experiments, ZVP was found to only minimally inhibit caspase-8 and the serine proteases trypsin and chymotrypsin (data not shown).

Sample preparation

Cells were pelleted and washed twice with ice-cold PBS. The pellet was lysed in buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 25 mM NaF, 25 mM ß-glycerophosphate, 2 mM Pefabloc, 0.011 mM Leupeptin, 0.2% Triton X-100, and 0.3% NP40, kept on ice for 10 min and stored at −80 °C until use.

Western blot analysis

For immunoblotting, samples were defrosted on ice, sonified and centrifuged for 20 min at 4 °C. The protein concentration in the supernatant was determined using the Advanced Protein Assay from Cytoskeleton (Denver, CO, USA). Thereafter, 20 μg of protein was mixed with sample buffer Roti-Load 1 (Carl Roth, Karlsruhe, Germany) according to the manufacturer’s suggested protocol, heated for 4 min at 95 °C and subjected to Laemmli SDS–PAGE. The fractionated proteins were either stained with Coomassie blue to confirm the uniformity of protein loading or electrophoretically transferred onto PVDF membranes using Towbin’s buffer (125 mM Tris, 95 mM glycine, 0.02% SDS, 20% methanol). The membranes were blocked in PBS-T containing 2.5% BSA and 2.5% nonfat milk powder for 5 h at room temperature and probed with the respective antibody overnight at 4 °C. Labelled proteins were visualized by enhanced chemiluminescence, following the manufacturer’s suggested protocol, using horseradish peroxidase-conjugated secondary antibody supplied with the kit at a 1:20,000 dilution (Amersham, Freiburg, Germany) or by the chromogenic immunodetection system WesternBreeze from Invitrogen. To adjust for differences in protein loading and Western transfer efficiency, the efficiency of batches of antibodies used, and the time of exposure, appropriate control samples were included in each gel. Densitometric analyses of immunoblots were performed using an electronic camera and the EASY program from Herolab (Wiesloch, Germany).

Determination of cell membrane damage

Cell damage was assessed by calculating the ratio of lactate dehydrogenase (LDH) activity in the incubation medium to total activity contained in the lysed cell pellet and in the incubation medium using LDH test kit and Synchron LX 20 analyzer (Beckman Coulter, Krefeld, Germany). To separate the incubation medium from cells, a sample of cell suspension was centrifuged through a 2:1 mixture (vol/vol) of dibutyl phthalate and bis (3,5,5-trimethylhexyl) phthalate. Total LDH activity was determined after lysis of cells in buffer containing 130 mM Tris, 75 mM NaCl, 10 mM CaCl2, and 0.2% Triton X-100, pH 8.0.

Isolation of RNA

Total RNA was isolated from fresh acini using the RNeasy kit from Qiagen (Hilden, Germany), according to the manufacturer’s protocol. The RNA quality was verified by electrophoresis using 1% agarose-formaldehyde gels, and the quantity was determined from absorbance measurements at 260 and 280 nm.

Reverse transcription-polymerase chain reaction analysis

Expressions of both ubiquitous calpain isoforms and calpastatin were monitored by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Briefly, 0.5 μg of total RNA was used and the sequence amplified by the SuperScript One-Step RT-PCR system from Life Technologies according to the manufacturer’s suggested protocol. The PCRs were individually optimized, and ß-actin was used as a loading control. The sequences of the forward and reverse primers were as follows: μ-calpain: 5′-GGT CAG CCT GTG CAC TTG AAG CG-3′ and 5′-TTG TTG GGC TCG AAG GTG GAG GG-3′; m-calpain: 5′-GAC AAC CCG AGC CAG GGA GCG-3′ and 5′-TTG TTG GGC TCG AAG GTG GAG GG-3′; calpastatin: 5′-CAA ACT CTT AAG CAT GC-3′ and 5′-CTA CTG GTC CAG TAT ATG GTG-3′; ß-actin: 5′-CCA CAC CTT CTA CAA TGA GCT GCG TGT GGC-3′ and 5′-GCC TGG ATA GCA ACG TAC ATG GCT G-3′. The following reaction conditions were established: μ-calpain: one cycle of 94 °C (2 min); 35 cycles of 94 °C (30 s), 62 °C (60 s), 72 °C (60 s) and one cycle of 72 °C (7 min); m-calpain: one cycle of 94 °C (2 min); 30 cycles of 94 °C (30 s), 62 °C (60 s), 72 °C (60 s) and one cycle of 72 °C (7 min); calpastatin: one cycle of 94 °C (2 min); 30 cycles of 94 °C (30 s), 54 °C (60 s), 72 °C (60 s) and one cycle of 72 °C (7 min); ß-actin: one cycle of 94 °C (2 min); 20 cycles of 94 °C (30 s), 61 °C (60 s), 72 °C (60 s) and one cycle of 72 °C (7 min). The PCR products were separated by 2% agarose gel electrophoresis containing ethidium bromide (0.1 mg/ml) and imaged by UV transillumination.

Electron-microscopic evaluation

For electron-microscopic investigations, cells were fixed for 1 h in 4% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.0), washed in the same buffer, and postfixed in 1% osmium tetroxide. Thereafter, the specimens were embedded in 0.1% liquid agar. Small cubes of the samples were dehydrated in a graded series of ethanolic solutions and embedded in Durcupan ACM. Ultrathin sections were prepared, stained with uranyl acetate and lead citrate, and examined with an electron microscope EM 902 A (Zeiss, Oberkochen, Germany). The evaluation was performed by a pathologist blinded to the experimental protocol.

Morphometry was performed on a total of 10 randomly selected electron micrographs of pancreatic acinar cells with a cytoplasmic area of 922.75 μm2 in each case (magnification x5050). The analysis of acinar cells included the estimation of number of vacuoles and the measurement of their areas using the software package iTEM, version 5, from OSIS (Muenster, Germany).

Fluorescence labelling of F-actin in isolated acini and confocal laser scanning microscopy

F-actin labelling was performed as we have previously described (Siegmund et al. 2004). Briefly, acini were fixed with 4% paraformaldehyde dissolved in HEPES buffer (in mM: 250 HEPES, 10 EGTA, and 4 MgCl2, pH 7.7) for 30 min. The fixed cells were incubated with 50% acetone and 1% BSA each for 10 min. Thereafter, they were treated with 1% Triton X-100 in imidazole buffer (in mM: 20 imidazole, 2 MgCl2, 80 KCl) containing 2 mM EGTA for 5 min and with imidazole buffer containing 1 mM EGTA for another 5 min. The last two steps were performed on ice. F-actin was stained with Bodipy FL phallacidin (5 U/ml) for 20 min in the dark following the manufacturer’s suggested protocol. Acini were washed four times with PBS and once with water, centrifuged on 3-aminopropyltriethoxysilane-coated coverslips and embedded in fluorescence mounting medium. Actin filament distribution was visualized using a confocal laser scanning microscope LSM 410 (Carl Zeiss, Jena, Germany) equipped with a 488 nm argon-ion laser, an emission filter LP 515 and a 63x oil immersion objective (Plan- Neofluar). Laser energy and parameters of intensity detection were kept constant during all measurements.

Statistical methods

Mean values of normally distributed data with equal variance were compared by Student‘s t-test. If the normality test failed, the data were compared by Mann–Whitney Rank Sum test. The statistical software package SigmaStat 3.1 from Jandel Corporation (Erkrath, Germany) was used. A P < 0.05 was considered as statistically significant.

Results

Time course of CCK-induced calpain activation in rat pancreatic acini

Calpain activation involves autoproteolytical truncation of the large 80-kDa subunit at the NH2 terminus (Saido et al. 1992; Melloni et al. 1996). Therefore, to investigate the time course of calpain activation in response to supraphysiological stimulation of pancreatic acini with CCK, we used antibodies directed against the corresponding 80-kDa catalytic calpain subunit on Western blot that only recognize latent but not the active calpain. The results show that CCK led to activation of both μ-calpain and m-calpain as indicated by the processing of the corresponding 80-kDa band (Figures 1a and 2a, each with lanes 2, 5 and 8; Figures 1b and 2b). The results also show that CCK-mediated calpain activation was a very early and rapid process. Thus, in the acinar cells taken immediately following CCK application (time point 0 min), calpain was already cleaved by an autoproteolytical process before the cells could be incubated in lysis buffer on ice.

Figure 1.

Figure 1

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Time course of CCK-induced μ-calpain activation in isolated rat pancreatic acini. Isolated rat pancreatic acini were incubated with or without the calpain inhibitor Z-Val-Phe methyl ester (100 μM, ZVP) for 30 min and stimulated with cholecystokinin (0.1 μM, CCK). Cells were harvested at the times indicated and calpain activation was identified by immunoblotting. (a) Representative immunoblot probed with an antibody directed against the 80-kDa subunit of μ-calpain. CCK-induced activation of μ-calpain is indicated by the processing of the 80-kDa calpain subunit and the reduced processing of this band in the presence of ZVP. (b) Corresponding densitometric quantification of four independent experiments on different acini preparations. The results are expressed as mean + SD. Statistical comparisons were performed by Mann–Whitney Rank Sum test. (c) Following extraction of total RNA, μ-calpain RNA expression was determined by RT-PCR. RT-PCR of ß-actin was used as loading control.

graphic file with name iep0090-0387-f2.jpg

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Time course of CCK-induced m-calpain activation in isolated rat pancreatic acini. Experimental design as in Figure 1. (a) Representative m-calpain immunoblot probed with an antibody directed against the 80-kDa subunit of m-calpain. CCK-induced activation of m-calpain is indicated by the processing of the 80-kDa band and the reduced processing of the band in the presence of ZVP. (b) Corresponding densitometric quantification of four independent experiments on different acini preparations. The results are expressed as mean + SD. Statistical comparisons were performed by Mann–Whitney Rank Sum test. (c) Following extraction of total RNA, m-calpain RNA expression was determined by RT-PCR. RT-PCR of ß-actin was used as loading control.

Incubation of acini with the calpain inhibitor ZVP before CCK administration diminished the calpain activation as detected by the reduced processing of the corresponding 80-kDa band in CCK-stimulated cells pretreated with ZVP in relation to CCK-treated cells without inhibitor preconditioning (Figures 1a and 2a, each with lanes 3, 6 and 9; Figures 1b and 2b).

To investigate whether CCK-induced calpain activity is regulated at the transcriptional level, RT-PCR was performed with ß-actin as control gene. As demonstrated in Figures 1c and 2c (each with lanes 2, 4 and 6), the increase in calpain activity was not accompanied by an increase in calpain mRNA expression. In fact, there were no differences in the RNA levels between the resting and stimulated cells during the 30-min observation period.

Effect of CCK hyperstimulation on the calpastatin expression in rat pancreatic acini

To gain insight into the possibility that a decrease in the endogenous calpain inhibitor calpastatin may play a role in CCK-induced calpain activation, RT-PCR and immunoblotting of the inhibitor were performed. The results demonstrate that CCK hyperstimulation of acini did not lead to changes in the calpastatin expression pattern neither at the transcriptional nor at the protein level (Figure 3a,b).

Figure 3.

Figure 3

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Supramaximal CCK concentration has no effect on calpastatin expression in isolated rat pancreatic acini. Pancreatic acini stimulated without or with 0.1 μM CCK were harvested at the times indicated. (a) Whole cell lysates were subjected to Western blot analysis using an antibody directed against calpastatin. Shown is a representative immunoblot from four independent experiments on different acini preparations. (b) Following extraction of total RNA, calpastatin RNA expression was determined by RT-PCR. RT-PCR of ß-actin was used as loading control.

Involvement of calpain in CCK-mediated damage of rat pancreatic acini

To elucidate the role of calpain in CCK-induced acinar cell damage, we investigated whether inhibition of calpain by ZVP preconditioning exerts protection against CCK-induced cellular damage. Stimulation of acini with CCK led to a time-dependent damage of the plasma membrane as indicated by the release of LDH into the incubation medium (Figure 4). Pretreatment of cells with ZVP nearly completely prevented the cytoplasmic LDH increase within the first 15 min, whereas the beneficial effect disappeared thereafter. There was no significant difference in the LDH release between CCK-stimulated cells both with and without ZVP treatment.

Figure 4.

Figure 4

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Involvement of calpain in CCK-induced plasma membrane damage of rat pancreatic acini. Experimental design as in Figure 1. At times indicated, the percentage of total cellular LDH activity released into the incubation medium was measured spectrophotometrically. Data are presented as mean + SD from six separate experiments. *P = 0.015;**P = 0.014; ***P = 0.001, untreated cells vs. cells stimulated with CCK; +P = 0.065, ++P = 0.009, CCK-stimulated cells vs. CCK-stimulated cells pretreated with ZVP; #P = 0.019, untreated cells vs. CCK-stimulated cells pretreated with ZVP. Statistical comparisons were performed by Student’s t-test and Mann–Whitney Rank Sum test.

Acute formation of numerous cytoplasmic vacuoles of various sizes hallmarks morphological acinar cell damage in response to CCK hyperstimulation (compare Figure 5a,b). Preconditioning of acinar cells with ZVP protected against this damage. Indeed, as demonstrated by electron-microscopic analysis, the degree of vacuolization was strongly diminished in CCK-stimulated cells pretreated with ZVP compared with CCK-stimulated cells without ZVP preconditioning when examined after 15 min (data not shown) and 30 min (compare Figure 5b,c). This observation was supported by a morphometric analysis revealing that size and number of acinar vacuoles decreased by 67% and 49%, respectively (Table 1). In particular, ZVP treatment clearly reduced the number of large vacuoles (>1.0 μm2) and midsize vacuoles (>0.05–1.0 μm2) 4.5-fold and twofold respectively, whereas the effect on the smaller vacuoles (≤0.05) was marginal only (Figure 6).

Figure 5.

Figure 5

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Involvement of calpain in CCK-induced ultrastructural damage of rat pancreatic acini. Experimental design as in Figure 1. At 30 min after incubation with CCK, cells were harvested for electron-microscopic investigations. The evaluation was performed by a pathologist blinded to the experimental protocol. Representative electron micrographs of an untreated cell (a) and of CCK-stimulated cells pretreated either without (b) or with ZVP (c).

Table 1.

Comparison of number and size of cytoplasmic vacuoles in pancreatic acini following CCK hyperstimulation in the presence or absence of ZVP

Mean number of vacuoles Mean area of vacuoles [μm2]
CCK 33.3 ± 6.9 11.9 ± 1.7
CCK/ZVP 16.9 ± 4.3 (51)* 3.9 ± 1.3 (33)*
P 0.058 0.001
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Experimental design as in Figure 1. At 30 min after incubation with CCK, cells were harvested for electron-microscopic investigations. For morphometrical analysis of acinar cell vacuoles, a total of 10 electron micrographs with a cytoplasmic area of 922.75 μm2 in each group were evaluated as described in Material and methods. Data are means ± SEM.

*

Values in parentheses are expressed as percentage of CCK. Statistical comparisons were performed by Student’s t-test.

Figure 6.

Figure 6

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Protective effect of calpain inhibitor ZVP on acinar cell vacuolization induced by CCK hyperstimulation. Experimental design as in Table 1. For morphometric analysis of acinar vacuoles, a total of 10 electron micrographs with a cytoplasmic area of 922.75 μm2 were evaluated in each group as described in Material and methods. Data are the number of vacuoles divided into five fractions of different sizes in each group.

To investigate whether calpain activation contributes to CCK-induced disorganization of the actin cytoskeleton likewise characterizing CCK-mediated acinar cell damage, the filamentous actin was stained with Bodipy FL phallacidin and analyzed by confocal laser scanning microscopy. The fluorescence images show that in resting acini, the actin filaments were mainly organized surrounding the luminal membrane, whereas only slight filament bands were visible along the basolateral region (Figure 7a–c). Exposure of acini to CCK caused time-dependent changes in the actin distribution with an actin filament decrease at the apical cell pole and an increase at basolateral areas within 30 min (Figure 7d–f). Additionally, the cell–cell contacts appeared to be disintegrated. There were substantial spaces between adjacent cells, which had also lost their normal pyramidal shape. Numerous cells were completely dissociated from the cell assembly. Preconditioning of cells with ZVP reduced the extent of CCK-induced actin filament alterations (Figure 7g–i). In detail, F-actin was restored to the apical membrane. But the fluorescence intensity beneath the lateral and basolateral membrane was of stronger degree compared with the resting cells. The cell-cell contacts appeared intact.

Figure 7.

Figure 7

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Involvement of calpain in CCK-induced changes in the actin cytoskeleton organization of rat pancreatic acini. Experimental design as in Figure 1. At the times indicated, cells were harvested, stained for F-actin with Bodipy FL phallacidin and evaluated by confocal laser scanning microscopy. Representative fluorescence micrographs of untreated acini (a–c) and of CCK-stimulated acini pretreated either without (d–f) or with ZVP (g–i). Note the bright actin labelling along the acinar luminal portion (arrow) and the weak actin labelling at the basolateral plasma membrane (arrowhead) (Figure 7c). The cell–cell contacts seem to be disrupted (Figure 7f; arrow).

To identify potential mechanisms responsible for calpain-induced acinar cell damage, immunoblot analyses for the cytoskeleton-associated proteins αII-spectrin, vinculin and E-cadherin were performed depending on time. Western blot analysis of αII-spectrin detected the parent protein at 240/280-kDa and the formation of spectrin breakdown products of 150 and 145-kDa immediately and at 15 min after CCK hyperstimulation respectively, that are specific for calpain (Figure 8a lanes 2, 5 and 8; Figure 8b). No αII-spectrin fragment at 120-kDa known to be specific for caspase 3 was observed. Investigating the effect of CCK on vinculin, a decrease in the parent protein band at 117-kDa accompanied by the formation of a calpain-specific vinculin fragment at 90-kDa was visible first after 15 min (Figure 9a, lanes 5 and 8; Figure 9b). In addition, the E-cadherin antibody identified the parent protein at 120-kDa that showed a decrease immediately after challenge (Figure 10a, lanes 2, 5 and 8; Figure 10b). Incubation of acini with ZVP before CCK administration decreased the formation of the 145-kDa spectrin fragment (Figure 8a, lanes 6 and 9; Figure 8b). In addition, ZVP also reduced the CCK-induced processing of vinculin and E-cadherin as demonstrated in Figure 9a, lanes 6 and 9; Figure 9b and in Figure 10a, lanes 3, 6 and 9; Figure 10b respectively.

Figure 8.

Figure 8

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Involvement of calpain in CCK-mediated proteolysis of αII-spectrin. Experimental design as in Figure 1. Whole cell lysates were subjected to Western blot analysis using an antibody directed against αII-spectrin. (a) Representative immunoblot demonstrating CCK-induced generation of spectrin breakdown products that is reduced when ZVP was given before CCK hyperstimulation. (b) Corresponding densitometric quantification of the spectrin fragments of four independent experiments on different acini preparations. The results are expressed as mean + SEM. Statistical comparisons were performed by Mann–Whitney Rank Sum test.

Figure 9.

Figure 9

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Involvement of calpain in CCK-mediated decrease in vinculin. Experimental design as in Figure 1. Whole cell lysates were subjected to Western blot analysis using an antibody directed against vinculin. (a) Representative immunoblot demonstrating CCK-induced processing of vinculin that is reduced when ZVP was given before CCK hyperstimulation. (b) Corresponding densitometric quantification of five independent experiments on different acini preparations. The results are expressed as mean + SD. Statistical comparisons were performed by Mann–Whitney Rank Sum test.

Figure 10.

Figure 10

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Involvement of calpain in CCK-mediated E-cadherin decrease. Experimental design as in Figure 1. Whole cell lysates were subjected to Western blot analysis using an antibody directed against E-cadherin. (a) Representative immunoblot showing CCK-induced decrease in the E-cadherin expression that is reduced when ZVP was given before CCK hyperstimulation. (b) Corresponding densitometric quantification of four independent experiments on different acini preparations. The results are expressed as mean + SD. Statistical comparisons were performed by Mann–Whitney Rank Sum test.

Discussion

Our results demonstrate that supramaximal CCK stimulation of rat pancreatic acini leads to activation of both ubiquitous calpain isoforms. Consequently, we were able to confirm our previous findings in intact pancreatic tissue showing that cerulein hyperstimulation of the rat causes activation of calpain as well (Weber et al. 2004). Using the in vitro model, we were able to show that the activation of calpain is a very rapid process occurring immediately following CCK hyperstimulation of acini.

Investigating whether the increase in calpain activity is associated with an increase in calpain RNA, we found no effect. This indicates that the regulation of calpain occurs at the post transcriptional level. Our finding is consistent with the concept that most cellular enzymes synthesized as pro-enzymes like calpain are regulated by conversion of the pro-enzyme into the active enzyme.

Several lines of evidence suggest that CCK may induce calpain activation via a Ca2+-dependent mechanism. Calpain is known to be regulated by an increase in the cytosolic Ca2+concentration in a variety of cellular systems (Saido et al. 1992; Melloni et al. 1996). We previously showed that Ca2+is also required for calpain activation in pancreatic acinar cells. Indeed, we observed a dose-dependent increase in calpain proteolytical activity following incubation of acinar cells with the Ca2+ionophore ionomycin that was completely prevented by quenching of intracellular Ca2+using the Ca2+chelator BAPTA-AM (Weber et al. 2002). In addition, stimulating isolated pancreatic acini with CCK in physiological concentration that is known to induce a rapid periodic rise in the acinar cytosolic Ca2±concentration, we observed a small increase in calpain proteolytical activity immediately following administration of the secretagogue (Ward et al. 1995; Weber et al. 2002). In this study, stimulating the cells with a supramaximal CCK dose that provokes an immediate and sustained Ca2±elevation, we found an activation of calpain as well also occurring immediately upon CCK administration (Raraty et al. 2000). Thus, the time course of CCK-induced Ca2+increase and CCK-induced calpain activation shows a close correlation suggesting that Ca2+may be responsible for calpain activation in response to CCK.

Further mechanism underlying CCK-induced calpain activation may be an inadequate level of the endogenous calpain inhibitor calpastatin (Melloni et al. 1992). We previously observed proteolytical degradation of the inhibitor in the pancreatic tissue of rats with cerulein-induced acute pancreatitis (Weber et al. 2004). In this study, however, we found no changes in the calpastatin expression pattern neither at the RNA level nor at the protein level indicating that a reduced inhibitory activity cannot be a mechanism for calpain activation in response to CCK. We assume that the degradation of calpastatin observed in the animal model has to be a consequence of the disease rather than an initial calpain activating event. It seems possible that the digestive pancreatic proteases or the proteasome likewise activated in the course of acute pancreatitis may be responsible for this (Grady et al. 1998; Doumit & Koohmaraie 1999; Saluja et al. 1999; Letoha et al. 2007).

CCK-induced calpain activation results in pancreatic acinar cell damage. We conclude this from the observation that inhibition of calpain by ZVP is associated with protection against CCK-induced morphological damage. In particular, ZVP clearly reduced the degree of CCK-induced vacuole formation. Interestingly, we observed a clear decrease in the number of large and midsize vacuoles, whereas the effect on small vacuoles was marginal. In line with this, only the large vacuoles have been reported to be a sign of cellular injury induced by CCK hyperstimulation (Jonas et al. 1998). The present results are supported by our previous observation that ZVP reduces the severity of cerulein-induced rat pancreatitis (Weber et al. 2004). Further support is provided by a study demonstrating a protective effect of calpain I inhibitor in cerulein-induced acute pancreatitis of the mouse (Virlos et al. 2004). The finding that ZVP nearly completely prevents the plasma membrane damage in the early stage following CCK hyperstimulation whereas the beneficial effect decreases later on may reflect the increasing influence of other pathological events such as the activation of the digestive enzymes (Doumit & Koohmaraie 1999; Grady et al. 1998, Saluja et al. 1999). In accordance with this assumption, the protective effect of ZVP against CCK-induced acinar cell vacuolization and actin filament disorganization was partial as well.

To understand molecular mechanisms underlying calpain-provoked cell damage, it is important to identify cellular targets of the protease. Acute pancreatitis induced by supramaximal secretagogue stimulation is characterized by a very early and rapid disruption of the apical actin cytoskeleton. The actin cytoskeleton is a highly dynamic network composed of actin filaments and a variety of actin-associated proteins playing a crucial role in the maintenance of the cellular structural and functional integrity including exocytosis (O’Konski & Pandol 1990; Elliget et al. 1991; Jungermann et al. 1995). Several lines of evidence suggest that calpain may contribute to acinar cell damage during acute pancreatitis through alterations of the actin cytoskeleton. Thus, some of the many calpain substrates have been reported to include actin and various actin-associated proteins (Croall & DeMartino 1991; Elliget et al. 1991). Furthermore, oxidative stress, an important event in the development of acute pancreatitis, induces both activation of calpain and changes in the spatial and temporal actin filament organization (Rosado et al. 2002; Weber et al. 2005). Inhibition of the oxidant-provoked calpain activity by different calpain inhibitors was found to be associated with a decrease in oxidant-induced changes in the actin filament organization suggesting a role of the protease in this injurious process (Weber et al. 2005). In line with this, oxidant-induced calpain activation in hepatocytes has been reported to cause degradation of the cytoskeletal proteins α-actinin and talin subsequently leading to cell damage (Miyoshi et al. 1996). Further evidence that the actin cytoskeleton may be a target of calpain is proved by our previous study showing that prophylactic application of ZVP exerts protection against changes in the pancreatic actin filament network in cerulein-induced acute rat pancreatitis (Weber et al. 2004). In this study, using the in vitro model of secretagogue-induced pancreatitis, we obtained comparable results. Thus, we found that inhibition of CCK-induced calpain activity by ZVP protects against ultrastructural damage of pancreatic acini including disassembly of the actin filaments. In support of this, various studies demonstrated that calpain inhibitors attenuate cleavage of actin and actin-associated proteins, resulting in cytoprotection against toxic injury, ischaemia-reperfusion injury and apoptosis in different systems (Elliget et al. 1991; Blomgren et al. 1995; Villa et al. 1998).

The actin-associated protein spectrin belongs to the preferential substrates of calpain. It is cleaved by the protease into fragments corresponding to apparent molecular weights of 150-and 145-kDa. The detection of these products has been widely used as biochemical markers for proteolytical calpain activity (Robert-Lewis & Siman 1993; Nath et al. 1996; Wang 2000; Goll et al. 2003). Like in a variety of epithelial cells, in the pancreatic acinar cell, spectrin is localized beneath the inner surface of the cell membrane stabilizing it through interactions with the actin cytoskeleton (Hesketh 1986; Bennett 1990). In addition, spectrin has been reported to participate in the establishment of specialized membrane domains and contribute to the stabilization of the adherens junctions (Beck & Nelson 1998; Pradhan et al. 2001;Simonovic et al. 2006). Our immunoblot analysis of αII-spectrin shows for the first time that degradation of this protein is a very early event in CCK-induced acinar cell damage and that calpain may contribute to this. The observation of the calpain-specific spectrin fragments supports the view that the processing of the 80-kDa calpain band really results in proteolytical activity of the protease. Calpain inhibitor ZVP reduces the expression of the 145-kDa breakdown product further supporting our conclusion that calpain contributes to its formation. The non-finding of any inhibitory effect on the 150-kDa spectrin fragment suggests that beside calpain other proteases may also be contributing to the formation of this fragment. In this regard, interleukin-1β-converting enzyme (caspase-1) and caspase-3 have been reported to cleave spectrin into a fragment of 150-kDa (Nath et al. 1996; Warren et al. 2007). Both proteases also are known to be activated during the course of acute pancreatitis (Norman et al. 1997; Beil et al. 2002). Based on the absence of a caspase-3-specific 120-kDa spectrin fragment, however, we can rule out the involvement of caspase-3 in CCK-mediated spectrin degradation (Nath et al. 1996; Warren et al. 2007). In line with our observation, calpain-induced proteolysis of spectrin has been reported to occur during hypoxia/ischaemia in different cellular systems including alveolar epithelial, neuronal and myocardial cells resulting in cell injury (Blomgren et al. 1995; Yoshida et al. 1995; Pike et al. 2001; Bouvry et al. 2006).

The trans-membrane glycoprotein E-cadherin is a further actin cytoskeleton-associated protein that has been identified as calpain substrate (Goll et al. 2003). E-cadherin plays a role in the formation of the adherens junctions. The extracellular domain of the protein links neighboring cells whereas the intracellular domain serves as anchor for the actin cytoskeleton via interaction with several catenins (Leser et al. 2000). During the course of acute pancreatitis, E-cadherin is rapidly dissociated, internalized and degraded, which is supposed to be responsible for actin disruption (Lerch et al. 1997; Leser et al. 2000). Accordingly, our data show a decrease in the E-cadherin level upon CCK hyperstimulation of acini as well. Blocking the calpain activity with ZVP exerts protection against CCK-induced decrease in E-cadherin suggesting a role of calpain in this process. Similarly, in prostate epithelial cells, calpain has been shown to cleave E-cadherin into a stable fragment that down-regulates the endogenous E-cadherin consequently leading to a decrease in cell survival (Rios-Doria et al. 2003; Rios-Doria & Day 2005). Whether a similar calpain-induced mechanism may also be important in our cellular system remains to be investigated.

The actin filament-associated protein vinculin is likewise known to be a substrate of calpain (Goll et al. 2003). Vinculin is localized within the focal adhesion complex and functions in connecting the actin filaments with the plasma membrane along with several adapter proteins (Drenckhahn & Mannherz 1983). Proteolysis of vinculin by calpain produces a 90-kDa fragment that can no longer cross-link the adapter proteins (Goll et al. 2003; Liu & Schnellmann 2003). In highly differentiated cells such as renal cells, calpain-induced cleavage of vinculin is associated with increased plasma membrane permeability and cell death (Liu & Schnellmann 2003). Our results demonstrate the formation of a 90-kDa vinculin breakdown product upon CCK treatment of acini as well that is sensitive to ZVP. Thus, it seems conceivable that a similar calpain-mediated mechanism may also contribute to morphological damage in our cellular system.

In summary, our results suggest that the mechanism underlying CCK-induced pancreatic acinar cell damage requires activation of the cysteine protease calpain and that the actin cytoskeleton and actin cytoskeleton-associated proteins belong to the cellular targets of the protease. Our observations have potential therapeutic implications for ameliorating acinar cell injury. We propose that prophylactic administration of calpain inhibitors may be a promising strategy for alleviating pancreatic damage occurring during pancreas transplantation, including organ preservation.

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