Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+ entry in mouse pancreatic acinar cells

Juan A. Rosado,1 Pedro C. Redondo,1 Ginés M. Salido,1 Stewart O. Sage,2 and Jose A. Pariente1

1Department of Physiology, University of Extremadura, Cáceres, Spain; and 2Department of Physiology, University of Cambridge, Cambridge, United Kingdom

Submitted 13 May 2004 ; accepted in final form 4 September 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We recently reported that store-operated Ca2+ entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+ channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as "secretion-like coupling." As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+ entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+ influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+ store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry


THE STIMULATION OF PANCREATIC ACINAR CELLS by various agonists results in an increase in the intracellular free Ca2+ concentration ([Ca2+]i) that consists of two components: Ca2+ release from finite intracellular stores and influx of Ca2+ across the plasma membrane (6), which is often required for full activation of cellular functions, such as secretion (35). Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in nonexcitable cells, including pancreatic acinar cells, is a process controlled by the filling state of the intracellular Ca2+ stores (22); however, the mechanism by which the filling state of the intracellular stores is communicated to the plasma membrane is poorly understood. Several hypotheses have been postulated to explain the activation of SOCE that may be organized into two main categories: indirect coupling, which supports the generation of a messenger molecule that opens or inserts channels into the plasma membrane so facilitating Ca2+ entry, and direct or conformational coupling, which suggests a physical interaction between a Ca2+ channel in the plasma membrane and a protein in the endoplasmic reticulum (ER) (3, 19). A number of studies by us and others have provided evidence supporting a conformational coupling-based model as the mechanism involved in the activation of SOCE in several nonexcitable cells (20, 27), including pancreatic acinar cells (26), which shares properties with secretion. This secretion-like coupling model is based on trafficking of portions of the ER toward the plasma membrane to facilitate direct interaction between proteins in the two membranes. Transient receptor potential channels (TRPC) have been presented as candidates for the conduction of SOCE (4, 24, 41), and a functional coupling between TRPC and various inositol 1,4,5-trisphosphate (IP3) receptor isoforms has been demonstrated in transfected cells and in cells naturally expressing TRPC (4, 23, 30, 40). As with secretion, in this model the reorganization of the actin cytoskeleton, especially the cortical filament network, plays a key regulatory role in the activation of SOCE (11, 15, 29, 32), and the membrane-associated cytoskeleton needs to be reorganized to facilitate coupling (20, 31).

Recently, we further demonstrated parallels between the events mediating secretion and SOCE in human platelets. Studies designed to investigate the involvement in SOCE of classically recognized secretory proteins, collectively termed soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), concluded that the synaptosome-associated protein (SNAP-25) is required for the activation and maintenance of SOCE in these cells, whereas vesicle-associated membrane proteins (VAMPs) are not necessary for Ca2+ entry in platelets (25). A number of studies have reported the presence of SNARE proteins in the pancreas, and a functional role for SNAREs in regulated exocytosis in these cells has been demonstrated in several studies (for a review, see Ref. 36). SNAREs mediate the specificity of vesicle trafficking and also support the formation of stable complexes between adjacent membranes (7, 38). To further investigate whether SNARE proteins are involved in SOCE in nonexcitable cells, we examined the role of SNAP-25 and VAMP in Ca2+ influx in mouse pancreatic acinar cells, a more specialized secretory cell type, in which our laboratory previously presented evidence for a secretion-like coupling model for the activation of SOCE (26).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Fura-2 and calcein acetoxymethyl esters (AMs) were obtained from Molecular Probes (Leiden, The Netherlands). Basal medium Eagle (BME) vitamin mixture, BME amino acid mixture, bovine serum albumin (BSA), tetanus toxin (TeTx), trypsin inhibitor, N-ethylmaleimide (NEM), phenylmethylsulfonyl fluoride (PMSF), leupeptin, thapsigargin (TG), and cholecystokinin octapeptide (CCK-8) were obtained from Sigma (Madrid, Spain). Collagenase CLSPA was obtained from Worthington Biochemicals (Lakewood, NJ). Botulinum toxin A (BoNT A) was purchased from Calbiochem (Madrid, Spain). Anti-SNAP-25 antibody (C-18), anti-VAMP antibody (FL-118), horseradish peroxidase-conjugated donkey anti-goat IgG antibody, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-VAMP-2 and anti-VAMP-3 antibodies were obtained from Affinity Bioreagents (Golden, CO). Enhanced chemiluminescence detection reagents were purchased from Pierce (Cheshire, UK). Hyperfilm ECL and Phadebas reagent test were obtained from Amersham (Arlington Heights, IL). All other reagents were purchased from Panreac (Barcelona, Spain).

Preparation of isolated pancreatic acinar cells. Adult male Swiss mice were obtained from the Animal Farm, Faculty of Veterinary Sciences, University of Extremadura. Mice were killed by cervical dislocation, the pancreas was rapidly removed, and the acinar cells were isolated as described previously (26). Briefly, the pancreas was incubated in the presence of collagenase for 5–10 min at 37°C under gentle agitation. The enzymatic digestion of the tissue was followed by gentle pipetting of the cell suspension through tips of decreasing diameter for mechanical dissociation of the acinar cells. After centrifugation, cells were resuspended in HEPES-buffered saline (HBS) containing (in mM) 10 HEPES, 140 NaCl, 4.7 KCl, 1 MgCl2, 10 glucose, and 1 CaCl2 (pH 7.4), supplemented with 1% (wt/vol) trypsin inhibitor, 1% (vol/vol) vitamin mixture, and 1% (vol/vol) amino acid mixture. In experiments performed in Ca2+-free medium, cells were resuspended in HBS containing 100 µM CaCl2, and 200 µM EGTA was added at the time of the experiment. All experimental procedures were approved by the local ethics committee.

Cell viability. Cell viability was assessed using calcein and Trypan blue. For calcein loading, cells were incubated for 30 min with 5 µM calcein-AM at 37°C and centrifuged, and the pellet was resuspended in fresh HBS. Fluorescence was recorded from 2-ml aliquots by using a spectrophotometer (Varian, Madrid, Spain). Samples were excited at 494 nm, and the resulting fluorescence was measured at 535 nm. After treatment with toxins for the times indicated, cells were centrifuged and resuspended in fresh HBS. The calcein fluorescence remaining in the cells was the same as in controls, suggesting that under our conditions there was no cellular damage. The results obtained with calcein were confirmed using the Trypan blue exclusion technique. Ninety-five percent of cells were viable after treatment with the toxins, similar to the observation in our resting acinar cell suspensions.

Western blotting. Cell stimulation was terminated by mixing with an equal volume of 2x Laemmli's buffer (14) with 10% dithiothreitol, followed by heating for 5 min at 95°C. One-dimensional SDS-electrophoresis was performed with 10 or 15% SDS-PAGE (50 µg total protein loaded/sample), and separated proteins were electrophoretically transferred, for 2 h at 0.8 mA/cm2, in a semidry blotter (Hoefer Scientific, Newcastle, UK) onto nitrocellulose for subsequent probing. Blots were incubated overnight with 10% (wt/vol) BSA in Tris-buffered saline with 0.1% Tween 20 (TBST) to block residual protein binding sites. Blocked membranes were then incubated with the anti-SNAP-25 antibody or the anti-VAMP antibody diluted 1:1,500 in TBST for 1 h. The primary antibody was removed, and blots were washed six times for 5 min each with TBST. For detection of the primary antibody, blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibody or horseradish peroxidase-conjugated donkey anti-goat IgG antibody diluted 1:10,000 in TBST, washed six times in TBST, and exposed to enhanced chemiluminescence reagents for 1 min. Blots were then exposed to photographic films, and the optical density was estimated using scanning densitometry.

Measurement of intracellular free Ca2+ concentration. Freshly isolated mouse pancreatic acinar cells were loaded with fura-2 by incubation with fura-2 acetoxymethyl ester (2 µM) at room temperature (23–25°C) for 40 min in accordance with previously established methods (6). Fluorescence was recorded from 2-ml aliquots of magnetically stirred cell suspensions (106 cells/ml) at 37°C by using a fluorescence spectrophotometer with excitation wavelengths of 340 and 380 nm and emission at 505 nm. Changes in [Ca2+]i were monitored using the fura-2 340/380 fluorescence ratio and calibrated according to the method of Grynkiewicz et al. (10).

Ca2+ entry in TG- and CCK-8-treated cells was estimated using the integral of the rise in [Ca2+]i for 2.5 min after addition of CaCl2 (27). TG- and CCK-8-induced Ca2+ release was estimated using the integral of the rise in [Ca2+]i for 6 min after their addition in cells suspended in a Ca2+-free medium (200 µM EGTA was added at the time of the experiment).

Amylase release. Amylase release was measured using the procedure published previously (13). Amylase activity, unless otherwise stated, was determined after a 30-min incubation with 1 nM CCK-8 using the Phadebas reagent. Amylase release was calculated as the percentage of the amylase activity in the acini at the beginning of the incubation that was released into the extracellular medium during the incubation.

Analysis of cytoskeleton-associated VAMPs. Pancreatic acinar cells were incubated in the absence or presence of TG (1 µM) in a Ca2+-free medium and were immediately lysed with an equal volume of 2x Triton buffer containing 2% Triton X-100, 2 mM EGTA, 100 mM Tris·HCl (pH 7.2), 100 µg/ml leupeptin, 2 mM PMSF, 10 mM benzamidine, and 2 mM Na3VO4 at 4°C for 30 min. Cell lysate was centrifuged at 16,000 g for 5 min. The supernatant was removed, and the pellet (cytoskeleton-rich fraction) was solubilized into the original volume in Laemmli's buffer (14), boiled for 5 min, and subjected to Western blotting as described previously using the anti-VAMP-2 and anti-VAMP-3 antibodies.

Statistical analysis. Analysis of statistical significance was performed using Student's t-test. The significance level was P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cleaving of SNAP-25 and VAMP by BoNT A and TeTx. The SNARE proteins SNAP-25 and VAMP, with molecular masses of 25 and 18 kDa, respectively (2), have been shown to be expressed in pancreatic acinar cells (5, 8, 12). These proteins are sensitive to cleavage by botulinum and tetanus toxins, respectively (16, 21). The efficacy of BoNT A and TeTX on SNAP-25 and VAMP, respectively, was tested under our conditions on the basis of their effect on amylase secretion and protein cleavage. As shown in Table 1, pretreatment of pancreatic acini for 1 h at 37°C in the presence of different concentrations (10–300 nM) of BoNT A or TeTx resulted in a concentration-dependent inhibition of both basal and CCK-8-stimulated amylase release. To confirm the inhibitory effect of BoNT A on SNAP-25 and of TeTx on VAMP in pancreatic acinar cells, we examined their effects on protein cleaving. As shown in Fig. 1, treatment of pancreatic acinar cells for 1 h at 37°C with 300 nM BoNT A, the concentration that significantly reduced basal and CCK-8-stimulated amylase secretion, resulted in a lower molecular mass immunoreactive band for SNAP-25, consistent with the removal of nine carboxyl-terminal amino acids, which accounts for the reduction in size of this protein after treatment with BoNT A (16, 33). In addition, treatment of pancreatic acinar cells with TeTx resulted in a significant loss of VAMP immunoreactivity, consistent with the centrally targeted cleaving of VAMPs by TeTx, yielding two small fragments that are undetectable at the gel resolution used (Fig. 1; n = 4), in agreement with previous studies (21).


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Table 1. Effect of BoNT A and TeTx on amylase release in pancreatic acinar cells

 


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Fig. 1. Cleavage of synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) by botulinum (BoNT A) and tetanus (TeTx) toxins. Pancreatic acinar cells were treated for 1 h at 37°C with 300 nM BoNT A or 300 nM TeTx and then lysed. Whole cell lysate proteins were separated using 15% SDS-PAGE (50 µg total protein loaded/sample) and transferred onto a nitrocellulose membrane for subsequent analysis. Membranes were analyzed by performing Western blotting (WB) using either anti-SNAP-25 antibody (top) or anti-VAMP antibody (bottom) as described in MATERIAL AND METHODS. Results are representative of 5 independent experiments.

 
Role of SNAP-25 and VAMP in activation of TG- and agonist-induced SOCE. SNAREs are membrane-associated proteins sensitive to NEM. We found that treatment of pancreatic acinar cells with NEM reduces both TG- and CCK-8-induced SOCE (data not shown), although NEM might affect other pathways. Hence, we tested the involvement of SNAP-25 and VAMP in SOCE using BoNT A and TeTx, which, as described, specifically inactivate these SNARE proteins. To determine whether the activity of SNAP-25 is required for the activation of SOCE in mouse pancreatic acinar cells, we examined the effect of BoNT A. As presented in Fig. 2, A and B, treatment of pancreatic acinar cells with 1 µM TG or 1 nM CCK-8 in a Ca2+-free medium resulted in a prolonged increase in [Ca2+]i due to Ca2+ release from intracellular stores. The subsequent addition of Ca2+ to the external medium induced a sustained elevation in [Ca2+]i indicative of SOCE (Fig. 2, A and B). Pretreatment of pancreatic acinar cells for 1 h at 37°C with 300 nM BoNT A decreased TG-induced SOCE by 61.0 ± 4.4% (Fig. 2A; n = 5; P < 0.01). Preincubation with BoNT A did not modify Ca2+ release by TG, suggesting that treatment with this toxin did not release Ca2+ from the stores.



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Fig. 2. Effect of BoNT A on the activation of thapsigargin (TG)- or cholecystokinin octapeptide (CCK-8)-evoked Ca2+ entry in pancreatic acinar cells. A: fura-2-loaded pancreatic acinar cells were incubated for 1 h in the presence of 300 nM BoNT A at 37°C and then stimulated with TG (1 µM) in a Ca2+-free medium (200 µM EGTA was added). CaCl2 (final concentration 1 mM) was added to the medium 6 min later to initiate Ca2+ entry. B: pancreatic acinar cells were incubated for 1 h at 37°C with 300 nM BoNT A and then stimulated with CCK-8 (1 nM) in a Ca2+-free medium (200 µM EGTA was added). CaCl2 (final concentration 1 mM) was added to the medium 6 min later to initiate Ca2+ entry. C: pancreatic acinar cells were incubated for 1 h at 37°C with 300 nM BoNT A and then stimulated with CCK-8 (1 nM) in a medium containing 1 mM CaCl2. Elevations in intracellular free Ca2+ concentration ([Ca2+]i) were monitored using the 340/380 fluorescence ratio, and traces were calibrated in terms of [Ca2+]i. Traces shown are representative of 5 separate experiments.

 
We further investigated the involvement of SNAP-25 in SOCE evoked by the physiological agonist CCK-8. Pretreatment of pancreatic acinar cells with 300 nM BoNT A for 1 h at 37°C decreased SOCE induced by CCK-8 (1 nM) by 53.2 ± 7.3% (Fig. 2B; n = 5; P < 0.01). Treatment with BoNT A did not modify CCK-8-evoked release of Ca2+ from stores, which confirms that this treatment did not affect the ability of pancreatic acinar cells to store Ca2+ in intracellular pools. In addition, our results indicate that BoNT A does not interfere with CCK-8-activated intracellular Ca2+ release pathways and that it does not affect receptor binding.

Figure 2C shows that treatment of pancreatic acinar cells with 300 nM BoNT A for 1 h at 37°C resulted in a substantial inhibition of the elevation in [Ca2+]i evoked by CCK-8 (1 nM) in a medium containing 1 mM Ca2+. The initial peak [Ca2+]i elevation above basal after agonist was significantly reduced from 348 ± 19 to 288 ± 9 nM (Fig. 2C; n = 5; P < 0.001). Because BoNT A does not affect CCK-8-induced Ca2+ release, the effect of this toxin on the CCK-8 response was entirely mediated by inhibition of SOCE. Taken together, these results suggest that the activity of SNAP-25 is at least partially necessary for the activation of SOCE in pancreatic acinar cells.

We next investigated the possible role of VAMP in SOCE induced by either TG or the physiological agonist CCK-8 in pancreatic acinar cells. Mouse pancreatic acinar cells were preincubated for 1 h at 37°C with 300 nM TeTx, a treatment that, as shown in Fig. 1, induces massive cleavage of VAMP. Pretreatment of pancreatic acinar cells with TeTx significantly decreased TG- and CCK-8-induced SOCE by 25.1 ± 9.5 and 38.6 ± 7.0%, respectively (Fig. 3, A and B; n = 5–7; P < 0.05). As with BoNT A, treatment with TeTx did not modify TG- or CCK-8-induced Ca2+ release, which indicates that this treatment did not affect the ability of these cells to store Ca2+ or interfere with the Ca2+ release mechanisms activated by CCK-8. To confirm the involvement of VAMPs on CCK-8-induced SOCE, we tested the effect of TeTx on CCK-8-induced Ca2+ mobilization in the presence of 1 mM extracellular Ca2+. As shown in Fig. 3C, treatment of pancreatic acinar cells with 300 nM TeTx for 1 h at 37°C significantly reduced the elevation in [Ca2+]i evoked by CCK-8 (1 nM) in a medium containing 1 mM Ca2+. The initial peak [Ca2+]i elevation above basal after agonist was significantly reduced from 332 ± 15 to 282 ± 11 nM (Fig. 3C; n = 5; P < 0.001).



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Fig. 3. Effect of TeTx on activation of TG- or CCK-8-induced Ca2+ entry in pancreatic acinar cells. A: fura-2-loaded pancreatic acinar cells were incubated for 1 h in the presence of 300 nM TeTx at 37°C and then stimulated with TG (1 µM) in a Ca2+-free medium (200 µM EGTA was added). CaCl2 (final concentration 1 mM) was added to the medium 6 min later to initiate Ca2+ entry. B: pancreatic acinar cells were incubated for 1 h at 37°C with 300 nM TeTx and then stimulated with CCK-8 (1 nM) in a Ca2+-free medium (200 µM EGTA was added). CaCl2 (final concentration 1 mM) was added to the medium 6 min later to initiate Ca2+ entry. C: pancreatic acinar cells were incubated for 1 h at 37°C with 300 nM TeTx and then stimulated with CCK-8 (1 nM) in a medium containing 1 mM CaCl2. Elevations in [Ca2+]i were monitored using the 340/380 fluorescence ratio, and traces were calibrated in terms of [Ca2+]i. Traces shown are representative of 5–7 separate experiments.

 
Role of SNAP-25 and VAMP in the maintenance of SOCE. SNAREs, such as SNAP-25 and VAMP, have been shown to participate in the machinery that leads to membrane fusion bringing the membranes into close apposition and maintaining stable membrane contacts (7). The latter is proposed to form the basis of the secretion-like coupling model for the maintenance of SOCE, in which the actin cytoskeleton has been shown to support the interaction between the ER and the plasma membrane (26, 27). To investigate the involvement of SNAP-25 in the maintenance of SOCE, we examined the effect of BoNT A on Ca2+ entry once SOCE had been activated using TG. As depicted in Fig. 4B, 300 µM BoNT A or the vehicle (HBS) was added 6 min after TG (1 µM) in pancreatic acinar cells. At the time the toxin was added, Ca2+ entry had already been activated (Fig. 4A, control t = 6 min). Cells were then incubated further for 1 h before Ca2+ was added to the medium to initiate Ca2+ entry. Addition of BoNT A after activation of SOCE significantly reduced the maintenance of Ca2+ entry by 42.9 ± 2.0% in pancreatic acinar cells (Fig. 4C; n = 5; P < 0.01). These observations suggest a role for SNAP-25 in both the activation and the maintenance of SOCE in these cells.



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Fig. 4. Effect of BoNT A on maintenance of TG-induced Ca2+ entry in pancreatic acinar cells. Fura-2-loaded pancreatic acinar cells were suspended in a Ca2+-free medium (200 µM EGTA was added) as described in MATERIALS AND METHODS. Cells were then stimulated with 1 µM TG, and 6 min later, 300 nM BoNT A or vehicle (HBS) was added as indicated (B). CaCl2 (final concentration 1 mM) was added to the medium at the same time, as a control (A; control, t = 6 min), or 1 h after BoNT A (B; control, t = 66 min), to initiate Ca2+ entry. C: histograms indicate the percentage of Ca2+ entry after treatment with BoNT A relative to its control (t = 66 min). Ca2+ entry was determined as described in MATERIALS AND METHODS. Values are means ± SE of 5 independent experiments. *P < 0.05 compared with control.

 
To investigate the involvement of VAMP in the maintenance of SOCE, we examined the effect of TeTx on Ca2+ entry once SOCE had been activated using TG. TeTx (300 µM) or the vehicle was added 6 min after TG, and cells were then incubated further for 1 h before Ca2+ (1 mM) was added to the medium to initiate Ca2+ entry. As shown in Fig. 5, addition of TeTx once SOCE had been initiated did not impair the maintenance of Ca2+ entry (n = 6). These observations suggest that VAMP is required for the activation but not the maintenance of SOCE in pancreatic acinar cells.



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Fig. 5. Effect of TeTx on maintenance of TG-induced Ca2+ entry in pancreatic acinar cells. Fura-2-loaded pancreatic acinar cells were suspended in a Ca2+-free medium (200 µM EGTA was added) as described in MATERIALS AND METHODS. Cells were then stimulated with 1 µM TG, and 6 min later, 300 nM TeTx or vehicle (HBS) was added as indicated (B). CaCl2 (final concentration 1 mM) was added to the medium at the same time, as a control (A, control t = 6 min), or 1 h after TeTx (control, t = 66 min), to initiate Ca2+ entry. C: histograms indicate the percentage of Ca2+ entry after treatment with TeTx relative to its control (t = 66 min). Ca2+ entry was determined as described in MATERIALS AND METHODS. Values are means ± SE of 6 independent experiments. *P < 0.05 compared with control.

 
VAMPs involved in SOCE in pancreatic acinar cells. Recent studies have reported that VAMP-1 is not expressed in pancreatic acinar cells (36); therefore, we investigated the expression of VAMP-2 and VAMP-3 in mouse pancreatic acini by performing SDS-PAGE and Western blotting using specific antibodies. Immunoblotting of whole cell lysates from pancreatic acinar cells with the anti-VAMP-2 or anti-VAMP-3 antibodies revealed the presence of both proteins in these cells (Fig. 6A; n = 4). To ascertain the VAMPs isoforms required for the activation of SOCE, we investigated the possible involvement of these proteins by testing their association with the cytoskeleton, which, as SNARE proteins, is involved in the vesicular transport during exocytosis (17) and SOCE (26). The cytoskeletal fraction was probed for the presence of VAMP-2 and VAMP-3 by performing Western immunoblot analysis on resting and Ca2+ store-depleted pancreatic acinar cells that had been treated with TG (1 µM). Only a small amount of these proteins (7 ± 2 and 14 ± 3% of total VAMP-2 and VAMP-3, respectively) was detected associated with the cytoskeleton of resting cells (Fig. 6B). When pancreatic acinar cells were treated with TG, the amount of VAMP-2 and VAMP-3 associated with the cytoskeleton significantly increased to 94 ± 6 and 82 ± 8% of total VAMP-2 and VAMP-3, respectively (Fig. 6B; n = 4).



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Fig. 6. VAMP-2 and VAMP-3 associate with the actin cytoskeleton upon store depletion in pancreatic acinar cells. A: pancreatic acinar cells were lysed, and whole cell lysate proteins were separated using 15% SDS-PAGE and transferred onto a nitrocellulose membrane for subsequent analysis. Membranes were analyzed by performing Western blotting using either anti-VAMP-2 or anti-VAMP-3 antibodies, as described in MATERIALS AND METHODS. B: pancreatic acinar cells were incubated for 6 min in the absence or presence of TG (1 µM) in a Ca2+-free medium (200 µM EGTA was added) and lysed with Triton buffer. Cell lysate was centrifuged, and proteins in the pellet (Triton X-100-insoluble cytoskeleton-rich fraction) and supernatant (Triton X-100 soluble) were subjected to Western blotting (50 µg total protein loaded/sample) as described in MATERIALS AND METHODS. Results are representative of 4 independent experiments.

 
In Fig. 1 we showed that TeTx cleaved most of the VAMP proteins detected with the general anti-VAMP antibody in pancreatic acinar cells. We have now investigated the sensitivity of VAMP-2 and VAMP-3 to TeTx, which might be helpful in identifying whether both proteins are involved in SOCE in these cells. As shown in Fig. 7, treatment of mouse pancreatic acinar cells for 1 h with 300 nM TeTx induced complete loss of VAMP-2 immunoreactivity. In contrast, VAMP-3 immunoreactivity was reduced by only 10%. These findings indicate that the effects observed after treatment with TeTx were mostly due to inactivation of VAMP-2.



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Fig. 7. Differing sensitivities of VAMP-2 and VAMP-3 to cleavage by TeTx. Pancreatic acinar cells were treated for 1 h at 37°C with 300 nM TeTx and then lysed. Whole cell lysate proteins were separated using 15% SDS-PAGE (50 µg total protein loaded/sample) and transferred onto a nitrocellulose membrane for subsequent analysis. Membranes were analyzed by performing Western blotting using either anti-VAMP-2 (left) or anti-VAMP-3 antibody (right) as described in MATERIALS AND METHODS. Results are representative of 3 independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Since the discovery of SOCE, different hypotheses, including direct "conformational" and indirect coupling, have been proposed to account for its activation. Recently, a number of studies suggested a modification of the conformational coupling hypothesis, the secretion-like coupling model, in several nonexcitable cells, including platelets (27, 31), smooth muscle cell lines (20), pancreatic acinar cells (26), astrocytes (34), or corneal endothelial cells (37). These studies reported an inhibitory effect of the cortical actin filament network on the activation of SOCE, a regulatory mechanism similar to that previously reported for secretion (17). In human platelets, our group (25) recently provided evidence for the involvement of SNAP-25, a SNARE protein, in the activation and maintenance of SOCE, which further supports the parallels between SOCE and secretion. In the present study, we investigated whether the secretory proteins SNAP-25 and VAMP are necessary for SOCE in a more specialized secretory cell type, the pancreatic acinar cell.

The functional involvement of SNAREs in SOCE in pancreatic acinar cells was investigated using highly specific botulinum and tetanus toxins, which effectively cleaved SNAP-25 and VAMPs, respectively, and which significantly blocked amylase secretion. In agreement with the findings reported in Xenopus oocytes (39) and platelets (25), our results indicate that preincubation of pancreatic acinar cells with BoNT A reduced SOCE evoked by either TG or the physiological agonist CCK-8. A similar effect was observed when pancreatic acinar cells were preincubated with TeTx to inactivate VAMPs. However, as reported in platelets (25) and Xenopus oocytes (39), we found that complete cleavage of SNAP-25 or VAMP reduced Ca2+ entry only partially. These proteins might play a role in enhancing SOCE by facilitating the coupling between elements in the plasma membrane and the ER. Alternatively, there might be an independent pathway also involved in the activation of SOCE in acinar cells, as we have previously shown in human platelets where two different Ca2+ stores activate two independent Ca2+ entry mechanisms (28). Our results clearly show that BoNT A and TeTx inhibited SOCE without having any effect on the release of Ca2+ from the intracellular stores, which suggests that their effect was entirely mediated by the modulation of Ca2+ entry.

Addition of BoNT A to pancreatic acinar cells after the activation of SOCE partially reversed Ca2+ entry already activated by TG or agonists, suggesting that SNAP-25 is partially required both for the activation and maintenance of SOCE in these cells. In contrast to the results observed with SNAP-25, we found that TeTx was without effect in pancreatic acinar cells when added once SOCE had been activated, which indicates that VAMPs are not required for the maintenance of SOCE in these cells. The lack of effect of TeTx on the maintenance of SOCE indicates that this toxin does not act as a Ca2+ channel blocker or a Ca2+ chelator.

Our results suggest that in pancreatic acinar cells SNAP-25 and VAMPs participate in the establishment of a stable contact between the membranes of the ER and the plasma membrane. However, we found a substantial difference between pancreatic acinar cells and platelets. In platelets, the regulation of SOCE by SNARE does not seem to involve VAMPs or else involves a TeTx-insensitive VAMP isoform (9, 25). However, in pancreatic cells, whereas SNAP-25 and VAMPs are initially required, perhaps to lead to a precise contact between membrane domains containing IP3 receptors and Ca2+ channels, only SNAP-25 seems to be required to maintain Ca2+ entry once the stores are depleted, perhaps as a component of the scaffold that supports the close apposition of the membranes. Because several VAMP isoforms have been described, we performed a series of experiments to identify the possible VAMPs involved in SOCE in pancreatic acinar cells. Our results indicate that both VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. At present, no pharmacological tools have been characterized that specifically inhibit one of these isoforms; therefore, we investigated the functional involvement of both isoforms by testing their association with the cytoskeleton, which is actively involved in the activation of SOCE and provides a support for vesicular trafficking (26, 27). Interestingly, we found that both proteins associate with the cytoskeletal fraction upon Ca2+ store depletion. These findings suggests that store depletion in mouse pancreatic acinar cells induces the association of both VAMP isoforms with the cytoskeleton. We found that whereas VAMP-2, under experimental protocol, is cleaved by TeTx, VAMP-3 seems to be insensitive to inactivation by this toxin, suggesting that the effect of the treatment with TeTx on SOCE is mostly due to inactivation of VAMP-2. Several studies recently reported the existence of TeTx-insensitive VAMP isoforms in several cell types, including neurons and epithelial cells (9, 18). Therefore, VAMP-2 associates with the cytoskeleton upon store depletion, where it might direct the transport of portions of the ER toward the plasma membrane to facilitate a precise contact between elements in both membranes, possibly the IP3 receptor in the ER and a Ca2+ channel in the plasma membrane. The physiological relevance of the cytoskeletal association of VAMP-3 deserves further attention.

As mentioned previously, a cytoskeleton-modulated secretion-like coupling is the model that best describes the mechanism of activation of SOCE in a number of cell types, including pancreatic acinar cells (26). The role of the actin cytoskeleton on Ca2+ influx has been extensively studied, and the results obtained are variable depending on the cell type investigated, ranging from a complete requirement of the actin filament reorganization (11, 29, 32) to a lack of effect (1, 37). The reason for these differences is not clear, and the distribution of actin network might be involved. Our present results suggest that SNAP-25 and VAMP-2 are partially required for SOCE in mouse pancreatic acinar cells. These observations provide further evidence supporting the secretion-like coupling mechanism for the activation of SOCE by showing parallels between SOCE and secretion in a secretory cell type. According to this model, store depletion by physiological agonists might induce the activation of a mechanism for the transport of portions of the ER toward the plasma membrane involving SNAP-25 and VAMP-2, similar to the exocytotic machinery, to direct a precise localization of both membranes into close apposition to facilitate the coupling between Ca2+ channels in the plasma membrane and IP3 receptors in the ER.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Ministerio de Ciencia y Tecnologia-DGI Grant BFI2001-0624. P. C. Redondo is supported by a DGESIC fellowship (BFI2001-0624).


    ACKNOWLEDGMENTS
 
We thank Mercedes Gómez Blázquez for technical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Rosado, Dept. of Physiology, Univ. of Extremadura, Av. Universidad s/n, Cáceres 10071, Spain (E-mail: jarosado{at}unex.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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