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
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ABSTRACT |
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synaptosome-associated protein; vesicle-associated membrane protein; pancreatic acinar cells; cytoskeleton; calcium entry
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).
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MATERIALS AND METHODS |
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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 510 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 (2325°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.
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RESULTS |
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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 = 57; 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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DISCUSSION |
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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.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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