The vesicle-associated membrane protein family of proteins in rat pancreatic and parotid acinar cells

GASTROENTEROLOGY 1996;111:1661–1669

The Vesicle-Associated Membrane Protein Family of Proteins in Rat Pancreatic and Parotid Acinar Cells HERBERT Y. GAISANO,* LAURA SHEU,* GILLES GRONDIN,‡ MENISHA GHAI,§ ANTONY BOUQUILLON,§ ANSON LOWE,x ADRIEN BEAUDOIN,‡ and WILLIAM S. TRIMBLE§ *Department of Medicine, University of Toronto and Toronto Hospital, Toronto, Ontario, Canada; ‡De´partement de Biologie, Universite´ de Sherbrooke, Sherbrooke, Quebec, Canada; §Departments of Physiology and Biochemistry, University of Toronto and Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada; and xDepartment of Medicine and Digestive Disease Center, Stanford University, Stanford, California

See editorial on page 1770. Background & Aims: The vesicle-associated membrane protein (VAMP) family of proteins may play an important role in regulating enzyme secretion from pancreatic and parotid acini. The purpose of this study was to characterize the isoforms produced in pancreatic and parotid acini and determine their subcellular locations. Methods: Using a battery of specific antisera and recombinant tetanus toxin light chain (which cleaves VAMP-2 and cellubrevin), the presence of each VAMP molecule in the acini was determined by immunoblotting of subcellular membrane fractions; their localization was determined by confocal immunofluorescence microscopy and immunogold electron microscopy. Results: Both VAMP-2 and cellubrevin were present on both the zymogen granule membrane and plasma membrane. VAMP1 was not present in the acinar cell but was found in the nerve endings innervating the acini. As expected, pancreatic acinar VAMP-2 and cellubrevin were sensitive to cleavage by recombinant tetanus toxin. Conclusions: VAMP-2 and cellubrevin may play integral roles in exocytosis of the pancreatic and parotid acinar cells, whereas VAMP-1 is restricted to nerves that innervate the acini and may function to modulate exocrine activity.

T

he acinar cell is a classic model for the study of regulated secretion in nonexcitable cells.1 Secretion in these cells is controlled by circulating hormones2 and by direct neural regulation.3 Receptors for these secretagogues activate second-messenger pathways that ultimately lead to secretion.2 Acinar enzyme secretion occurs by both constitutive and regulated pathways.4 However, little is known about the specific molecular mechanisms that directly mediate exocytotic fusion of the secretory (zymogen) granule (ZG) to the plasma membrane (PM).1,4 We have hypothesized that similar molecules that regulate the docking and fusion of the neuronal / 5E14$$0013

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synaptic vesicle to the plasma membrane, as proposed by the ‘‘SNARE hypothesis,’’5 are also used in acinar cells.6 This hypothesis essentially proposes that soluble N-ethylmalamide–sensitive factor attachment protein receptor (SNARE) on the donor vesicle (v-SNARE) interacts with those on the target membrane (t-SNARE) to form a multimolecular complex believed to be necessary for vesicular docking and exocytotic fusion.5,7 Vesicle-associated membrane proteins (VAMPs) are a family of well-characterized synaptic vesicle proteins.7 – 9 Three mammalian isoforms of the VAMP family have been sequenced to date. VAMP-1 and -2 are 18-kilodalton proteins initially believed to be restricted to neuronal synaptic vesicles8,9 and to function as v-SNAREs for synaptic vesicle exocytosis.5,7 Cellubrevin is a 17-kilodalton member of the VAMP family that has a nearly ubiquitous expression pattern and is believed to function as a vSNARE for constitutive membrane recycling.10 Tetanus toxin (TeTx), a potent neurotoxin that causes spastic paralysis, was recently shown to block neuroexocytosis by cleavage of VAMP-2.11 It was subsequently shown that TeTx could also cleave cellubrevin.10 We recently reported that a VAMP-2 immunoreactive protein identical in size to the brain isoform was present on the pancreatic acinar ZG membrane (ZGM).6 We also found that TeTx light chain (TeTx-LC) cleaved this ZGM-associated VAMP-2 and inhibited Ca2/-evoked amylase release in streptolysin O-permeabilized acini.6 These studies indicated a role of VAMP-2 as a v-SNARE in ZG exocytosis in the acinar cell. However, using a different VAMP antisera, a VAMP isoform was identified Abbreviations used in this paper: BoNT/B, botulinum neurotoxin type B; PM, plasma membrane; SDS-PAGE, sodium dodecyl sulfate– polyacrylamide gel electrophoresis; SNARE, soluble N-ethymalamide–sensitive factor attachment protein receptor; TeTx, tetanus toxin; TeTx-LC, TeTx light chain; t-SNARE, target SNARE; VAMP, vesicle-associated membrane protein; VAMP-2fp, VAMP-2 fusion protein; v-SNARE, vesicle SNARE; ZG, zymogen granule; ZGM, ZG membrane. q 1996 by the American Gastroenterological Association 0016-5085/96/$3.00

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in acini that was insensitive to botulinum toxin subtype B (BoNT/B).12 Because BoNT/B and TeTx cleave VAMP-2 at an identical site,11 this raised the possibility that additional toxin-insensitive VAMP isoforms are present in the acinar cells.6,12 In particular, it was proposed that VAMP-1, which in rats does not contain the TeTx and BoNT/B cleavage site, might be present in these cells.12 This possibility gained strength from the recent observation that VAMP-1 expression has been detected in other nonneural cell types.13 In this study, we attempted to clarify which isoforms of the VAMP family of proteins are present in pancreatic and parotid acinar cells. In addition, we used subcellular fractionation, Western blotting, confocal immunofluorescence microscopy, and immunogold electron microscopic techniques to determine the subcellular location of each VAMP isoform. The results indicate that VAMP2 and cellubrevin probably play integral roles in ZG exocytosis, whereas VAMP-1 probably functions in nerves that modulate exocrine function.

Materials and Methods Preparation of Dispersed Acini, ZGMs, and PMs Dispersed acini from male Sprague–Dawley rats (150– 175 g) were prepared using a serial enzymatic and mechanical dissociation technique described by us previously.14 Highly purified ZGMs and PMs were prepared from pancreata of normal or streptozotocin-induced diabetic male Sprague–Dawley rats (250–300 g) as previously described.6,14

Generation of Polyclonal Antibodies and Immunoblotting Rabbit antibodies for the VAMP-1 and -2 isoforms were generated as previously described.6 Crude antisera were affinity purified by passing immune serum over Sulfolink coupling columns (Pierce Chemicals, Rockford, IL) to which the peptides had been conjugated. The columns were washed with 20 column volumes of each of the following buffers in this order: phosphate-buffered saline (PBS), 2 mol/L NaCl in 10 mmol/L phosphate (pH 7), 0.1 mol/L sodium borate (pH 9.1), and 0.15 mol/L NaCl in phosphate (pH 4.5). Affinity-purified antibodies were then eluted in 2 column volumes of 20 mmol/ L glycine-HCl (pH 2.5) and immediately buffered to neutrality with an equal volume of 50 mmol/L Tris (pH 8.5). A third antisera previously described12 was generated against the entire VAMP-2 fusion protein (called anti–VAMP-2fp in this study). A rabbit polyclonal anticellubrevin antibody was a gift from T. Sudhof.10 Then 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of the membrane proteins was performed according to Laemmli.15 Immunoblots were performed using the following primary antisera: VAMP1, 1:300; VAMP-2, 1:700; VAMP-2fp, 1:2000; or anti-cellubrevin, 1:1000. Detection of the antigen was performed with

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goat anti-rabbit immunoglobulin (Ig) G or goat anti-mouse IgG coupled to horseradish peroxidase and visualization with a chemiluminescent horseradish peroxidase substrate (ECL; Amersham, Arlington Heights, IL) followed by exposure of the membranes to Kodak BMR film (Eastman Kodak, Rochester, NY).

Generation of Recombinant TeTx-LC Recombinant TeTx-LC containing a COOH terminal His6 tag in Qiagen Express plasmid QE3 (kindly provided by H. Niemann)16 was expressed in Escherichia coli and purified by binding to ProBond Ni-charged sepharose resin (Invitrogen, San Diego, CA) according to the manufacturer’s protocols. L chains were concentrated by ultrafiltration on Amicon Centricon-3 concentrators (W. R. Grace & Co., Beverly, MA) before use. Protein concentrations were then determined. The toxin was activated by incubation with 10 mmol/L dithiothreitol and 1 mmol/L ZnCl2 for 2 hours at 257C performed immediately before exposure to membranes. The final dithiothreitol concentrations in the membranes were °2 mmol/L. The membranes with the activated toxin were incubated for 2 hours at 377C followed by the addition of sample buffer and boiling for 3 minutes.

Confocal Immunofluorescence Microscopy The pancreas and the parotid gland were excised from a normal rat and immediately frozen in liquid nitrogen. These tissues were embedded in Tissue-Tek O.C.T. (Miles Inc., Elkhart, IN); 5-mm sections were then obtained, washed in PBS for 5 minutes, fixed in 0.5% formaldehyde, washed with 25 mmol/L NH4Cl (in PBS), and blocked with 0.1% saponin and 5% normal goat serum in PBS for 30 minutes. The tissue was then incubated in primary antibody (containing affinitypurified rabbit anti–VAMP-1, 1:50; anti–VAMP-2, 1:150; anti–VAMP-2fp, 1:50; anti-cellubrevin, 1:10; or mouse monoclonal antiamylase [Calbiochem, La Jolla, CA], 1:100; or anti-synaptophysin [Boehringer Mannheim, Indianapolis, IN], 1:100) overnight at 47C. The sections were rinsed four times with 0.1% saponin in PBS, treated with secondary antibody (diluted 1:500 for both fluorescein isothiocyanate and Texas red conjugated to goat anti-mouse and anti-rabbit IgG, respectively) for 1 hour, and rinsed four times with 0.1% saponin in PBS. Coverslips were mounted on slides in a fading retarder plus 0.1% p-phenylenediamine (ICN, Cleveland, OH) in glycerol and examined using a laser scanning confocal imaging system (Carl Zeiss, Thornwood, NY).

Immunogold Electron Microscopy Pancreata were excised from normal rats and dissected into small lobules of approximately 1 mm in diameter. Lobules were fixed by immersion in 2% paraformaldehyde and 0.25% glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.4, for 2 hours at room temperature. Samples were then dehydrated and embedded in Epon 812 (Electron Microscopy Sciences, Fort Washington, PA). Twelve-nanometer gold particles were prepared according to Slot and Geuze17 and coupled to protein A

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(Pharmacia, Uppsala, Sweden) as recommended by Horrisberger and Clerc.18 Thin sections were mounted on a 200mesh gold grid and incubated on a drop of 2% chicken albumin fraction V (Sigma Chemical Co., St. Louis, MO) for 5 minutes and incubated with the primary antisera (anti-GP2, 1:200; anti-amylase, 1:200; and anti–VAMP-2, 1:50) for 50 minutes at room temperature.19 After washing in PBS, the buffer sections were labeled with the protein A–gold complex at a dilution of 0.4 at optical density of 520 nm in the same buffer. After thoroughly washing with PBS and water, the sections were stained with uranyl acetate and lead citrate. Three controls were used in these experiments: incubation without primary antibody, incubation with antibody preabsorbed to the peptide antigen, and incubation with preimmune serum. Under all these control conditions, very few gold particles were detected and those detected were dispersed randomly throughout the tissue sections. Gold particles were counted from 20 randomly chosen micrographs.

Results VAMP-2 and Cellubrevin Are Present in Pancreatic Acinar ZGM and PM Previous studies have shown that ZGMs of rat pancreatic acinar cells express proteins immunologically related to the synaptic vesicle protein VAMP.6,12 However, the number and nature of the different VAMP isoforms expressed in these cells are unclear. To clarify this issue, we used affinity-purified antibodies specific for individual VAMP isoforms on immunoblots of subcellular fractions prepared from acini. As shown in Figure 1 (top

Figure 1. VAMP-2 and cellubrevin are present in rat pancreatic PMs and ZGMs. Pancreatic acinar fractions (10 mg of protein) were electrophoresed on a 15% SDS-PAGE and immunoblotted with an affinitypurified antibody specific to VAMP-2 (top ) and cellubrevin (bottom ) as described in Materials and Methods. These fractions include dispersed acini, ZGs, ZGMs, and pancreatic PMs obtained from normal and diabetic (DM) rats.

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Figure 2. VAMP-2 is an integral membrane protein in rat pancreatic acinar PMs and ZGMs. Ten micrograms of protein of each sample per lane was electrophoresed on a 15% SDS-PAGE and immunoblotted with an affinity-purified VAMP-2–specific antisera as described in Materials and Methods. The ZGM and PM fractions were washed with 0.25 mol/L NaBr (/) in water to remove adhering peripheral proteins from the membranes and compared with membrane fractions that were washed with distilled water (0).

panel), immunoreactive VAMP-2 protein was found not to be an abundant protein in dispersed acini or in the ZG fractions, but this Ç18-kilodalton protein was enriched in the ZGM and PM fractions. VAMP-2 appeared as a single immunoreactive band in these membrane fractions. VAMP-2 on the PM is not likely to be a contaminant because we have previously shown our PM fraction to be enriched for the PM cholecystokinin receptors.14 Nevertheless, these signals could be contributed by the VAMP-2 proteins known to be present in pancreatic islets.12 To rule out this possibility, we prepared PMs from rats that were treated with streptozotocin and had been diabetic (blood glucose level, ú18 mmol/L) for at least 1 week. We noted that the VAMP-2 signal in the pancreatic PM obtained from the diabetic rats was not diminished compared with normal rats. To show that VAMP-2 is an integral membrane protein, we washed the ZGM and PM fractions with 0.25 mol/L NaBr to remove peripherally adhering proteins (Figure 2). Level of amylase, the most abundant ZG content protein, was diminished (data not shown), but there was no change in the VAMP-2 signal in both the ZGM and PM fractions. These results indicate that VAMP-2 is an integral component of both ZGM and PM. Although cellubrevin is a ubiquitous protein believed to be involved in constitutive membrane recycling,10 we explored the possibility that cellubrevin has other roles in exocytosis by probing for its presence in the ZGM and PM. Figure 1 (bottom panel) also shows that an Ç17-kilodalton immunoreactive cellubrevin isoform similar in size to the one previously described10 is present in both PM and ZGM. A larger but weaker nonspecific band was inconsistently observed on the ZGM fraction. VAMP-1, present in the rat brain homogenate, was not present in either the ZGM or PM fractions (Figure 3; full immunoblots not shown). This indicates that the toxin-insensitive VAMP protein we previously detected with the VAMP-2fp antibody12 was not VAMP-1. WBS-Gastro

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nate, consistent with a previous report.10 Smaller proteolytic fragments resulting from TeTx cleavage of VAMP-2 and cellubrevin were not detected as reported previously.10,11 Remarkably, Figure 3 also shows that recombinant TeTx-LC at a concentration of 1 mmol/L completely cleaved rat brain VAMP-1. This result is surprising because VAMP-1 of the rat, unlike VAMP-2 and cellubrevin, lacks the TeTx cleavage site.11 Nevertheless, this result is consistent with a recent report showing that similarly high TeTx concentrations also cleaved recombinant VAMP-1.16 Location of VAMPs in Pancreatic and Parotid Acini Figure 3. Recombinant TeTX-LC cleaves VAMPs in rat pancreatic acinar cells and rat brain. Crude rat brain homogenate (2 mg protein/ lane), pancreatic acinar PM (10 mg protein/lane), and ZGM (10 mg protein/lane) were incubated with activated recombinant TeTX-LC (1 mmol/L) for 2 hours at 377C (TeTx-LC/) and compared with samples subjected to a control incubation at the same conditions but without the toxin (TeTx-LC0). These samples were divided and separated on a 15% SDS-PAGE and immunoblotted with affinity-purified VAMP-1 and VAMP-2 antisera and crude VAMP-2fp and cellubrevin antisera as described in Materials and Methods.

Recombinant TeTx-LC Cleaves Pancreatic Acinar VAMP Isoforms Previous studies have shown that clostridial neurotoxins cleave the VAMP proteins.10,11,16 We therefore examined the effects of recombinant TeTx-LC on each of the acinar VAMP proteins (Figure 3). Recombinant TeTx-LC was able to cleave the VAMP proteins identified by the VAMP-2 and cellubrevin antisera. These results indicate that the proteins contain the TeTx cleavage site.10,11 In a previous study, the ZGM-associated VAMP protein detected with the VAMP-2fp antisera was found to be insensitive to low concentrations of native BoNT/ B,12 which cleaves VAMP-2 at the same site as TeTx.11 Figure 3 shows that high concentrations of recombinant TeTx-LC completely cleaved the VAMP proteins detected by the VAMP-2fp antisera. Furthermore, both the VAMP-2 and VAMP-2fp antisera detected identical size 18-kilodalton proteins on the PM and ZGM, which were of the same molecular weight as that found in the brain. These results indicate that the toxin ‘‘insensitive’’ protein previously detected12 may be the same VAMP-2 immunoreactive protein detected by the VAMP-2–specific antibody. The cellubrevin immunoreactive proteins in both PM and ZGM were also cleaved by recombinant TeTxLC, indicating that the pancreatic acinar isoform may be the same as the previously reported ubiquitous isoform.10 Cellubrevin was not abundant in the rat brain homoge/ 5E14$$0013

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To ascertain the subcellular location of each VAMP protein, we performed confocal immunofluorescence microscopy of pancreatic (Figure 4A–E) and parotid (Figure 4F–I) tissue sections. Because VAMP-2 was enriched in both ZGM and PM (Figures 1–3), we compared its location(s) to amylase, a ZG content protein. Pancreatic sections double-labeled with rabbit anti– VAMP-2 (Figure 4B) and mouse antiamylase (Figure 4A) antisera showed that VAMP-2 colocalized with amylase located at the apical pole of pancreatic acinar cells. VAMP-2 antisera that was preabsorbed (30 minutes at 257C) with a 16–amino acid synthetic peptide (3 mg/ mL), corresponding to the variable NH2 -terminal region of VAMP-2 to which this antisera was generated against,6 resulted in a complete blockade of the VAMP-2 signal in pancreatic acini (Figure 4C). Furthermore, no immunofluorescence signals were detected when the acini were incubated with the primary or appropriate secondary antisera used alone or when the VAMP-2 was used along with an inappropriate secondary antisera (anti-mouse) (data not shown). Pancreatic sections double-labeled with rabbit anti– VAMP-2fp (Figure 4E) and mouse antiamylase antisera (Figure 4D) also showed colocalization of these proteins. Of note, anti–VAMP-2fp (Figure 4E) but not antiamylase (Figure 4D) labeled structures that corresponded to the luminal or apical PM of the acini (Figure 4E, arrowheads). These results were consistent with those previously reported with this antiserum.12 It should also be noted that anti–VAMP-2fp, unlike anti–VAMP-2, was generated against the full-length fusion protein of VAMP-2.12 We have found parotid acinar cells to generally contain more ZGs (Figure 4I) than pancreatic acinar cells (Figure 4A and D). VAMP-2 (labeled with anti–VAMP-2fp; Figure 4F) appeared to be also colocalized with amylase (Figure 4I) on the ZG located at the apical pole of parotid WBS-Gastro

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Figure 4. Localization of VAMP-1, VAMP-2, amylase, and synaptophysin in rat pancreatic and parotid acini by confocal immunofluorescence microscopy. (A–E ) Rat pancreatic tissue sections were double labeled with rabbit (B ) anti–VAMP-2 or (E ) anti–VAMP-2fp antisera against (A and D ) mouse antiamylase antisera. Arrowheads in D and E mark the position of apical PMs of pancreatic acini. (C ) The anti–VAMP-2 antibody was preabsorbed with an amino-terminal peptide of VAMP-2 (see Results for details), which blocked the VAMP-2 immunofluorescence signal. (F–I ) Rat parotid tissue sections were probed with (F ) rabbit anti–VAMP-2fp, (I ) mouse antiamylase antisera, (G ) rabbit anti–VAMP-1, and (H ) mouse antisynaptophysin antisera, respectively. Double labeling shows that (G ) VAMP-1 immunoreactivity overlaps with (H ) synaptophysin present in the nerve fibers (arrows ) situated along the basal surface of the acinar PM. (G and H ) Note the varicosities typically observed along the nerve fiber (bar in C Å 25 mm).

acinar cells. We also observed a VAMP-2 signal on the apical PM of the parotid acinar cell (Figure 4E), similar to that observed in pancreatic acini (Figure 4E). Labeling of the apical PM of either parotid or pancreatic acinar cells was inconsistently observed with the anti–VAMP/ 5E14$$0013

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2 antisera (not shown). These studies, showing VAMP2 on the ZG and the PM, are consistent with our immunoblotting studies of purified ZGMs and PMs, as shown in Figures 1–3. Figure 4G shows that VAMP-1, although not present WBS-Gastro

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in the parotid acinar cell, is present in structures that seem to straddle the extracellular portion of the basal PM region of parotid acini. These structures contain varicosities normally observed with nerves. Indeed, double labeling with the antisynaptophysin antiserum showed an identical distribution (Figure 4H). Synaptophysin is one of the most abundant proteins on the neuronal synaptic vesicle membrane,7 and we have previously shown it to be absent from our ZGM preparations.6 Similar nerve structures stained by both anti–VAMP-1 and antisynaptophysin antisera were also found in pancreatic tissues (data not shown). Antibodies to cellubrevin were unable to show subcellular localization despite several attempts (data not shown). VAMP-2 Is Located on the Cytoplasmic Surfaces of the ZGM All ZGM proteins characterized to date, including GP2 (Figure 5B), are found on the luminal side of the ZG and have no direct role in regulating exocytosis.1 For VAMP-2 to act as a v-SNARE of the ZG and effect exocytosis, it should be located on the cytoplasmic surface of the ZG to interact with as yet undefined t-SNAREs on the cytoplasmic surface of the PM. Furthermore, if VAMP-2 is translocated to the PM after exocytosis, it may accumulate on the cytoplasmic surface of the apical PM. We therefore performed immunogold electron microscopy with our VAMP-2 antibody on pancreatic tissues (Figure 5A). As shown in Figure 5A (arrowheads) and Table 1, we found a uniform distribution of approximately two VAMP-2 gold grains per section of ZG (2.1 { 0.3 vs. 0.5 { 0.1 per ZG for preimmune control serum). The specific VAMP-2 labeling of the ZGM was highly significant with 86 { 12 gold grains/100 mm ZGM compared with control preimmune serum of only 3.4 { 1.5 gold grains/100 mm ZGM. Furthermore, these gold grains seem to be on the cytoplasmic surface of the ZG. Variable numbers of gold grains were found over the cytoplasmic surface of the apical PM (small arrows) and dispersed over the over Golgi area (big arrows). However, the labeling of the apical PM and Golgi by the VAMP-2 antibody was too variable to provide statistical significance. These results were consistent with the immunofluorescence studies (Figure 4B). We have been unsuccessful in performing the electron microscopy studies with the VAMP-2fp antibody.12 Negative control experiments including preimmune serum (Figure 5C and Table 1) or preadsorption with peptide antigen (data not shown) confirmed the significance of this low level of immunogold staining (see Materials and Methods). No gold grains were observed along the basal PM domains of the acinar cell (data not shown). For comparison, sections / 5E14$$0013

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labeled with anti-GP2 and antiamylase antibodies are shown in Figure 5B and D, respectively. GP2 was found on the inner side of the ZGM, the luminal side of the apical PM, and the acinar lumen. Amylase, a secretory protein, was found abundantly inside the ZG and acinar lumen.

Discussion The SNARE hypothesis postulates that corresponding v- and t-SNAREs should only be present in the donor vesicle and target membrane, respectively, to ensure the fidelity of vectorial vesicular transport.5 Consistent with this hypothesis, we have extended our previous observation to show that VAMP-2 is a component of the ZGM.6 We also previously reported that VAMP2 is not an abundant protein of the ZGM, and ZGM purification was required to detect a signal by Western blotting. Our immunogold electron microscopy results confirm this relative rarity because we typically detected only approximately two VAMP-2 gold particles per section of the ZG. In this study, we also observed a VAMP-2 signal in pancreatic PM at approximately the same levels as those found on the ZGM. One explanation for this result is that ZG exocytotic fusion with the PM may result in incorporation of ZGM to the PM and therefore ‘‘translocation’’ of the ZGM-associated VAMP-2 to the PM. The presence of VAMP-2 on the cytoplasmic aspect of the apical PM shown in this study (Figures 4E and 5A) coupled to previous reports showing an expansion of the apical PM during the early stages of stimulated exocytosis4 support this possibility. This would occur if the kinetics of endocytosis is slower than exocytosis in the acinar cells, and, therefore, VAMP-2 in the PM may still be in the process of endocytic recycling. The inconsistent finding of VAMP-2 in the apical PM and Golgi (Figure 5A) supports this possibility of active VAMP-2 recycling. More studies are necessary to map out the recycling pathways of SNAREs in the acinar cell. Our results support the previous contention that the VAMP molecules identified by the VAMP-2–specific and VAMP-2fp antibodies12 appear to be VAMP-2. The proteins identified by both antisera were similarly sensitive to TeTx cleavage. Furthermore, we subsequently found that the VAMP-2fp antibody detected only recombinant VAMP-2 and had little, if any, cross-reactivity to VAMP-1. We have also found that more prolonged incubation of the ZGM with higher concentrations of BoNT/B (20 nmol/L) result in partial cleavage of the VAMP molecule (data not shown). The relative resistance of acinar VAMP-2 to cleavage by these toxins could be explained by the fact that the VAMP-2 in the acini may WBS-Gastro

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Figure 5. Immunogold electron microscopy localization of VAMP-2 in the rat pancreatic acinar cell. (A ) VAMP-2. This protein is found on the cytoplasmic sides of the ZGM (arrowheads ) and apical PM (small arrows ) as well as on the Golgi area (big arrows ) (original magnification 53,0001). (B ) GP2. This protein is found on the inner side of the ZGM (arrowheads ), the luminal side of the apical PM (arrows ), and the acinar lumen (big arrows ) (original magnification 53,0001). (C ) Preimmune serum was used as a control; nonspecific labeling is shown by the arrowhead (original magnification 41,0001). (D ) Amylase. Intense labeling of the ZG content (arrowhead ) and acinar lumen (arrow ) can be seen (original magnification 41,0001; bar Å 0.5 mm).

be complexed to as yet undefined SNARE proteins that render the cleavage site inaccessible.20 Furthermore, the cleavage sensitivity of the different isoforms by the different toxins is dependent not only on the presence of specific cleavage sites but also on the NH2 -terminal sequences distal to the cleavage sites.16 In the original studies, VAMP-1 in rats was found to be much less / 5E14$$0013

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sensitive to proteolysis by low concentrations of native TeTx; this was considered to be caused by the Gln to Val substitution at amino acid 76.11 Nevertheless, our results support the recent observation16 that under the conditions used, rat brain VAMP-1 can be efficiently cleaved by recombinant TeTx-LC. However, immunolocalization studies using the anti– WBS-Gastro

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Table 1. Quantitative Determination of VAMP-2 Immunoactivity No. of gold grains

Total/ZG ZGM/100 mm ZG matrix/mm2

VAMP-2

Preimmune serum

2.1 { 0.3 86 { 12 0.5 { 0.1

0.5 { 0.1 3.4 { 1.5 0.7 { 0.3

NOTE. The intensity of the labeling obtained with the anti–VAMP-2 antibody was quantitatively evaluated over the apical area and more specifically the ZG. Labeling of the ZG was estimated from 20 random micrographs. Taking into consideration the sizes of gold grains and IgG, gold grains located at a distance of 160 nm from the center of the membrane were considered to be associated with the latter. Results are expressed as per unit length of membrane and per square meter. Control experiments were performed with preimmune serum at the same dilution.

VAMP-2 and anti–VAMP-2fp antisera were not identical. Both antisera detected denatured VAMP-2 proteins in pancreatic PM and ZGM in the immunoblotting studies (Figures 1–3), but only the anti–VAMP-2fp antisera consistently labeled the apical PM by confocal immunofluorescence microscopy (Figure 4E). One possibility is that the fixation procedure used in the immunofluorescence studies may selectively alter the PM VAMP-2 antigen. In support of this possibility is our observation with electron microscopy, which has distinct fixation conditions, which showed that anti–VAMP-2 antiserum inconsistently recognized the apical PM. It is possible that the PM VAMP-2 epitope is inaccessible or sequestered by protein-protein interactions and that this epitope is occasionally unmasked in immuno electron-microscopic preparations. In contrast, anti–VAMP-2fp was generated against the entire VAMP-2 protein12 and would be able to recognize other epitopes of VAMP-2 that were not recognized by the anti–VAMP-2 antisera; this antisera therefore consistently detected the PM VAMP-2 signal in the immunofluorescence study. Unfortunately, the anti– VAMP-2fp antibody was sensitive to the aldehyde fixatives used for the electron-microscopic study.12 Cellubrevin was initially proposed to play a role in targeting vesicles that carry recycling transferrin receptors to the cell surface.10,21 Its presence in the acinar PM suggests that this process may involve translocation of cellubrevin from these vesicles to the PM. However, its presence on the ZG, a secretory granule of the classic regulated exocytotic pathway, is surprising. This may in part be explained by the fact that acinar cells are believed to have complex constitutive exocytotic pathways that coexist with the regulated pathway.4 In the classic constitutive exocytotic pathway, vesicles containing secretory proteins arise directly from the trans-Golgi network.4 / 5E14$$0013

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However, an alternative paragranular pathway has also been postulated to exist in the acinar cell in which small vesicles containing secretory proteins arise from maturing ZGs and undergo constitutive exocytosis at the apical PM.4,22 It is possible that cellubrevin in the acinar cells mediates constitutive exocytosis by either classical or paragranular pathways. Both pancreatic and parotid acini are richly innervated by nerve fibers.3 These neurons release cholinergic as well as peptidergic neurotransmitters,3 but the exocytotic machinery regulating this release is virtually unknown. Our observation that VAMP-1 is present in the nerve terminals of the peripheral nervous system indicates that it may function to regulate exocrine function of the pancreas and the parotid gland. These nerves are well positioned to release neurotransmitters along the axonal varicosities that abut the surface of the basolateral PM to bind and activate PM receptors of the acinar cell. The above results do not rule out the possibility of other toxin-insensitive VAMP isoforms that would not be detected by our antibodies. The existence of such molecules could explain our previous results in which TeTx-LC caused only partial inhibition of amylase release (Ç30%) in permeabilized acini.6 Nevertheless, the current findings together with our previous studies6,12 indicate important roles for all three known members of the VAMP family of v-SNARE proteins in the complex exocytotic pathways of pancreatic and parotid acinar cells. Insights gained from these studies may serve as the foundation for studies aimed at probing for additional molecules that may interact with these v-SNAREs to regulate exocytosis.

References 1. Wagner ACC, Williams JA. Pancreatic zymogen granule membrane proteins: molecular details begin to emerge. Digestion 1994;55: 191–199. 2. Williams JA, Yule DI. Stimulus-secretion coupling in pancreatic acinar cells. In: Go VLW, DiMagno E, Gardner JD, Lebenthal E, Reber HA, Scheele GA, eds. The Pancreas. 2nd ed. New York: Raven, 1991:167–189. 3. Holst JJ. Neural regulation of pancreatic exocrine function. In: Go VLW, DiMagno E, Gardner JD, Lebenthal E, Reber HA, Scheele GA, eds. The pancreas. Ist ed. New York: Raven, 1986:287– 300. 4. Beaudoin AR, Grondin G. Secretory pathways in animal cells: with emphasis on pancreatic acinar cells. J Electr Microsc Tech 1991; 17:51–69. 5. Rothman JE, Warren G. Implications of the SNARE hypothesis for intracellular membrane topology and dynamics. Curr Biol 1994;4:220–233. 6. Gaisano HY, Sheu L, Foskett JK, Trimble WS. Tetanus toxin light chain cleaves a vesicle-associated membrane protein (VAMP) isoform 2 in rat pancreatic zymogen granules and inhibits enzyme secretion. J Biol Chem 1994;269:17062–17066. 7. Bennett MK, Scheller RH. A molecular description of synaptic

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vesicle membrane trafficking. Annu Rev Biochem 1994;63:63– 100. Elferink LA, Trimble WS, Scheller RH. Two vesicle-associated membrane protein genes are differentially expressed in the rat central nervous system. J Biol Chem 1989;264:11061–1064. Trimble WS, Gray TS, Elferink LA, Wilson MD, Scheller RH. Distinct patterns of expression of two VAMP genes within the rat brain. J Neurosci 1990;10:1380–1387. McMahon MT, Ushkaryov YA, Edelmann L, Link E, Binz T, Niemann H, Jahn R, Sudhof TC. Cellubrevin is a ubiquitous tetanus toxin substrate homologous to a putative synaptic vesicle fusion protein. Nature 1993;364:346–349. Schiavo G, Benfenati, F, Poulain B, Rosetto O, Polverino de Laureto P, DasGupta BR, Montecucco C. Tetanus toxin and botulinum-B neurotoxins block neurotransmitter release by proteolytic cleavage of snyaptobrevin. Nature 1992;359:832–835. Braun JEA, Fritz BA, Wong SME, Lowe AW. Identification of a vesicle-associated membrane protein(VAMP)-like membrane protein in zymogen granules of the rat exocrine pancreas. J Biol Chem 1994;269:5326–5335. Ralston E, Beushausen S, Ploug T. Expression of the synaptic vesicle proteins VAMPs/synaptobrevins 1 and 2 in non-neuronal tissues. J Biol Chem 1994;269:15403–15406. Gaisano HY, Klueppelberg UG, Pinon DI, Pfenning MA, Powers SP, Miller LJ. Novel tool for the study of cholecystokinin-stimulated pancreatic enzyme secretion. J Clin Invest 1989;83:321– 325. Laemmili UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680–685. Yamazaki S, Braumeister G, Binz T, Blasi J, Link E, Cornille F, Roques B, Fykse EM, Sudhof TC, Jahn R, Niemann H. Cleavage of members of the synaptobrevin/VAMP family by types D and F botulinal neurotoxins and tetanus toxin. J Biol Chem 1994;269: 12764–12772.

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17. Slot JW, Geuze HJ. A new method of preparing gold probes for multiple labelling cytochemistry. Eur J Cell Biol 1985;38:87–93. 18. Horrisberger M, Clerc MF. Labelling of colloidal gold with protein A: a quantitative study. Histochemistry 1985;82:219–225. 19. Beaudoin AR, Grondin G, Laperche Y. Imunocytochemical localization of L-glutamyl transpeptidase, GP2 and amylase in the rat exocrine pancreas. The concept of zymogen granule membrane recycling after exocytosis. J Histochem Cytochem 1993;41:225– 233. 20. Hayashi T, McMahon H, Yamasaki S, Binz T, Hata Y, Sudhof TC, Niemann H. Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly. EMBO J 1994;13:5051– 5061. 21. Galli T, Chilcote T, Mundigl O, Binz T, Niemann H, De Camilli P. Tetanus toxin–mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor containing vesicles in CHO cells. J Cell Biol 1994;125:1015–1024. 22. Arvan P, Castle JD. Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas. J Cell Biol 1987;104:243–252.

Received August 18, 1995. Accepted July 25, 1996. Address requests for reprints to: Herbert Y. Gaisano, M.D., Room 7226, Medical Sciences Building, University of Toronto, Toronto, Ontario, Canada M5S 1A8. Fax: (416) 978-8765. Supported by grants from the Medical Research Council of Canada (to W.S.T. and H.Y.G.), the Natural Sciences and Engineering Research Council (to W.S.T.), and the Fraser Elliott Foundation (to H.Y.G.); by American Gastroenterology Association/Industry (Pharmacia and Upjohn) Research Scholar Award (to H.Y.G.); and by funds from the Alzheimer’s Association of Ontario (to W.S.T.). The authors thank Allen Volchuk and Amira Klip for helpful discussions and technical assistance.

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