Vesicle-associated membrane protein 2 is essential for cAMP-regulated exocytosis in rat parotid acinar cells. The inhibition of cAMP-dependent amylase release by botulinum neurotoxin B.

Amylase exocytosis of the parotid gland is mediated by intracellular cAMP. To investigate whether cAMP-dependent secretion has a mechanism similar to that of regulated neuroexocytosis, we examined the expression of synaptosome-associated proteins. In rat parotid acinar cells, we found 25 (p25) and 18 kDa (p18) proteins reacted with antibodies against Rab3A and vesicle-associated membrane protein 2 (VAMP-2), respectively. On the other hand, syntaxin 1 and SNAP-25, which interact with VAMP-2 at synapses, were undetectable. Rab3A-like p25 and VAMP-2-like p18 were also expressed in other exocrine acinar cells. The latter was localized at secretory granule membranes, and the former was detected in secretory granule and cytosolic fractions. The antibody against VAMP-2 used in this study did not react with cellubrevin, and p18 was cleaved with botulinum neurotoxin B. Thus, we identified p18 as VAMP-2. Botulinum neurotoxin B inhibited the cAMP-induced amylase release from streptolysin O-permeabilized acinar cells. Therefore, VAMP-2 is required for cAMP-regulated amylase release in rat parotid acinar cells. This is the first report that VAMP-2 is involved in regulated exocytosis that is independent of Ca2+.

tors induces the activation of adenylate cyclase, followed by intracellular cAMP accumulation (5). Intracellular signal transduction is mediated by cAMP to cause exocytosis of amylase without an elevation of cytosolic Ca 2ϩ (6,7). In contrast, cholinergic or ␣-adrenergic receptor stimulation increases the cytosolic free Ca 2ϩ level (8) and elicits the production of the primary secretory fluid (water and ion transport). The increase in cytosolic Ca 2ϩ also induces amylase secretion, but the amount of released amylase is less than that induced by cAMP (4). The cAMP-dependent protein kinase is activated by cAMP. However, proteins involved in cAMP-regulated amylase release have not been identified.
The molecular mechanisms involved in neurotransmitter exocytosis are becoming clearer, and various proteins that function in the membrane fusion have been identified (9). These proteins form complexes and play important roles in the fusion of the synaptic vesicles with the plasma membranes (10 -14). Several of their components are highly conserved throughout various steps of the secretory pathway in eukaryotes, from yeast to mammals (15,16). Vesicle-associated membrane proteins (VAMPs) 1 are expressed in various tissues, and they are considered to be important for exocytosis (17)(18)(19)(20)(21). However, these proteins can be regulated and function in a different manner in each system. We considered that these proteins also function in cAMP-dependent secretion.
Subcellular Fractionation and Preparation of Secretory Granule Membranes-From dispersed acinar cells, we prepared fractions of the 1,000 ϫ g, 10,000 ϫ g, and 100,000 ϫ g pellets and the 100,000 ϫ g supernatant, and we refer to them here as nuclear-, mitochondrial-, and microsomal-rich and cytosol fractions.
Secretory granules of the rat parotid glands were isolated by Percolldensity gradient centrifugation as described by Gasser and Hopfer (34) with some modifications. The parotid glands were removed, finely minced in ice-cold homogenizing buffer, composed of 250 mM sucrose, 20 mM HEPES (pH 7.2), 1 mM EGTA, 0.1 mM MgCl 2 , and 1 mM phenylmethylsulfonyl fluoride, and dispersed by eight strokes in a glass-glass homogenizer. The homogenate was centrifuged at 150 ϫ g for 5 min, and the supernatant was centrifuged at 1,500 ϫ g for 10 min. The pellet was suspended in the homogenizing buffer containing 40% Percoll and centrifuged at 20,000 ϫ g for 20 min. Secretory granules were collected from the bottom of the tube, diluted with the homogenizing buffer, and centrifuged at 1,500 ϫ g for 10 min. The pellet was resuspended in 20 mM HEPES buffer (pH 7.2) containing 1 mM EGTA, 0.1 mM MgCl 2 , and 1 mM phenylmethylsulfonyl fluoride and homogenized in a Teflon-glass homogenizer. The homogenate was centrifuged at 100,000 ϫ g for 60 min. The pellet was used as a granule membrane fraction.
Cleavage of p18 with Activated Botulinum Neurotoxin B-Activated and inactivated botulinum neurotoxin B (BNT-B) were prepared by an incubation at 37°C for 30 min with 10 M dithiothreitol in the absence or presence of 10 M EDTA, respectively. The 100,000 ϫ g pellet from rat parotid acinar cells was incubated with activated or inactivated BNT-B at 37°C for 60 min. Thereafter, the samples were boiled with buffer containing SDS and used for Western blotting analysis using anti-VAMP-2 antibody. Amylase Release from Streptolysin O-permeabilized Acinar Cells-The incubation medium was composed of 100 mM KCl, 19.2 mM NaCl, 25 mM HEPES-NaOH (pH 7.2), 1 mM MgSO 4 , 0.96 mM NaH 2 PO 4 , 10 M propranolol, 10 M atropine, 0.1% bovine serum albumin, and 0.1 mg/ml trypsin inhibitor (intracellular buffer). The desired free Ca 2ϩ concentrations of this intracellular buffer were obtained by adjusting the EGTA:Ca 2ϩ ratio as described previously (35). A lyophilized powder of streptolysin O (SLO) was dissolved in water and activated by a 15-min incubation on ice with 10 M dithiothreitol. One hemolytic unit of SLO causes 50% lysis of 1 ml of a 2% rabbit red blood cell suspension in 40 min at 37°C.
Parotid acinar cells were prepared by an incubation with collagenase (0.35 units/ml) and hyaluronidase (75 g/ml) in Hanks' balanced salt solution buffered with 20 mM HEPES-NaOH (pH 7.4), containing 0.5% bovine serum albumin, at 37°C in 100% O 2 . Thereafter, dispersed acinar cells were filtered through nylon mesh, and washed twice with intracellular buffer. An acinar cell suspension (600 l) was pipetted into a tube containing 12 l of 2500 units/ml SLO, with or without 15 l of activated neurotoxins, and rotated for up to 30 min at 25°C at 280 rpm. Thereafter, 100 l of the acinar cell suspension were reacted with cAMP. After the reaction, 900 l of intracellular buffer were added, and the medium was collected by passage through filter paper. To measure total amylase activity, the acinar cells were sonicated. Amylase activity was measured according to Bernfeld (36), and the released amylase activity is described as the relative value to that of the basal release in the absence of cAMP and neurotoxins.

Rat Parotid Acinar Cells Contain Vesicle-associated Mem-
brane Protein-2-like p18 and Rab3A-like p25-The expression of the synaptosome-associated proteins in rat parotid acinar cells was compared to that in the catecholamine-secreting cell line, PC12. All proteins tested in this study were detected in PC12 cells: synaptotagmin I (65 kDa), synaptophysin (38 kDa), syntaxin1 (35 kDa), SNAP-25 (25 kDa), Rab3A (25 kDa), and VAMP-2 (18 kDa). Rab3A was detected in both the supernatant and pellet fractions, at almost a 1:1 ratio (Fig. 1E), and other proteins were observed in the pellet fraction (Fig. 1, A-D  VAMP-2 Is Essential for cAMP-dependent Exocytosis F). The anti-syntaxin 1 antibody recognized two bands at a molecular mass of about 35 kDa that may correspond to syntaxins 1A and 1B (Fig. 1C). In rat parotid acinar cells, anti-Rab3A and anti-VAMP-2 antibodies detected a protein band in the supernatant (Fig. 1E) and pellet fractions (Fig. 1F), respectively. The electrophoretic mobility of the protein bands detected with antibodies against Rab3A and VAMP-2 in rat parotid acinar cells was the same as those in PC12 cells (25 and 18 kDa, respectively). Synaptotagmin I, synaptophysin, syntaxin 1, and SNAP-25 were not detected in the rat parotid acinar cells under these conditions (Fig. 1A-D).
Rab3A-like p25 and VAMP-2-like p18 Are Expressed Commonly in Exocrine Glands-The 100,000 ϫ g supernatant and pellet fractions of exocrine gland acinar cells were prepared from various exocrine glands, including the parotid, submandibular lacrimal gland, and pancreas. Immunoblot analysis was performed using antibodies against Rab3A and VAMP-2. A Rab3A-like p25 was expressed in the submandibular and lacrimal acinar cells, but not in the pancreas (Fig. 2, top panel). A 25-kDa protein has been detected in the pancreas using the antibody against Rab3 (37). Because the antibody used in this study does not react with other Rab3 homologues, the Rab3 in the pancreas may be another isoform(s) such as Rab3B, C, or D.
The antibody against VAMP-2 reacted with an 18-kDa protein expressed by all the exocrine glands examined (Fig. 2,  bottom panel). These results suggest that Rab3 and VAMP-2 have common roles in various exocrine glands. On the other hand, syntaxin 1, synaptotagmin I, synaptophysin, and SNAP-25 were not detected in the exocrine gland acinar cells under the same conditions (data not shown).
The Protein p18 Is Located at the Secretory Granule Membrane, and p25 Is in the Cytosol and Granule Membrane Fractions-To investigate the localization of Rab3A-like p25 and VAMP-2-like p18, we prepared nuclear-, mitochondrial-, microsomal-, and secretory granule membrane-rich and cytosol fractions of rat parotid acinar cells as described under "Experimental Procedures." Immunoblotting revealed that Rab3A was mainly localized in the cytosol fraction, and very little was detected in the granule membrane fraction (Fig. 3, top panel). About 99% of the Rab3A was detected in the cytosol, and less than 1% was localized at granule membranes. Rab3A was not detected in the other membrane fractions. In contrast, VAMP-2 is specifically localized at the granule membranes in rat parotid acinar cells (Fig. 3, bottom panel) Botulinum Neurotoxin B Cleaved the p18 -Neurotoxins are zinc endopeptidases that cleave synaptosome-associated proteins such as syntaxin1 or SNAP-25. One of them, BNT-B, specifically cleaves VAMP-2. To ascertain whether or not p18 is VAMP-2, we cleaved the protein with BNT-B. Activated BNT-B was mixed with the 100,000 ϫ g pellet fraction of rat parotid acinar cells, and cleavage of the p18 in the pellet fraction was examined. The p18 band disappeared, and a novel 9-kDa band appeared (Fig. 4, lane 2). Thus, BNT-B cleaved p18. The 9-kDa proteins band may be the N-terminal fragment of the p18. This proteolytic activity was diminished in the presence of 10 mM EDTA because of the chelation of zinc ions (Fig. 4, lane 3). BNT-B also cleaves the VAMP homologue, cellubrevin (38). However, the antibody used in this experiment does not react with cellubrevin. Therefore, p18 was identified as VAMP-2.
Amylase Release from SLO-permeabilized Cells Was Inhibited by BNT-B-The BNT-B-induced cleavage of synaptic vesicle VAMP-2 is considered to be the molecular basis of the BNT-B-induced neuroexocytotic blockade. Thus, we examined the effects of BNT-B on amylase secretion from SLOpermeabilized parotid acinar cells. In rat parotid acinar cells, cAMP stimulated amylase secretion (Fig. 5, lane 2). A prior incubation with BNT-B did not affect the amount of basal amylase secretion in the absence of cAMP (Fig. 5, compare  lanes 1 and 3), but reduced the amount of amylase released by stimulation with cAMP down to 48% of the level in the absence of BNT-B (Fig. 5, lanes 2 and 4). Under these conditions, BNT-B cleaved only about 50% of VAMP-2 in the permeabilized cells (data not shown), corresponding to an inhibition efficiency of 48%. These results showed that VAMP-2 is involved in the cAMP-dependent pathway. In contrast, Ca 2ϩstimulated amylase secretion was not affected by BNT-B (data not shown).
Other neurotoxins, BNT-A and BNT-C1, reportedly cleave SNAP-25 and syntaxin 1, respectively (39). In rat parotid acinar cells, VAMP-2 was detected, but SNAP-25 and syntaxin were not. We then investigated the effects of these neurotoxins on the cAMP-dependent amylase secretion. Although BNT-B inhibited the cAMP-dependent secretion, neither BNT-A nor BNT-C1 affected the basal or signal-dependent secretion (Fig.  5B, lanes 5-8). This result supports the notion that SNAP-25 and syntaxin 1 were not detected in the rat parotid acinar cells. DISCUSSION This is the first report that VAMP-2 is involved in the regulated exocytosis that is independent of Ca 2ϩ elevation. In other systems for example, that of neuronal cells and chromaffin, VAMP-2 is considered to play an important role in Ca 2ϩ - evoked exocytosis. Tetanus toxin and BNT-B inhibit Ca 2ϩdependent neurotransmitter release from synaptic vesicles (17,18), which is prevented by antibody against VAMP-2 (19). In pancreatic ␤-cells, BNT-B inhibits Ca 2ϩ -but not GTP␥S-induced insulin secretion (21). The amylase release from pancreatic exocrine cells is also Ca 2ϩ -dependent, and the secretion is inhibited by tetanus toxin, which also cleaves VAMP-2 (13). In rat parotid acinar cells, however, BNT-B inhibited cAMP-stimulated amylase release. Thus, VAMP-2 is essential for cAMPdependent exocytosis in the parotid gland.
Among synaptosome-associated proteins, we also found Rab3A in rat parotid acinar cells. Rab3A was mainly detected in the cytosol fraction. The amount of the protein localized at secretory granule membranes was less than 1%. In neuronal cells, Rab3A is mainly located at synaptic vesicles (40,41), and in this study, about half of the total Rab3A was found in the pellet fraction of PC12 cells. Rab3A-like GTP-binding protein is also located on the cytosolic face of zymogen granules in pancreatic exocrine cells (37). It is possible that cytosol-located Rab3A translocates to granule membranes in a signal-dependent manner. In a preliminary experiment, however, we did not find such large scale translocation in rat parotid acinar cells (data not shown). We have not yet determined the involvement of Rab3A in amylase secretion in the parotid gland.
In neuronal cells, the hypothesis is proposed that synaptic vesicles dock to a target membrane through the interaction of vesicular and target membrane proteins referred to as SNAP receptors (SNAREs) (11,12). VAMP-2 is a vesicle-docked SNARE (v-SNARE), and syntaxin1 and SNAP-25 are target membrane SNAREs (t-SNAREs). In rat parotid acinar cells, t-SNAREs were not detected, although the v-SNARE was de-tected at the secretory granules. Moreover, the proteases that cleave the t-SNAREs did not inhibit the amylase secretion from rat parotid acinar cells. It is suggested that VAMP-2 interacts with proteins other than syntaxin 1 and SNAP-25 in parotid acinar cells. These unidentified t-SNAREs may be subject to cAMP-dependent protein kinase in rat parotid acinar cells. Identification of a putative target protein of VAMP-2 in the parotid gland will be useful for investigating cAMP-dependent membrane traffic in other systems.
The next issue is to determine the target of VAMP-2 in cAMP-dependent exocytosis. The putative target can be required only for the cAMP-dependent pathway or it is common to Ca 2ϩ -dependent and cAMP-dependent systems. Either notion is possible. A VAMP-binding protein, VAP-33, has been identified in the Aplysia central nervous system, and an antibody against it inhibited synaptic transmission (42). VAP-33 locates at plasma membranes, and may play an essential role in membrane fusion by interacting with VAMP-2. Its amino acid sequence has no homology with those of syntaxin and SNAP-25 (42). Therefore, a protein with a function homologous to that of VAP-33 may also function independently of syntaxin and SNAP-25 in the mammalian parotid gland.