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Exocytosis is thought to result from the fusion of vesicle and plasma membranes caused by the formation of a trans-complex between proteins of the vesicle-associated membrane protein (VAMP) family on the vesicle with members of the syntaxin and synaptosomal-associated protein of 25 kd (SNAP-25) families on the plasma membrane. In the pancreatic acinar cell, synaptosomal-associated protein of 23 kd (SNAP-23) is the major SNAP-25 isoform expressed in pancreatic acinar cells, but its role in acinar cell exocytosis has not been determined.To examine the role of SNAP-23 in regulated exocytosis in acinar cells, we subcloned into adenoviral vectors SNAP-23, SNAP-25, and dominant negative mutants in which the C-terminal domains corresponding to the botulinum neurotoxin A cleavage sites are deleted.High-efficiency infection of rat pancreatic acini in culture with these adenoviruses by subcellular fractionation showed that the overexpressed SNAP-23, SNAP-25, and their truncated mutant proteins were uniformly targeted to the zymogen granules and plasma membrane. To maximally stimulate apical exocytosis from these infected acini, we used the cholecystokinin–phenylethyl ester analog (CCK-OPE), which does not show inhibition of secretion from maximal levels at high doses. CCK-OPE–stimulated amylase release from adenovirus–cytomegalovirus (AdCMV)–SNAP-23 or AdCMV–SNAP-25–infected acini to the same extent as from acini infected with the empty vector. In contrast, CCK-OPE–evoked enzyme secretion from AdCMV–SNAP-23ΔC8– and AdCMV–SNAP-25 1−197 –infected acini were inhibited by 60% and 40%, respectively. The identical targeting of the mutant SNAP-23 and SNAP-25 proteins to the same membrane compartments as SNAP-23 suggests that the inhibition of secretion was a result of their competition against endogenous SNAP-23. This is supported by the fact that this inhibition by the mutant proteins was partially reversed or rescued when the AdCMV–SNAP-25ΔC8– or AdCMV–SNAP-25 1−197 –infected acini were co-infected with wild-type SNAP-23 or SNAP-25.From these results, we conclude that SNAP-23 plays a role in CCK-evoked regulated exocytosis in the acinar cells.