title: Expression, localization, and functional role for synaptotagmins in pancreatic acinar cells.
abstract: Secretagogue-induced changes in intracellular Ca(2+) play a pivotal role in secretion in pancreatic acini yet the molecules that respond to Ca(2+) are uncertain. Zymogen granule (ZG) exocytosis is regulated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. In nerve and endocrine cells, Ca(2+)-stimulated exocytosis is regulated by the SNARE-associated family of proteins termed synaptotagmins. This study examined a potential role for synaptotagmins in acinar secretion. RT-PCR revealed that synaptotagmin isoforms 1, 3, 6, and 7 are present in isolated acini. Immunoblotting and immunofluorescence using three different antibodies demonstrated synaptotagmin 1 immunoreactivity in apical cytoplasm and ZG fractions of acini, where it colocalized with vesicle-associated membrane protein 2. Synaptotagmin 3 immunoreactivity was detected in membrane fractions and colocalized with an endolysosomal marker. A potential functional role for synaptotagmin 1 in secretion was indicated by results that introduction of synaptotagmin 1 C2AB domain into permeabilized acini inhibited Ca(2+)-dependent exocytosis by 35%. In contrast, constructs of synaptotagmin 3 had no effect. Confirmation of these findings was achieved by incubating intact acini with an antibody specific to the intraluminal domain of synaptotagmin 1, which is externalized following exocytosis. Externalized synaptotagmin 1 was detected exclusively along the apical membrane. Treatment with CCK-8 (100 pM, 5 min) enhanced immunoreactivity by fourfold, demonstrating that synaptotagmin is inserted into the apical membrane during ZG fusion. Collectively, these data indicate that acini express synaptotagmin 1 and support that it plays a functional role in secretion whereas synaptotagmin 3 has an alternative role in endolysosomal membrane trafficking.
authors.affiliation: Department of Nutritional Sciences, University of Wisconsin-Madison, USA.
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Falkowski
authors.forename: Diana D H
authors.initials: DD
authors.lastname: Thomas
authors.forename: Scott W
authors.initials: SW
authors.lastname: Messenger
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Martin
authors.forename: Guy E
authors.initials: GE
authors.lastname: Groblewski
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D008297:Male; D046790:Pancreas, Exocrine / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050600:SNARE Proteins / Q000502:physiology; D022142:Secretory Vesicles / Q000737:chemistry; D050857:Synaptotagmins / Q000032:analysis* / Q000378:metabolism / Q000502:physiology*; D050988:Vesicle-Associated Membrane Protein 2 / Q000032:analysis*
chemical_list: D050600:SNARE Proteins; D050988:Vesicle-Associated Membrane Protein 2; D050857:Synaptotagmins