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VAMPs are vesicle associated membrane proteins that are essential for secretion. A spliced isoform of rat VAMP-2, called VAMP-2B, is characterized in this study. The VAMP-2B transcript is the result of alternative RNA splicing in which an intron is retained. The predicted amino acid sequence of VAMP-2B differs from VAMP-2 at its carboxy-terminal end. Because recent studies have shown that VAMP's carboxy-terminal end influences the protein's sorting, the location of myc-epitope tagged VAMP-2B in PC12 cells was determined. Subcellular fractionation showed colocalization of myc-VAMP-2B and endogenous VAMP-2. Thus alternative RNA splicing does not affect VAMP-2 sorting in PC12 cells.

The vesicle-associated membrane protein (VAMP) family is essential to vesicle-mediated protein transport. Three mammalian isoforms, VAMP-1, VAMP-2, and cellubrevin, play a role in protein transport to the plasma membrane. In this study, we describe a new rat VAMP-1 isoform produced by alternative pre-mRNA splicing. Only one VAMP-1 isoform dominates in each tissue. Analysis of the nucleotide sequence for the newly discovered isoform, VAMP-1b, reveals that its expression is determined by whether an intron is retained or removed. The predicted amino acid sequences for the VAMP-1 isoforms differ at the carboxy-terminal end of the protein. A similar process has been described for VAMPs in Drosophila melanogaster and suggests a conserved function for the carboxy-terminal domain that can be modulated.

Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways.

Zymogen granules of the exocrine pancreas are the secretory organelles responsible for the regulated secretion of digestive enzymes. Several proteins are associated with or are integral components of the lipid bilayer that forms the zymogen granule membrane. These proteins likely represent important components in the regulated secretion of digestive enzymes. VAMPs (vesicle-associated membrane proteins)/synaptobrevins are a family of 18-kDa integral membrane proteins originally characterized in synaptic vesicles. Polyclonal antisera raised against either a VAMP/glutathione S-transferase (GST) fusion protein or rat brain synaptic vesicles, detected an 18-kDa immunoreactive protein in zymogen granule membranes that co-migrates electrophorectically with rat brain synaptic vesicle VAMP. Rat brain synaptic vesicle VAMP was detected by both antisera. Botulinum-B toxin treatment of zymogen granule membranes did not result in cleavage of zymogen granule membrane VAMP, indicating that exocrine pancreatic VAMP is either VAMP1 or a novel VAMP-isoform. Immunofluorescent studies demonstrated that exocrine pancreatic VAMP localized with GP2, a zymogen granule membrane protein, to the apical region of pancreatic acinar cells. No significant labeling was observed in basolateral regions of pancreatic acinar cells. These results establish the presence of a VAMP protein in the zymogen granule of the rat pancreas and suggest that VAMPs have a role in exocrine secretion.

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.

In response to an external stimulus, neuronal cells release neurotransmitters from small synaptic vesicles and endocrine cells release secretory proteins from large dense core granules. Despite these differences, endocrine cells express three proteins known to be components of synaptic vesicle membranes. To determine if all three proteins, p38, p65, and SV2, are present in endocrine dense core granule membranes, monoclonal antibodies bound to beads were used to immunoisolate organelles containing the synaptic vesicle antigens. [3H]norepinephrine was used to label both chromaffin granules purified from the bovine adrenal medulla and rat pheochromocytoma (PC12) cells. Up to 80% of the vesicular [3H]norepinephrine was immunoisolated from both labeled purified bovine chromaffin granules and PC12 postnuclear supernatants. In PC12 cells transfected with DNA encoding human growth hormone, the hormone was packaged and released with norepinephrine. 90% of the sedimentable hormone was also immunoisolated by antibodies to all three proteins. Stimulated secretion of PC12 cells via depolarization with 50 mM KCl decreased the amount of [3H]norepinephrine or human growth hormone immunoisolated. Electron microscopy of the immunoisolated fractions revealed large (greater than 100 nm diameter) dense core vesicles adherent to the beads. Thus, large dense core vesicles containing secretory proteins possess all three of the known synaptic vesicle membrane proteins.