VAMP-2 and cellubrevin are expressed in pancreatic beta-cells and are essential for Ca(2+)-but not for GTP gamma S-induced insulin secretion.

Abstract

VAMP proteins are important components of the machinery controlling docking and/or fusion of secretory vesicles with their target membrane. We investigated the expression of VAMP proteins in pancreatic beta‐cells and their implication in the exocytosis of insulin. cDNA cloning revealed that VAMP‐2 and cellubrevin, but not VAMP‐1, are expressed in rat pancreatic islets and that their sequence is identical to that isolated from rat brain. Pancreatic beta‐cells contain secretory granules that store and secrete insulin as well as synaptic‐like microvesicles carrying gamma‐aminobutyric acid. After subcellular fractionation on continuous sucrose gradients, VAMP‐2 and cellubrevin were found to be associated with both types of secretory vesicle. The association of VAMP‐2 with insulin‐containing granules was confirmed by confocal microscopy of primary cultures of rat pancreatic beta‐cells. Pretreatment of streptolysin‐O permeabilized insulin‐secreting cells with tetanus and botulinum B neurotoxins selectively cleaved VAMP‐2 and cellubrevin and abolished Ca(2+)‐induced insulin release (IC50 approximately 15 nM). By contrast, the pretreatment with tetanus and botulinum B neurotoxins did not prevent GTP gamma S‐stimulated insulin secretion. Taken together, our results show that pancreatic beta‐cells express VAMP‐2 and cellubrevin and that one or both of these proteins selectively control Ca(2+)‐mediated insulin secretion.

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…Direct analysis of membrane capacitance has directly shown that GppNHp induces exocytosis [36] and it is likely that the same final steps in the exocytotic pathway are activated by Ca 2+ or by GppNHp. Previous work on insulin-secreting cells had shown that secretion induced by GTPTS was insensitive to tetanus and botulinum B neurotoxins despite extensive cleavage of VAMP, and inhibition of Ca2+-induced secretion, leading to the suggestion that VAMP is essential for the CaZ+-induced secretion but not that induced by non-hydrolysable GTP analogues [29]. The present data from chromaffin cells show, in contrast, that both stimuli are neurotoxin D-sensitive and therefore act via a VAMPdependent pathway.…”

“…We have shown here that recombinant His6-tagged botulinum neurotoxin D light chain cleaves VAMP but not syntaxin or SNAP-25 in permeabilised chromaffin cells and substantially inhibits both Ca2+-induced and GppNHp-induced catecholamine release suggesting that one or more VAMPs are essential in both cases. This differs from the situation in insulin-secreting cells where it has been suggested that VAMPs are not required for GTPTS-induced secretion [29]. While botulinum neurotoxin E is believed to be specific for SNAP-25, neurotoxin C1 has recently been shown to cleave both syntaxin and SNAP-25 in chromaffin cells [46].…”

“…Insulin-secreting cells are also sensitive to these two classes of neurotoxins [29][30][31] and studies on these cells have suggested that VAMP is essential for Ca2+-induced secretion but not for insulin release activated by the non-hydrolysable GTP analogue, GTPTS, since this was insensitive to tetanus toxin and botulinum B neurotoxin [29]. These data on insulin-secreting cells, coupled with studies on constitutive vesicle traffic in epithelial cells in which basolateral but not apical exocytosis is inhibited by botulinum neurotoxins [32], appear to suggest that exocytosis could occur by either SNARE-dependent or SNARE-independent pathways.…”

Novel Rabphilin-3-like Protein Associates with Insulin-containing Granules in Pancreatic Beta Cells

“…4B, lanes 2 and 4). Synaptotagmin III is localized in the insulin granules (19), whereas VAMP-2 exists in both granules and SLMVs in beta cells (31). These results indicate an association of granuphilin-a with insulin-containing dense-core granules in beta cells.…”

“…4A, upper panel), whereas synaptophysin was enriched at fractions 6 and 7 (lower panel). Synaptophysin, an integral membrane protein of SSVs in neurons, is absent in the insulin granules, but present in the SLMVs in beta cells (12,19,31). Thus, the two types of secretory vesicles were clearly separated.…”