SNAP-25 is expressed in islets of Langerhans and is involved in insulin release.

Abstract

Abstract. SNAP-25 is known as a neuron specific molecule involved in the fusion of small synaptic vesicles with the presynaptic plasma membrane. By immunolocalization and Western blot analysis, it is now shown that SNAP-25 is also expressed in pancreatic endocrine cells. Botulinum neurotoxins (BoNT) A and E were used to study the role of SNAP-25 in insulin secretion. These neurotoxins inhibit transmitter release by cleaving SNAP-25 in neurons.Cells from a pancreatic B cell line (HIT) and primary rat islet cells were permeabilized with streptolysin-O to allow toxin entry. SNAP-25 was cleaved by BoNT/A and BoNT/E, resulting in a molecular mass shift of ~ol and 3 kD, respectively. Cleavage was accompanied by an inhibition of Ca÷÷-stimulated insulin release in both cell types. In HIT cells, a concentration of 30-40 nM BoNT/E gave maximal inhibition of stimulated insulin secretion of '~60%, coinciding with essentially complete cleavage of SNAP-25. Half maximal effects in terms of cleavage and inhibition of insulin release were obtained at a concentration of 5-10 nM. The A type toxin showed maximal and halfmaximal effects at concentrations of 4 and 2 nM, respectively. In conclusion, the results suggest a role for SNAP-25 in fusion of dense core secretory granules with the plasma membrane in an endocrine cell typethe pancreatic B cell. IN pancreatic B cells, proinsulin is sorted in the transGolgi network for delivery to secretory granules where it is processed to insulin (23,40). Insulin is packed and stored in large dense core granules (LDCG) ~ and is released when exocytosis is stimulated by secretagogues such as glucose (23). Current understanding of B cell stimulussecretion coupling suggests that nutrient stimuli cause depolarization of the cell membrane, which leads to an influx of Ca ++ triggering the fusion of granules with the plasma membrane (48). Although sensitivity to glucose is a peculiarity of the pancreatic B cell, the other steps leading from the trans-Golgi network to LDCGs and the fusion of granules with the plasma membrane are, most probably, Please address all correspondence to Dr. Karin Sadoul, Laboratoires de Recherche Louis Jeantet, Centre Medical Universitaire, 1, rue MichelServet, CH-1211 Geneva 4, Switzerland. Tel.: 41 22 7025537. Fax.: 41 22 3473334. Abbreviations used in this paper:BoNT, botulinum neurotoxin; LDCG, large dense core granule; LDCV, large dense core vesicle; NSE N-ethylmaleimid-sensitive factor; SLMV, synaptic-like microvesicle; SNAP, soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kD; SSV, small synaptic vesicle.common to all cells possessing the regulated secretory pathway.The molecular mechanism for docking and fusion of LDCGs in endocrine cells has not so far been studied. Conversely, recent data from S611ner et al. have established a detailed model for docking and fusion of small synaptic vesicles (SSV) in neuronal cells (57,58). Using cosedimentation and immunoprecipitation techniques they could identify a 20-S fusion complex. The complex is...

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