In:
American Journal of Physiology-Cell Physiology, American Physiological Society, Vol. 281, No. 3 ( 2001-09-01), p. C740-C750
Abstract:
Previous reports showed that cleavage of vesicle-associated membrane protein-2 (VAMP-2) and synaptosomal-associated protein of 25 kDa (SNAP-25) by clostridial neurotoxins in permeabilized insulin-secreting β-cells inhibited Ca 2+ -evoked insulin secretion. In these reports, the soluble N-ethylmaleimide-sensitive factor attachment protein target receptor proteins might have formed complexes, which preclude full accessibility of the putative sites for neurotoxin cleavage. In this work, VAMP-2 and SNAP-25 were effectively cleaved before they formed toxin-insensitive complexes by transient transfection of insulinoma HIT or INS-1 cells with tetanus toxin (TeTx) or botulinum neurotoxin A (BoNT/A), as shown by immunoblotting and immunofluorescence microscopy. This resulted in an inhibition of Ca 2+ (glucose or KCl)-evoked insulin release proportionate to the transfection efficiency (40–50%) and an accumulation of insulin granules. With the use of patch-clamp capacitance measurements, Ca 2+ -evoked exocytosis by membrane depolarization to −10 mV was abolished by TeTx (6% of control) but only moderately inhibited by BoNT/A (30% of control). Depolarization to 0 mV to maximize Ca 2+ influx partially overcame BoNT/A (50% of control) but not TeTx inhibition. Of note, cAMP activation potentiated Ca 2+ -evoked secretion by 129% in control cells but only 55% in BoNT/A-transfected cells and had negligible effects in TeTx-transfected cells. These results indicate that, whereas VAMP-2 is absolutely necessary for insulin exocytosis, the effects of SNAP-25 depletion on exocytosis, perhaps on insulin granule pool priming or mobilization steps, could be partially reversed by higher levels of Ca 2+ or cAMP potentiation.
Type of Medium:
Online Resource
ISSN:
0363-6143
,
1522-1563
DOI:
10.1152/ajpcell.2001.281.3.C740
Language:
English
Publisher:
American Physiological Society
Publication Date:
2001
detail.hit.zdb_id:
1477334-X
SSG:
12
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