Ca(2+) influx and cAMP elevation overcame botulinum toxin A but not tetanus toxin inhibition of insulin exocytosis.

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 beta-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.

• Authors: X Huang, Y H Kang, E A Pasyk, L Sheu, M B Wheeler, W S Trimble, A Salapatek, H Y Gaisano
• Journal: American journal of physiology. Cell physiology (Am J Physiol Cell Physiol) Vol. 281 Issue 3 Pg. C740-50 (Sep 2001) ISSN: 0363-6143 [Print] United States
• PMID: 11502551 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Insulin
• Membrane Proteins
• Nerve Tissue Proteins
• R-SNARE Proteins
• Recombinant Proteins
• Synaptosomal-Associated Protein 25
• Tetanus Toxin
• Cyclic AMP
• Botulinum Toxins, Type A
• Glucose
• Calcium

Topics

• Animals
• Botulinum Toxins, Type A (pharmacology)
• Calcium (metabolism)
• Cell Line
• Cell Membrane Permeability
• Clone Cells
• Cyclic AMP (metabolism)
• Exocytosis (drug effects, physiology)
• Glucose (pharmacology)
• Insulin (metabolism)
• Insulin Secretion
• Insulinoma
• Islets of Langerhans (drug effects, metabolism, physiology)
• Kinetics
• Membrane Potentials (drug effects, physiology)
• Membrane Proteins (genetics, physiology)
• Nerve Tissue Proteins (genetics, physiology)
• Pancreatic Neoplasms
• Patch-Clamp Techniques
• R-SNARE Proteins
• Recombinant Proteins (metabolism)
• Synaptosomal-Associated Protein 25
• Tetanus Toxin (pharmacology)
• Transfection
• Tumor Cells, Cultured