The Effect of Botulinum Neurotoxins on the Release of Insulin from the Insulinoma Cell Lines HIT-15 and RINm5F (*)
- From the From Speywood Pharmaceuticals Ltd., Porton House, 1 Bath Road, Maidenhead, Berkshire SL4 4UH, United Kingdom and Division of Biologics, Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, United Kingdom
- § To whom correspondence should be addressed. Tel.: 44 628 771447; Fax: 44 628 770199.
Abstract
Western blotting of the insulin-secreting β-cell lines HIT-15 and RINm5F with anti-SNAP-25 (synaptosomal associated protein
of 25 kDa), anti-synaptobrevin, and anti-syntaxin 1 antibodies revealed the presence of proteins with the same electrophoretic
mobility as found in neural tissue. Permeabilization of both of these insulinoma cell lines to botulinum neurotoxin A by electroporation
resulted, after 3 days of culture, in the loss of 
90% of SNAP-25 immunoreactivity. A similar permeabilization of these cells with botulinum neurotoxin B resulted in the cleavage
of 
90% of the synaptobrevin-like immunoreactivities. Botulinum neurotoxin F also cleaved 
90% of the synaptobrevin-like immunoreactivity in RINm5F cells. The permeabilization of both insulinoma cells to neurotoxin
A resulted in a >90% inhibition of potassium-stimulated, calcium-dependent insulin release. By contrast, permeabilization
of the insulinoma cell lines to neurotoxin B resulted in only a 
60% inhibition of potassium-stimulated insulin release in HIT-15 cells, and neither neurotoxin B nor F caused inhibition in
RINm5F cells. Thus HIT-15 and RINm5F cells contain the components of the putative exocytotic docking complex described in
cells derived from the neural crest. In HIT-15 cells both SNAP-25 and synaptobrevin appear to be involved in calcium-dependent
insulin secretion, whereas in RINm5F cells SNAP-25 but not synaptobrevin is involved.
Footnotes
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- SNAP-25
-
synaptosomal associated protein of 25 kDa
- BoNT
-
botulinum neurotoxin
- DMEM
-
Dulbecco's modified Eagle's medium
- HK-KRB
-
high potassium Krebs-Ringer bicarbonate buffer
- LK-KRB
-
low potassium Krebs-Ringer bicarbonate buffer
- v-SNARE and t-SNARE
-
vesicle and target-soluble, respectively, N-ethylmaleimide-sensitive factor-associated protein receptor.
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- Received March 31, 1995.
- Revision received May 26, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
