The effect of botulinum neurotoxins on the release of insulin from the insulinoma cell lines HIT-15 and RINm5F.
Western blotting of the insulin-secreting beta-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 approximately 90% of SNAP-25 immunoreactivity. A similar permeabilization of these cells with botulinum neurotoxin B resulted in the cleavage of approximately 90% of the synaptobrevin-like immunoreactivities. Botulinum neurotoxin F also cleaved approximately 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 approximately 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.
Boyd
R S
RS
Speywood Pharmaceuticals Ltd., Maidenhead, Berkshire, United Kingdom.
Duggan
M J
MJ
Shone
C C
CC
Foster
K A
KA
D000595:Amino Acid Sequence; D015153:Blotting, Western; D001905:Botulinum Toxins / Q000494:pharmacology*; D018274:Electroporation; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007340:Insulinoma / Q000378:metabolism; D007515:Islets of Langerhans / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000276:immunology / Q000302:isolation & purification*; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D014407:Tumor Cells, Cultured
D007328:Insulin; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D050825:Synaptosomal-Associated Protein 25; D001905:Botulinum Toxins