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 R INm5F with anti SNAP-25 (synaptosomal associated protein of 25 kDa), a nti-synaptobrevin, and anti-syntaxin 1 antibodies revealed the presenc e of proteins with the same electrophoretic mobility as found in neura l tissue. Permeabilization of both of these insulinoma cell lines to b otulinum neurotoxin A by electroporation resulted, after 3 days of cul ture, in the loss of similar to 90% of SNAP-25 immunoreactivity. A sim ilar permeabilization of these cells with botulinum neurotoxin B resul ted in the cleavage of similar to 90% of the synaptobrevin-like immuno reactivities. Botulinum neurotoxin F also cleaved similar to 90% of th e synaptobrevin-like immunoreactivity in RINm5F cells. The permeabiliz ation of both insulinoma cells to neurotoxin A resulted in a >90% inhi bition of potassium-stimulated, calcium-dependent insulin release. By contrast, permeabilization of the insulinoma cell lines to neurotoxin B resulted in only a similar to 60% inhibition of potassium-stimulated insulin release in HIT-15 cells, and neither neurotoxin B nor F cause d inhibition in RINm5F cells. Thus HIT-15 and RINm5F cells contain the components of the putative exocytotic docking complex described in ce lls derived from the neural crest. In HIT-15 cells both SNAP-25 and sy naptobrevin appear to be involved in calcium-dependent insulin secreti on, whereas in RINm5F cells SNAP-25 but not synaptobrevin is involved.