Inhibition of calcium-dependent release of noradrenaline from PC12 cells by botulinum type-A neurotoxin. Long-term effects of the neurotoxin on intact cells.

(a) Clostridium botulinum type-A neurotoxin (BoNTA) inhibited the calcium-dependent release of noradrenaline from PC12 cells in a dose-dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37 degrees C, a neurotoxin concentration of approximately 0.12 +/- 0.03 microM was required to inhibit 50% of the calcium-dependent noradrenaline release. (b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose-dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I-labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high-affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non-specific binding to the cell membrane or via a low-concentration low-affinity acceptor molecule. (c) A study of the long-term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0.3 microM) for 24 h was reduced to less than 20% of control values over a subsequent 4-day period. After 8 days, release levels were significantly lower (60-65%) than control values, despite a more than 10-fold increase in the cell mass. (d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94-98%) to be associated with the cell membrane fraction. Of this, 50-80% of the BoNTA was associated with the nuclear and cell debris fraction and 11-25% was recovered in the large-granule-vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar. (e) Examination of the form of the cell-associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non-reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells.(ABSTRACT TRUNCATED AT 400 WORDS)