INHIBITION BY CLOSTRIDIAL NEUROTOXINS OF CALCIUM-INDEPENDENT [H-3] NORADRENALINE OUTFLOW FROM FREEZE-THAWED SYNAPTOSOMES - COMPARISON WITH SYNAPTOBREVIN HYDROLYSIS

Clostridial neurotoxins are known to inhibit regulated, i.e. calcium-d ependent exocytosis. In the present study we have investigated their p otential role in also inhibiting calcium-independent exocytosis. Synap tosomes from rat forebrain were preloaded with [H-3]noradrenaline and permeabilized reversibly by freezing in Ca2+-free potassium glutamate containing dimethyl sulfoxide and the toxins to be assayed, Subsequent ly, outflow of radioactivity was measured in isotonic calcium-free pot assium glutamate. The synaptic vesicle protein synaptobrevin-2/VAMP-2 and its toxin-dependent degradation were analysed by Western blotting. The light chain of tetanus toxin reduced the synaptosomal outflow of radioactivity, whereas the activity of the heavy chain was at the dete ction limit. The respective activities of the dichain toxins from Clos tridium tetani and C. botulinum A, B and E were enhanced by pretreatme nt with dithiothreitol. Reduced single-chain tetanus toxin was less po tent than reduced dichain tetanus toxin. Pretreatment with ethylene di amine tetraacetic acid as an inhibitor of Zn2+-proteases abolished the actions of the tetanus toxin light chain and of the reduced dichain t oxins. Hydrolysis of synaptobrevin-2/VAMP-2 was obtained with tetanus toxin light chain, reduced dichain tetanus toxin and C. botulinum B to xin. Its hydrolysis by single-chain tetanus toxin was less pronounced, and it was absent with botulinum toxins A and E. It is concluded that clostridial neurotoxins can not only inhibit calcium-dependent releas e but also affect calcium-independent outflow from synaptosomes. Since this is accompanied by selective intrasynaptosomal proteolysis of syn aptobrevin, calcium-independent outflow may at least in part involve t he vesicular release apparatus.