Ca2(+)-dependent noradrenaline release from permeabilised PC12 cells is blocked by botulinum neurotoxin A or its light chain.
Permeabilisation of PC12 cells with digitonin allowed a direct study of the intracellular action of botulinum neurotoxin A, one of a group of dichain proteins produced by Clostridium botulinum that causes the fatal neuroparalytic condition, botulism. Release of [3H]noradrenaline from these permeabilised cells could be evoked by Ca2+ and this was inhibited specifically by the neurotoxin in a dose-dependent manner (half-maximal dose approximately 2 nM under the conditions used). Inclusion of the reducing agent dithiothreitol (up to 10 mM) had no effect on the level of inhibition. Moreover, electrophoretic analysis showed that this treatment of the toxin in the native state caused negligible reduction of inter-chain disulphide bonds. Toxin-induced blockade of neurotransmitter release was incomplete and could not be overcome by increased Ca2+ concentration (100 microM). The observed toxin-insensitivity of the release from intact PC12 cells must result from inefficient toxin uptake, relative to that in peripheral cholinergic neurones. Refolded light chain alone inhibited exocytosis to the same degree and with similar potency to that of the intact neurotoxin, an effect not altered by the heavy chain. This inhibitory activity of the light chain in PC12 cells accords with observations made in permeabilised chromaffin cells [(1989) J. Biol. Chem. 264, 10354-10360; (1989) FEBS Lett. 255, 391-394] but contrasts with invertebrate neurones, where intracellular injection of the same preparations of both chains were necessary for inhibition of quantal release of acetylcholine [(1988) Proc. Natl. Acad. Sci. USA 85, 4090-4094]. These collective findings may signify an interesting difference in the release process in such diverse systems or denote a dissimilarity in the transport or processing of the toxin when applied into intact neurones or cells permeabilised by detergent or streptolysin.
McInnes
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Department of Biochemistry, Imperial College of Science, Technology and Medicine, South Kensington, London, UK.
Dolly
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JO
D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000494:pharmacology* / Q000633:toxicity; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D051379:Mice; D009638:Norepinephrine / Q000378:metabolism*; D010673:Pheochromocytoma; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D001905:Botulinum Toxins; D004072:Digitonin; D011188:Potassium; D002118:Calcium; D004229:Dithiothreitol; D009638:Norepinephrine