Beta‐bungarotoxin stimulates the synthesis and accumulation of acetylcholine in rat phrenic nerve diaphragm preparations.

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Abstract
The effects of .beta.-bungarotoxin on acetylcholine (ACh) synthesis, tissue content and release were studied in the rat diaphragm. A gas chromatographic mass spectrometric assay was used to measure ACh and choline. Within 30 min, .beta.-bungarotoxin (0.14 or 1.4 .mu.g/ml) caused a significant increase in tissue ACh content. This increase was apparent prior to the final inhibition by .beta.-bungarotoxin of evoked (10 Hz) ACh release. The toxin enhanced the incorporation of [2H4]Ch into [2H4]ACh in both resting and stimulated preparations. Hemicholinium-3 blocked the rise in diaphragm ACh normally produced by .beta.-bungarotoxin. .beta.-Bungarotoxin did not directly activate choline acetyltransferase in muscle homogenates. The toxin-induced rise in tissue ACh was largely absent Ca2+-free solutions which contained either EGTA [ethylene glycol bis (.beta.-aminoethyl ether) tetraacetate] (1 mM) or SrCl2 (2 or 10 mM). Non-neurotoxic phospholipases A2, fatty acids and the neurotoxic phospholipase A2, notexin, did not cause ACh accumulation in the diaphragm. .beta.-Bungarotoxin did not stimulate ACh synthesis in denervated muscle. The extra ACh which accumulated after .beta.-bungarotoxin did not contribute to enhanced release by nerve impulses even when 4-aminopyridine was added to the medium. High K+ solution and black widow spider venom were ineffective in increasing output from toxin-treated diaphragms relative to controls that had not been treated with .beta.-bungarotoxin. Prior injection of a rat with botulinum toxin prevented the accumulation of ACh due to .beta.-bungarotoxin. Tubocurarine, did not antagonize .beta.-bungarotoxin. .beta.-Bungarotoxin probably has a unique capacity to inhibit ACh release and stimulate ACh synthesis in diaphragm nerve endings. Results are discussed in terms of a possible action of .beta.-bungarotoxin to raise the level of ionized Ca in the nerve terminal cytosol.