N,O‐di andN,N,O‐tri [3H] acetyl α‐bungarotoxins as specific labelling agents of cholinergic receptors

Abstract

1 α-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [³H] acetic anhydride and N-[³H] acetyl imidazole. Tri-N-acetyl and hexa-N-acetyl derivatives were obtained from the former, and N,O-di, N,N,O-tri and N,N,N,O-tetraacetyl derivatives from the latter reaction, respectively. 2 There were parallel decreases in both neuromuscular blocking action in the phrenic nerve—diaphragm preparation of rats and depression of acetylcholine response of the rectus abdominis muscle of frogs with increased acetylation. Also, a parallel but greater decrease of toxicity in mice was found. 3 N,O-Di and N,N,O-triacetyl toxins were localized mostly in the motor endplate region of the rat diaphragm, whereas a slight nonspecific binding along the whole muscle fibre in addition to the peak in the endplate region was observed with N,N,N,O-tetraacetyl and tri-N-acetyl toxins. In contrast, there was a marked nonspecific binding with hexa-N-acetyl toxin and no peak was observed at the endplate zone. 4 The specific binding was saturable and irreversible. The number of toxin-receptive sites in one endplate was 1·9–2·2 × 10⁷ for all of the labelled toxins irrespective of their potency. 5 (+)-Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins. 6 Denervation of the rat diaphragm caused an increase of toxin-receptive sites beginning from the endplate zone at 1–2 days and then along the whole muscle fibre, reaching the maximum at about 18 days. The total receptive sites increased by about 30-fold. 7 The significance of the findings is discussed and it is concluded that N,O-di and N,N,O-tri-[³H] acetyl α-bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.