N,O-di and N,N,O-tri ( 3 H) acetyl  -bungarotoxins as specific labelling agents of cholinergic receptors.
1. alpha-Bungarotoxin isolated from the venom of Bungarus multicinctus was acetylated with [(3)H] acetic anhydride and N-[(3)H] 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 x 10(7) 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-[(3)H] acetyl alpha-bungarotoxins are specific and irreversible labelling agents for the cholinergic receptors of skeletal muscle.
D000107:Acetylation; D000109:Acetylcholine / Q000037:antagonists & inhibitors; D000818:Animals; D001001:Anura; D002038:Bungarotoxins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000494:pharmacology / Q000633:toxicity; D003714:Denervation; D004558:Electric Stimulation; D004592:Electrophoresis, Starch Gel; D066298:In Vitro Techniques; D007928:Lethal Dose 50; D051379:Mice; D009119:Muscle Contraction / Q000187:drug effects; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology; D010276:Parasympatholytics / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011950:Receptors, Cholinergic / Q000187:drug effects; D011955:Receptors, Drug / Q000187:drug effects*; D012911:Snakes; D013997:Time Factors; D014316:Tritium; D014403:Tubocurarine / Q000494:pharmacology; D014688:Venoms / Q000494:pharmacology*
D002038:Bungarotoxins; D010276:Parasympatholytics; D011950:Receptors, Cholinergic; D011955:Receptors, Drug; D014688:Venoms; D014316:Tritium; D000109:Acetylcholine; D014403:Tubocurarine