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

Abstract

Summary 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. 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. 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. 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. (+)‐Tubocurarine protected effectively against the binding as well as the irreversible neuromuscular blocking effect of the toxins. 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. 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.

Influence of chronic neostigmine treatment on the number of acetylcholine receptors and the release of acetylcholine from the rat diaphragm

“…Number of acetylcholine receptors The number of acetylcholine receptors in one end-plate of diaphragms of rats weighing about 250 g has been determined to be 19-2-2 x 107 (Chang et al 1973) and 2-9 x 107 (Fambrough & Hartzell, 1972) by using various purified derivatives of ac-bungarotoxin. In the present experiment we obtained 2-11 + 0-12 x 107 (mean + S.D., n = 9) receptive sites per endplate with N, O-di[3H]acetyl ax-bungarotoxin.…”

“…Several changes of Tyrode solution were made in 2 hr in order to remove any neostigmiine retained by the tissue. The number of acetylcholine receptors in the motor end-plate was estimated from the irreversible binding of N, O-di[3H]acetyl ac-bungarotoxin 400 mci/m-mole (Chang et al 1973). Diaphragms were incubated with the toxin at 1-25 x 10-v M for 2 hr, a condition known to saturate the receptor, and then washed for 5 hr with twelve changes of Tyrode solution to remove the non-specific binding.…”

“…Recently, it has become possible to determine the number of acetylcholine receptors in the endplate (Miledi & Potter, 1971; Barnard, Wieckowski & Chiu, 1971;Fambrough & Hartzell, 1972;Chang, Chen & Chuang, 1973) by using isotopically labelled derivatives of c-bungarotoxin isolated from the venom of Bungarus multicinctus (Chang & Lee, 1963). Denervation of the rat diaphragm is thus shown to increase the number of receptors by about * Present address: Institute of Zoology, Academia Sinica, Taipei, Taiwan. 614 C. C. CHANG, T. F. CHEN AND SING-TAI CHUANG thirty-fold along the whole muscle fibre (Miledi & Potter, 1971;Chang et al 1973). In order to get further insight into the trophic effect of nerve on muscle, it was of interest to see what effect the prolonged inhibition of cholinesterase might produce, since this treatment may affect neuromuscular transmission in a way opposite to that of denervation.…”

Appearance of new acetylcholine receptors on the baby chick biventer cervicis and denervated rat diaphragm muscles after blockade with α‐bungarotoxin

“…The appearance of new binding sites in the baby chick muscle and denervated rat diaphragm where plenty of extrajunctional AChRs exist (Chang et al 1973(Chang et al , 1975b, but not in the innervated rat diaphragm, suggests that the presence of extrajunctional AChR may be related in some way with this phenomenon. Since the recovery of response after removal of a-bungarotoxin occurs only to exogenously applied ACh but not to nerve stimulation in both chick and rat muscles, these observations may indicate that the new toxin-binding sites are extrajunctional rather than junctional.…”

“…The toxin bound with the junctional AChR is practically non-dissociable on repetitive washings (Chang, Chen & Chuang, 1973). The response of the blocked muscle to exogenous ACh, however, can be partially restored when the toxin is removed by repetitive washings in the baby chick biventer cervicis (Chang & Tang, 1974) and in the denervated diaphragm muscles (Lapa, Albuquerque & Daly, 1974; Chiu, Lapa, Barnard & Albuquerque, 1974;Sakmann, 1975).…”