Brain Fipotinic Acetylohpline Feceptors Biochemical Characterization by Neosurugatoxin

Specific [3Hjnicotine binding to rat forebrain membranes was saturable and of high affinity, and it exhibited pharmacological specificity as well as stereoselectivity for nicotinic agents. There was a regional variation of specific [3Hjnicotine binding in rat brain. Low concentrations of neosurugatoxin markedly inhibited specific [3H]nicotine binding in rat forebrain (1C5() = 78 nM) and the competition curve by the toxin was biphasic (pseudo-Hill slope, 0.44). Approximately 50 % of [3Hjnicotine binding in rat forebrain was inhibited by low concentrations (0.3-100 nM) of neosurugatoxin and the residual binding was inhibited by higher concentrations (0.3-10 sM). In the presence of 3 nM, 100 nM, and 1 iM neosurugatoxin, there was a concentration-dependent (28, 54, and 71%, respectively) loss of [3Hjnicotine-binding sites (Bmax) in rat forebrain with little change in the dissociation constant (Kd). The blockade of brain [3Hjnicotine-binding sites induced by neosurugatoxin was not reversed by washing. Further, the toxin (10 tiM) considerably accelerated the dissociation of [3H]nicotine from its receptor sites initiated by nonlabeled (-)nicotine. These observations suggest that neosurugatoxin may alloster-ically regulate [3H]nicotine binding rather than competing directly. In contrast to a marked inhibition of [3Hjnicotine binding, neosurugatoxin (100 nM-10 tiM) had no effect on brain [3Hjquinuclidinyl benzilate binding. In conclusion, the present study has shown that [3Hjnicotine selectively labels nicotinic cholinergic receptors in rat brain and that neosurugatoxin is a potent noncompetitive antagonist of these receptors