Brain nicotinic acetylcholine receptors. Biochemical characterization by neosurugatoxin.
Specific [3H]nicotine 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 [3H]nicotine binding in rat brain. Low concentrations of neosurugatoxin markedly inhibited specific [3H]nicotine binding in rat forebrain (IC50 = 78 nM) and the competition curve by the toxin was biphasic (pseudo-Hill slope, 0.44). Approximately 50% of [3H]nicotine 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 microM). In the presence of 3 nM, 100 nM, and 1 microM neosurugatoxin, there was a concentration-dependent (28, 54, and 71%, respectively) loss of [3H]nicotine-binding sites (Bmax) in rat forebrain with little change in the dissociation constant (Kd). The blockade of brain [3H]nicotine-binding sites induced by neosurugatoxin was not reversed by washing. Further, the toxin (10 microM) considerably accelerated the dissociation of [3H]nicotine from its receptor sites initiated by nonlabeled (-)nicotine. These observations suggest that neosurugatoxin may allosterically regulate [3H]nicotine binding rather than competing directly. In contrast to a marked inhibition of [3H]nicotine binding, neosurugatoxin (100 nM-10 microM) had no effect on brain [3H]quinuclidinyl benzilate binding. In conclusion, the present study has shown that [3H]nicotine selectively labels nicotinic cholinergic receptors in rat brain and that neosurugatoxin is a potent noncompetitive antagonist of these receptors.
Yamada
S
S
Isogai
M
M
Kagawa
Y
Y
Takayanagi
N
N
Hayashi
E
E
Tsuji
K
K
Kosuge
T
T
D000818:Animals; D001285:Atropine / Q000378:metabolism; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D001931:Brain Mapping; D018738:Hexamethonium; D006584:Hexamethonium Compounds / Q000378:metabolism; D007700:Kinetics; D008978:Mollusk Venoms / Q000378:metabolism*; D009538:Nicotine / Q000378:metabolism; D010276:Parasympatholytics / Q000378:metabolism; D010277:Parasympathomimetics / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011978:Receptors, Nicotinic / Q000378:metabolism*
D006584:Hexamethonium Compounds; D008978:Mollusk Venoms; D010276:Parasympatholytics; D010277:Parasympathomimetics; D011978:Receptors, Nicotinic; D018738:Hexamethonium; D009538:Nicotine; D001285:Atropine; C036793:neosurugatoxin