Evidence that a nereistoxin metabolite, and not nereistoxin itself, reduces neuronal nicotinic receptors: studies in the whole chick ciliary ganglion, on isolated neurons and immunoprecipitated receptors.
Nereistoxin (100 microM, 2-10 min) blocks nicotinic receptors in the intact chick ciliary ganglion. This effect mimics blockade by the reducing agent dithiothreitol (2 mM, 20 min), which is not reversed until oxidation with dithiobisnitrobenzoic acid (1 mM, 5 min). After treating intact ganglia with either nereistoxin or dithiothreitol, the affinity alkylating agent bromoacetylcholine causes irreversible blockade that cannot be reversed by dithiobisnitrobenzoic acid. These data suggest that nereistoxin, or a metabolite, acts to reduce nicotinic receptors, although nereistoxin differs from dithiothreitol in that agonists only partially protect against nereistoxin reduction. In studies on chick retina, we previously proposed that a metabolite of nereistoxin (such as dihydronereistoxin) is the actual reducing agent for neuronal nicotinic receptors. Current findings in chick ciliary ganglion supporting this hypothesis include: 1) changing pH alters the minimal nereistoxin concentration needed for blockade in intact ganglia, but has little effect on the minimal concentration needed for dithiothreitol, 2) application of a quaternary analog of nereistoxin has little effect on intact ganglion, but a quaternary analog of dihydronereistoxin blocks nicotinic receptors by reduction, 3) nereistoxin weakly oxidizes rather than reduces immunoprecipitated receptors from chick brain and 4) in whole-cell patch-clamp studies, nereistoxin clearly does not reduce receptors on chick ciliary neurons, although dihydronereistoxin mimics receptor blockade by dithiothreitol, and requires oxidation by dithiobisnitrobenzoic acid for reactivation. Together, these data suggest that nereistoxin is not a direct reducing agent for neuronal nicotinic receptors.
Xie
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Y
Department of Pharmaceutical Sciences, Northeastern University, Boston, Massachusetts, USA.
McHugh
T
T
McKay
J
J
Jones
G S
GS
Loring
R H
RH
D000109:Acetylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens; D005726:Ganglia, Parasympathetic / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D008387:Marine Toxins / Q000378:metabolism / Q000494:pharmacology*; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure*; D010084:Oxidation-Reduction; D011233:Precipitin Tests; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013876:Thiophenes / Q000494:pharmacology
D008387:Marine Toxins; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; D013438:Sulfhydryl Compounds; D013876:Thiophenes; C097748:dihydromethylnereistoxin; C025633:nereistoxin; C097747:methylnereistoxin; D000109:Acetylcholine