Liu_1995_Pestic.Biochem.Physiol_52_170

[3H]Imidacloprid ([3H]IMI) is known to bind with high affinity to the nicotinic acetylcholine receptor (nAChR) agonist site in Musca domestica L. (Musca) head membranes. Physostigmine (PHY) is reported to act in vertebrates at the nAChR ion-channel complex as an activator at low concentrations and an open channel blocker at high levels. PHY inhibits [3H]IMI binding in Musca with an IC50 of 8 uM. The binding site for PHY appears to be associated with the nAChR since the potency of PHY is decreased 2.3- to 13-fold by (-)-nicotine but little if any by carbachol, each at 1 uM. Acetylcholine (ACh) serves as both a competitive inhibitor of [3H]IMI binding, reducing the rates of association and dissociation in a biphasic manner, and a substrate for acetylcholinesterase (AChE), which is in turn inhibited by PHY. PHY at 0.1 and 1 uM appears to be a competitive inhibitor of [3H]IMI binding, whereas at 100 uM it is noncompetitive and increases the Kd by 25-fold and Bmax by 2.2-fold. PHY at 0.1 uM increases the apparent potency of ACh as an inhibitor of [3H]IMI binding, due to AChE inhibition, whereas at 10 and 100 uM it does not alter the IC50s of ACh. These overall findings indicate an apparent allosteric interaction between the [3H]IMI and PHY binding sites. The high structural specificity of PHY in this region is established by finding that eseroline is also active (IC50 = 40 uM) and that over 140 methyl- and dimethylcarbamate and organophosphorus insecticides and related compounds are not inhibitory at 10 uM. [3H]IMI binding is also inhibited by the chloronicotinyl analgetic agent epibatidine (IC50 = 350 nM) and by six cyanoimine insecticides including chloronicotinyl and chlorothiazolyl analogs. The inhibitory potency of acetamiprid (IC50 = 3.2 nM) and other cyanoimines at the nAChR agonist site correlates well (r = 0.98, n = 6) with their intrinsic toxicity to Musca (i.e., their knockdown or lethal effects on injection and with a synergist to minimize oxidative detoxification). [3H]IMI is therefore a suitable radioligand for investigating the interaction of PHY and a variety of nicotinic agonists with chloropyridyl and chlorothiazolyl substituents at the insect nAChR.