1-(6-Chloronicotinyl)-2-nitroimino-imidazolidine (imidacloprid or IMI) is a potent insecticide of a new chemical class considered from earlier studies to act at the insect nicotinic acetylcholine receptor. In a direct approach to the mode of action of IMI, it was radiolabeled and [3H]IMI was examined in binding studies to elucidate its pharmacological profile. [3H]IMI undergoes high affinity specific binding in house fly head P2 membranes with 95% specific binding, a dissociation constant of 1.2 nM, and a maximal binding site capacity of 853 fmol/mg protein. The standard binding assay consisted of 1 nM [3H]IMI and 200 μg membrane protein in 50 mM NaCl, 10 mM sodium phosphate (pH 7.4) containing 0.1% Triton X-100 with incubation for 60 min at 22°C prior to filtration. The radioligand undergoes rapid biphasic association and dissociation consistent with a two-stage sequential reaction in each case. [3H]IMI binding is very sensitive to carbachol (IC50 1.9 μM) and other choline esters (IC50s 0.2 to 0.5 μM for acetylcholine, propionylcholine, and butyrylcholine in the presence of paraoxon as a cholinesterase inhibitor). The pharmacological profile for [3H]IMI binding indicates inhibition by both nicotinic and muscarinic agents with IC50s of 0.6 μM for (-)-nicotine, 2.2 μM for α-bungarotoxin, 30 μM for D-tubocurarine, 90 μM for atropine, 275 μM for quinuclidinyl benzilate, and 288 μM for dexetimide. Lineweaver-Burk plots establish competitive inhibition kinetics for [3H]IMI with acetylcholine, α-bungarotoxin, and quinuclidinyl benzilate. Detection of [3H]IMI binding in membrane preparations from several insects but not from the vertebrates examined is consistent with the selective toxicity of the nitromethylene and nitroimine insecticides.
All Science Journal Classification (ASJC) codes
- Agronomy and Crop Science
- Health, Toxicology and Mutagenesis