Amitraz and its metabolite differentially activate α- and ß-adrenergic-like octopamine receptors.

BACKGROUND: Amitraz is a formamidine acaricide and insecticide used to control ticks, mites and fleas. N2 -(2,4-Dimethylphenyl)-N1 -methyformamidine (DPMF), a metabolite of amitraz, is thought to be an active agent that exerts acaricidal and insecticidal effects by acting as an agonist on octopamine receptors. The emergence of cattle ticks resistant to amitraz is a serious problem that requires urgent attention. The objective of this research was to determine which type of octopamine receptor is the primary target of amitraz and thereby understand the molecular mechanisms of action and resistance to amitraz. RESULTS: Amitraz and DPMF potently activated Bombyx mori α- and ß-adrenergic-like octopamine receptors (α- and ß-AL OARs) that were stably expressed in HEK-293 cells. Notably, DPMF elevated intracellular cAMP levels, with an EC50 of 79.6 pm in ß-AL OARs, the transcripts of which were prevalently and widely localised in B. mori body parts. Furthermore, DPMF elevated the intracellular Ca2+ levels, with an EC50 of 1.17 nm in α-AL OARs. CONCLUSION: Although both amitraz and DPMF acted as OAR agonists, the metabolite DPMF was more potent than amitraz and differentially activated α- and ß-AL OARs. The present findings provide a basis for studies to examine the mechanism of amitraz resistance and to develop novel acaricides and insecticides. © 2016 Society of Chemical Industry.

Molecular cloning and pharmacological characterization of a Bombyx mori tyramine receptor selectively coupled to intracellular calcium mobilization.

Tyramine (TA) is a biogenic amine in invertebrates. cDNA encoding the TA receptor (TAR) BmTAR2 was cloned from the nerve tissue of the silkworm Bombyx mori. The receptor's functional and pharmacological properties were examined in BmTAR2-transfected HEK-293 cells. In [(3)H]TA binding assays, BmTAR2 showed considerably higher affinity for TA than for other biogenic amines, with an IC(50) value of 57.5 nM. Moreover, TA induced a dose-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in cells, with an EC(50) value of 11.6 nM, whereas octopamine and dopamine increased [Ca(2+)](i) only at concentrations above 100 microM. A few antagonists were found to inhibit the TA-induced increases in [Ca(2+)](i); the rank order of potency was yohimbine > chlorpromazine > mianserin. TA showed no effect on intracellular cAMP concentration. The data indicate that BmTAR2 belongs to the second class of TARs, which are selectively coupled to intracellular Ca(2+) mobilization. RT-PCR analysis revealed that BmTAR2 was expressed predominantly in the nervous tissue of B. mori larvae, suggesting that TA has neurotransmitter and neuromodulatory roles that are mediated by BmTAR2.