Presence of Muscarinic Acetylcholine Receptors in the Cattle TickBoophilus microplusand in Epithelial Tissue Culture Cells ofChironomus tentans

Abstract

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [ 3 H]quinuclidinyl benzilate (QNB) with high affinity (1.2 ± (0.13) nM ; B max 22.5 pmol mg protein -1 ) were detected that do not bind nicotinic compounds specifically. The estimated IC 50 values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [ 3 H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC 50 values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM, 0.8(±0.2) μM, 2.8(±0.6) μM and 78(±1.9) μM, respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [ 3 H]QNB were found. Muscarinic antagonists selectively displaced [ 3 H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC 50 2.13(±1.02) μM) among different subtype-specific mAChR antagonists (4-DAMP had IC 50 49.9(±9.13) μM and methoctramine had IC 50 121(±14.2) μM) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4.8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC 50 71.3(±19.6) μM) and carbachol (IC 50 253(±87.1) μM) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.