Desnitro-imidacloprid Activates the Extracellular Signal-Regulated Kinase Cascade via the Nicotinic Receptor and Intracellular Calcium Mobilization in N1E-115 Cells

Abstract

Imidacloprid (IMI) is the principal neonicotinoid (the only major new class of synthetic insecticides of the past three decades). The excellent safety profile of IMI is not shared with a metabolite, desnitro-IMI (DNIMI), which displays high toxicity to mammals associated with agonist action at the α4β2 nicotinic acetylcholine receptor (nAChR) in brain. This study examines the hypothesis that IMI, DNIMI, and (−)-nicotine activate the extracellular signal-regulated kinase (ERK) cascade via primary interaction with the α4β2 nAChR in mouse neuroblastoma N1E-115 cells. These three nicotinic agonists induce phosphorylation of ERK (p44/p42) in a concentration-dependent manner with an optimal incubation period of 30 min. DNIMI (1 μM)-induced ERK activation is blocked by nicotinic antagonist mecamylamine but not by α-bungarotoxin and muscarinic antagonist atropine. This activation is prevented by intracellular Ca 2+ chelator BAPTA-AM but not by removal of external Ca 2+ using EGTA and Ca 2+-free medium. 2-Aminoethoxy-diphenylborate, a blocker for inositol 1,4,5-trisphosphate (IP 3)-mediated Ca 2+ release from intracellular stores, inhibits DNIMI-induced ERK activation but a high level of ryanodine (to block ryanodine receptor-mediated Ca 2+ release) does not. The inhibitor U-73122 for phospholipase C (to suppress IP 3 production) prevents ERK activation evoked by DNIMI. Inhibitors for protein kinase C (PKC) (GF109203X) and ERK kinase (PD98059) block this activation whereas an inhibitor (H-89) for cyclic AMP-dependent protein kinase does not. Thus, neonicotinoids activate the ERK cascade triggered by primary action at the α4β2 nAChR with an involvement of intracellular Ca 2+ mobilization possibly mediated by IP 3. It is further suggested that intracellular Ca 2+ activates a sequential pathway from PKC to ERK.