10 research outputs found
Récepteurs nicotiniques neuronaux d'insectes et insecticides : Caractérisation de facteurs intracellulaires impliqués dans la modulation de l'efficacité des néonicotinoides (ThÚse de Doctorat d'Université)
L\u27utilisation des produitq phytosanitaires est indispensable pour augmenter la qualitĂ© des productions agricoles. Cependant les directives europĂ©ennes et nationales prĂ©conisent une rĂ©duction de 50% de leur utilisation pour prĂ©server l\u27environnement et limiter les effets secondaires sur les organismes non-cibles. Dans cette optique, et pour optimiser l\u27efficacitĂ© de ces produits tout en diminuant leur dose d\u27utilisation, il est nĂ©cessaire de caractĂ©riser les facteurs cellulaires et molĂ©culaires impliquĂ©s dans la modulation de l\u27effet insecticide de ces composĂ©s sur leurs cibles membranaires. Parmi les insecticides les plus utilisĂ©s en agriculture, les nĂ©onicotinoĂŻdes occupent une place importante sur le marchĂ©. Ils ont pour cible les rĂ©cepteurs Ă lâacĂ©tylcholine de type nicotinique (nAChRs) du systĂšme nerveux central des insectes. Chez la blatte Periplaneta americana, des cellules neurosecrĂ©trices
identifiées, les neurones DUM, expriment deux sous-types de nAChRs, (nAChR1 et nAChR2).
Alors que nAChR1 est sensible Ă lâimidaclopride (IMI), nĂ©onicotinoĂŻde de premiĂšre gĂ©nĂ©ration, cet insecticide ne prĂ©sente aucun effet sur nAChR2. Afin dâobtenir une meilleure comprĂ©hension du mode dâaction de ces insecticides sur des nAChRs insensibles, lâobjectif de ce travail de recherche a Ă©tĂ© de caractĂ©riser du point de vue Ă©lectro-pharmacologique les facteurs cellulaires et molĂ©culaires qui influencent lâefficacitĂ© dâun insecticide nĂ©onicotinoĂŻde de seconde gĂ©nĂ©ration, lâacĂ©tamipride (ACT) qui prĂ©sente des caractĂ©ristiques chimiques diffĂ©rentes de lâIMI.
GrĂące Ă la technique Ă©lectrophysiologique du patch-clamp, dans des conditions de potentiel imposĂ©, il a Ă©tĂ© possible de dĂ©montrer que lâeffet de lâACT sur nAChR2 dĂ©pend du potentiel de membrane. Une dĂ©polarisation de la membrane augmente lĂ©gĂšrement la sensibilitĂ© de nAChR2 pour lâACT alors quâune hyper polarisation produit un effet inverse significatif. Dans les deux cas, lâimplication du calcium intracellulaire a Ă©tĂ© dĂ©montrĂ©e. Pour des potentiels plus positifs que le potentiel de membrane (i.e., -50 mV), lâinhibition de lâinflux calcique via lâactivation des canaux calci ques Ă haut seuil dâactivation (High Voltage-Activated) par le chlorure de cadmium et lâÏ-conotoxine GVIA augmente la sensibilitĂ© de nAChR2 pour lâACT. Des effets similaires sont obtenus pour des potentiels de membrane plus hyperpolarisĂ©s lorsque la permĂ©abilitĂ© calcique est inhibĂ©e par le LOE 908, un inhibiteur des canaux TRPÎł. Dans ce dernier cas, lâutilisation dâo utils pharmacologiques spĂ©cifiques (e.g., forskoline, W7) a permis de rĂ©vĂ©ler que la voie de signalisation intracellulaire AMPcyclique/adenylate cyclase est impliquĂ©e dans la modulation de lâefficacitĂ© de lâACT sur nAChR2. Enfin, des mesures de la rĂ©sistance membran aire, rĂ©alisĂ©es en parallĂšle dans des conditions de courant imposĂ©, indiquent que lâĂ©tat conformationnel des nAChR2 joue Ă©galement un rĂŽle important dans la modulation de lâefficacitĂ© de lâACT. Lâensemble de ces rĂ©sultats qui ont permis dâidentifier de nouveaux facteurs cellulaires et molĂ©culaires impliquĂ©s dans la modulation de lâefficacitĂ© dâun nĂ©onicotinoide, lâACT ouvrent des perspectives trĂšs intĂ©ressantes pour optimiser lâefficacitĂ© dâun traitement insecticide
Transmembrane potential, calcium and cyclic AMP modulate selectively insect neuronal nAChR subtype sensitivity to the neurotoxic neonicotinoid insecticides
Modulation de la sensibilité des nAChRs aux insecticides néonicotinoïdes : implications du potentiel de membrane, du calcium, des voies de signalisation et des propriétés fonctionnelles des nAChRs
A fluorinated quinuclidine benzamide named LMA 10203 acts as an agonist of insect nicotinic acetylcholine receptors
In the present study, we take advantage of the fact that cockroach dorsal unpaired median neurons express different nicotinic acetylcholine receptor subtypes to demonstrate that simple quinuclidine benzamides such as the 2-fluorinated benzamide LMA 10203, could act as an agonist of cockroach alpha-bungarotoxin-insensitive nicotinic acetylcholine receptor subtype, called nAChR2. Indeed, 1 mM LMA 10203 induced ionic currents which were partially blocked by 0.5 muM alpha-bungarotoxin and methyllycaconitine and completely blocked by 5 muM mecamylamine. Moreover, the current-voltage curve revealed that the ionic current induced by LMA 10203 increased from -30 mV to +20 mV confirming that it acted as an agonist of alpha-bungarotoxin-insensitive nAChR2. In addition, 1 mM LMA 10203 induced a depolarization of the sixth abdominal ganglion and this neuroexcitatory activity was completely blocked by 5 muM mecamylamine. These data suggest that nAChR2 was also expressed at the postsynaptic level on the synapse between the cercal afferent nerve and the giant interneurons. Interestingly, despite LMA 10203 being an agonist of cockroach nicotinic receptors, it had a poor insecticidal activity. We conclude that LMA 10203 could be used as an interesting compound to identify specific insect nAChR subtypes
Transmembrane potential polarization, calcium influx, and receptor conformational state modulate the sensitivity of the imidacloprid-insensitive neuronal insect nicotinic acetylcholine receptor to neonicotinoid insecticides
Neonicotinoid insecticides act selectively on insect nicotinic acetylcholine receptors (nAChRs). Recent studies revealed that their efficiency was altered by the phosphorylation/dephosphorylation process and the intracellular signaling pathway involved in the regulation of nAChRs. Using whole-cell patch-clamp electrophysiology adapted for dissociated cockroach dorsal unpaired median (DUM) neurons, we demonstrated that intracellular factors involved in the regulation of nAChR function modulated neonicotinoid sensitivity. DUM neurons were known to express two alpha-bungarotoxin-insensitive nAChR subtypes: nAChR1 and nAChR2. Whereas nAChR1 was sensitive to imidacloprid, nAChR2 was insensitive to this insecticide. Here, we demonstrated that, like nicotine, acetamiprid and clothianidin, other types of neonicotinoid insecticides, acted as agonists on the nAChR2 subtype. Using acetamiprid, we revealed that both steady-state depolarization and hyperpolarization affected nAChR2 sensitivity. The measurement of the input membrane resistance indicated that change in the acetamiprid-induced agonist activity was related to the receptor conformational state. Using cadmium chloride, omega-conotoxin GVIA, and (R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-acetamide (LOE 908), we found that inhibition of calcium influx through high voltage-activated calcium channels and transient receptor potential gamma (TRPgamma) activated by both depolarization and hyperpolarization increased nAChR2 sensitivity to acetamiprid. Finally, using N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7), forskolin, and cAMP, we demonstrated that adenylyl cyclase sensitive to the calcium/calmodulin complex regulated internal cAMP concentration, which in turn modulated TRPgamma function and nAChR2 sensitivity to acetamiprid. Similar TRPgamma-induced modulatory effects were also obtained when clothianidin was tested. These findings bring insights into the signaling pathway modulating neonicotinoid efficiency and open novel strategies for optimizing insect pest control