Partial Agonist Activity of Neonicotinoids on Rat Nicotinic Receptors: Consequences over Epinephrine Secretion and In Vivo Blood Pressure

Abstract

Neonicotinoid insecticides are nicotine-derived molecules which exert acute neurotoxic effects over the insect central nervous system by activating nicotinic acetylcholine receptors (nAChRs). However, these receptors are also present in the mammalian central and peripheral nervous system, where the effects of neonicotinoids are faintly known. In mammals, cholinergic synapses are crucial for the control of vascular tone, blood pressure and skeletal muscle contraction. We therefore hypothesized that neonicotinoids could affect cholinergic networks in mammals and sought to highlight functional consequences of acute intoxication in rats with sub-lethal concentrations of the highly used acetamiprid (ACE) and clothianidin (CLO). In this view, we characterized their electrophysiological effects on rat α3β4 nAChRs, knowing that it is predominantly expressed in ganglia of the vegetative nervous system and the adrenal medulla, which initiates catecholamine secretion. Both molecules exhibited a weak agonist effect on α3β4 receptors. Accordingly, their influence on epinephrine secretion from rat adrenal glands was also weak at 100 μM, but it was stronger at 500 μM. Challenging ACE or CLO together with nicotine (NIC) ended up with paradoxical effects on secretion. In addition, we measured the rat arterial blood pressure (ABP) in vivo by arterial catheterization. As expected, NIC induced a significant increase in ABP. ACE and CLO did not affect the ABP in the same conditions. However, simultaneous exposure of rats to both NIC and ACE/CLO promoted an increase of ABP and induced a biphasic response. Modeling the interaction of ACE or CLO on α3β4 nAChR is consistent with a binding site located in the agonist pocket of the receptor. We present a transversal experimental approach of mammal intoxication with neonicotinoids at different scales, including in vitro, ex vivo, in vivo and in silico. It paves the way of the acute and chronic toxicity for this class of insecticides on mammalian organisms.

Keywords: acetamiprid; acute intoxication; blood pressure; clothianidin; epinephrine secretion; neonicotinoids; nicotine; α3β4 nAChR.

Figure 1

Molecular structures of acetylcholine (ACh), nicotine (NIC) and the neonicotinoids clothianidin (CLO) and acetamiprid (ACE). NIC, the molecular template of most neonicotinoids, is protonated at physiological pH, whereas CLO and ACE possess an electronegative nitro- and cyano- functional group, respectively [25].

Figure 2

Pharmacological profile of ACh, NIC, ACE and CLO on rat $\alpha$3$\beta$4 nAChRs expressed in Xenopus oocytes. (A) ACh (n = 12) and NIC (n = 7) elicit robust currents, while ACE (n = 8) and CLO (n = 7) induce much more modest currents. Due to the solubility limit in DMSO, ACE and CLO could not be used at concentrations upper than 2 × 10${}^{-5}$ M. (B) Concentration–response curves of ligand-evoked currents which are expressed as a % of ACh–elicited current amplitude. Two Y axis were used to visualize ACh and NIC (left), and CLO and ACE (right). Inset: Hexamethonium (HEX, 0.2 μM) an $\alpha$3-containing nAChRs antagonist, inhibits ACh-elicited current as expected. (C) Peak currents (±SEM) elicited by 20 μM ACh, NIC, ACE or CLO on rat $\alpha$3$\beta$4 nAChRs. Different lowercase letters above the graphs indicate significant differences between treatments according to Tukey’s multiple comparison post hoc test (p < 0.05).

Figure 3

Influence of NIC, ACE and CLO added separately or in combination on epinephrine release from rat adrenal gland slices. (A) Experimental design modified from [44]. (B) Concentration–response of epinephrine release following NIC stimulation ± HEX, an $\alpha$3-containing nAChR antagonist. (C–F) Epinephrine release following either ACE or CLO treatment in combination or not with NIC ± HEX. Data are mean ± SEM and values in brackets correspond to the number of biological replicates. Different lowercase letters above the graphs indicate significant differences between two histograms within each graph according to Dunn’s multiple comparison post hoc test (p < 0.05). Note that neonicotinoids are able to stimulate the secretion of epinephrine when applied to the medulla tissue alone.

Figure 4

Schematic protocol of rat arterial blood pressure (ABP) measurements. (A) Illustration of an anesthetized rat with a catheter inserted in right femoral artery for ABP measurements through a blood pressure transducer, and with a catheter inserted in left femoral vein for i.v. injections. Rat drawing modified from Watts et al. [47]. (B) Schematic time-course of the protocol. Each rat was challenged every 5 min with AngII, ACh and vehicle solution (VEH) before fixed concentrations of NIC, ACE, CLO or DMSO 2% (CTRL). (C) Analysis method of raw trace to collect the difference between mean arterial blood pressure ($\Delta$ABP). See Materials and Methods Section 4.7 for further explanation.

Figure 5

Influence of NIC, ACE and CLO on rat ABP. (A) Representative raw traces of ABP following ACE or CLO infusion (red arrow with doses used) compared to the CTRL group. As expected, protocol assessment molecules such as AngII (1.05 × 10${}^{-5}$ mg·kg${}^{-1}$) and ACh (0.5 × 10${}^{-5}$ mg·kg${}^{-1}$) induce increased and decreased ABP, respectively. Neither DMSO 0.5–3% (VEH) nor ACE or CLO affected ABP, whatever the dose tested. (B) Box and whisker plots of $\Delta$ABP data in the CTRL, CLO and ACE groups following i.v. injections, 0.93 mg·kg${}^{-1}$ for ACE and 1 mg·kg${}^{-1}$ for CLO. (C) Representative raw traces of ABP following infusions of NIC alone or NIC with either ACE or CLO. NIC was used at 0.33 mg·kg${}^{-1}$ (red arrow) in combination with a single dose of ACE (0.09 mg·kg${}^{-1}$) or CLO (0.33 mg·kg${}^{-1}$). AngII, ACh and VEH infusions triggered BP response as expected. In addition, 0.33 mg·kg${}^{-1}$ NIC induced a BP increase as expected, while its combination with either ACE or CLO was responsible for a biphasic response with low (o) and high (•) ABP. (D) Box and whisker plots of $\Delta$ABP data in the NIC, NIC+ACE and NIC+CLO groups following the various infusions cited above. Whiskers are minimum to maximum values and bar represents the median value. Different lowercase letters and § above the graphs indicate significant differences between treatments according to Tukey’s multiple comparison post hoc test (p < 0.05).

Figure 6

Homology modeling of rat $\alpha$3$\beta$4 nAChR and molecular docking of NIC, CLO and ACE. (A,B) Model of the receptor viewed from the membrane plane (A) and from the extracellular domain (B). The protein is shown in cartoon representation with a different color code for each polypeptide. The position of the membrane is represented by spheres. (C–E) Graphical representations of the orthosteric site of rat $\alpha$3$\beta$4 nAChR. The $\alpha$3$\beta$4 subunit interface in complex with: NIC (C); CLO (D); or ACE (E).