J. Pesticide Sci. 20, 57-64 (1995) Original Article Pharmacological Characteristics of Insect Nicotinic Acetyicholine Receptor with Its Ion Channel and the Comparison of the Effect of Nicotinoids and Neonicotinoids Motohiro TOMIZAWA, Hiroko OTSUKA, Toru MIYAMOTO, Mohyee E. ELDEFRAWI* and Izuru YAMAMOTO Department of Agricultural Chemistry, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156, Japan *Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, MD 21201, U. S. A. (Received July S, 1994; Accepted September 26, 1994) Using radioreceptor assay with [3H]ce-bungarotoxin (a-BGT) and [3H]phencyclidine (PCP) as probes for the nicotinic acetylcholine receptor (nAChR) in membranes obtained from honeybee heads, the effects of various nAChR ligands, nicotinoids and neonicotinoids were studied. The data indicated differences in pharmacological characteristics between Torpedo electric organ and honeybee brain nAChRs. [3H]a-BGT binds to the acetylcholine (ACh) recognition site of the nAChRs of vertebrate skeletal muscle, Torpedo electric organ and honeybee brain. In vertebrates, [3H]PCP binds to an allosteric site on the receptors ion channel and its binding is stimulated by receptor activation with agonists. However, the tested vertebrate cholinergic agonists inhibited [3H]a-BGT binding, but did not activate [3H]PCP binding to the honeybee nAChR. Saturation isotherms of the binding of [3H]a-BGT with or without PCP indicated that PCP interacted with the ACh recognition site on the nAChR. Furthermore, nicotine inhibited not only [3H]a-BGT binding but also [3H]PCP binding. Detailed study of [3H]PCP binding indicated that [3H]PCP bound to the honeybee brain membranes both at high and low affinity sites. The former corresponded to the verte- brate allosteric site on the nAChR and the latter to the ACh recognition site. Nicotine, anabasine and nitenpyram bound to both sites, while imidacloprid, 6-Cl-PMNI and acet- amiprid bound selectively to the ACh recognition site. In houseflies, nicotine and imida- cloprid produced excitation followed by paralysis, while PCP was anesthetic, even though PCP was as insecticidal as nicotine. was also demonstrated by the voltage-depend- INTRODUCTION ent blockade of the ACh-induced current by Pharmacological characteristics of the nico- historionicotoxin, 4 PCP5 and the analog of tinic acetylcholine receptor (nAChR) has fire ant venom, cis-2-methyl-6-undecylpiperi- been studied in Torpedo electric organ by using dine. 6 However, unlike binding to the [3H]perhydrohistorionicotoxin (H12-HTX)1 and Torpedo nAChR, the binding of [3H]H12-HTX [3H]phencyclidine (PCP)23 as probes of the and [3H]PCP to the insect nAChRs, was not allosteric site located in the receptors ion activated by cholinergic agonists. 7 Also, the channel. The presence of the allosteric site in binding of [3H] or [125I]a-bungarotoxin (a- the ion channel of the nAChR of the cockroach BGT), which binds to the ACh recognition site (Periplaneta americana) central nervous system of insect nAChRs, 89 was almost unaffected by 58 日本 農 薬 学 会 誌 第20巻 第1号 平 成7年2長 H12-HTX or PCP. 57 studies.) Other tested ligands were pur- Recently developed novel insecticides, imi- chased from Sigma Chemical or Wako Pure dacloprid and related compounds, were shown Chemical. to interact with the insect nAChR. IO-12 The similarity between imidacloprid related com- 2. nAChR Preparation from the Honeybee pounds and nicotinoids was indicated by Heads 16) structure-activity relationships. The term Honeybee (Apis mellifera) heads collected by neonicotinoids was proposed to cover imi- sieving of liquid nitrogen frozen honeybees dacloprid related compounds. 14 The effects were homogenized three times in ten volumes of neonicotinoids on the binding of [H] PCP to of 50mM Na2HPO4-HC1 buffer, pH 7.4, con- the nAChR in Torpedo electric organ were taining 320mM sucrose and 1mM EDTA in compared with that of nicotinoids. It was Polytron (setting 7) for 30 sec each with a 60 shown that neonicotinoids were weaker nAChR sec rest. The homogenate was filtered through agonists than nicotinoids. In the present four layers of cheesecloth to remove the de- study, the authors also found that the binding bris. The filtrate was centrifuged at 1000xg properties of [H] PCP to the nAChR from the for 10min, and the supernatant was centri- honeybee head were quite different from those fuged at 40,000xg for 30min. The pellet was to the Torpedo nAChR. Therefore, the phar- suspended in 1 mM Na2HPO4-HC1 buffer, pH macological characteristics of insect nAChR 7.4 (1g head/ml), and the suspension was with its ion channel were studied in detail frozen by immersion in a dry ice-methanol using various nAChR ligands, nicotinoids and mixture, lyophilized and stored at -80C. neonicotinoids. The lyophilized membranes contained ca. 0.3 mg protein /mg membranes. MATERIALSAND METHODS 1. Materials 3. [H]PCP Binding [Piperidyl-3, 4-H(N)]phencyclidine ([H] - The nAChR preparation (ca. 0.3mg protein PCP; 52 Ci/mmol), and N-[propionyl-H]pro- assay) was incubated at 25C for 30min (for pionylated a-bungarotoxin ([H]a-BGT; 68 saturation isotherm experiment) or 40 sec (for Cifmmol) were purchased from New England other experiments) with 5nM of [H]PCP and Nuclear Research Products and Amersham, with or without tested ligand at appropriate respectively. concentrations, in a total volume of 250 al of PCP and N- [1-(2-thienyl)cyclohexyl]piperi- 20 mM Tris-HC1 buffer, pH 7.4 containing 0.1 dine (TCP) were synthesized by the method of mM diisopropylfluorophosphate (to inhibit Kalir et al. and were handled under the acetylcholinesterase) and 0.02% sodium azide. license issued by governor of Tokyo metro- The reaction was terminated by rapid filtra- polis. tion on the GF JB filter (Whatman) that had 1-(6-Chloro-3-pyridylmethyl)-2-nitroimino- been presoaked in 0.05% polyethyleneimine. imidazolidine (imidacloprid), 1-(6-chloro-3-py- The GF/B filter was rinsed three times with 2. 5 ridylmethyl)-2-nitromethylene-imidazolidine ml of 0.9% NaCI and transferred into a count- (6-C1-PMNI) and 2-(nitromethylene)-tetra- ing vial. The radioactivity was counted after hydro-l, 3-thiazine (NMTHT) were obtained overnight incubation in 4 ml of scintillation from Nihon Bayer Agrochem. 1-[N-(6-Chloro- cocktail (Ready Protein, Beckman). Specific 3-pyridylmethyl)-N-ethyl]amino-1-methyl- binding was defined as the difference in radio- amino-2-nitroethene (nitenpyram) was ob- activity on the filter in the absence and tained from Takeda Chemical. N1-[(6-Chloro- presence of 100/GMunlabeled PCP. 3-pyridyl) methyl]-N2-cyano-N1-methylacet- amidine (acetamiprid) was obtained from 4. [H]c-BGT Binding Nippon Soda. l-Methylthioethyl-2-nitrometh- The nAChR preparation (ca. 0.4-0.5mg ylene-imidazolidine (MTENI) was obtained protein/assay) was incubated at 25C for 60 from Mitsui Toatsu Chemicals. Trimethaphan min with 2nM of [H]a-BGT, with or without camsylate was available from our earlier the tested ligand. The total volume was 250 Journal of Pesticide Science 20 (1) February 1995 59 ul of 20mM Na2HPO4-HC1buffer, pH 7.4 con- taining 1mM EDTA and 0.1mM phenyl- methylsulfonyl fluoride. Filtration and count- ing were the same as used for [3H]PCP binding. Specific binding was defined as the difference in counts in the absence and the presence of 2uM unlabeled a-BGT. 5. Data Analysis Saturation isotherms were obtained by dis- placing 10nM of [3H]PCP or 2nM of [8H]a- BGT binding with unlabeled PCP or a-BGT, respectively (at various concentrations), and analyzed by iterative nonlinear least-squares B regression using the LIGAND program with McPhersons modification for an IBM personal computer (Biosoft, Cambridge, U. K.). IC5o values (the molar concentration necessary for inhibiting 50% of specific binding) were deter- mined by iterative nonlinear least-squares regression using the SIGMAPLOT program (Jandel Scientific CA, U. S.A.). Inhibition constant (K;) was calculated by the equation: K1=IC5o/l+[L]/KD, where [L] is a concentration of the added Fig. 1 Effect of l-nicotine and PCP on the radiolabelled ligand and KD is a dissociation binding of [3H]a-BGT (A) and [3H]PCP (B) to the constant from a saturation isotherm. membranes from the honeybee heads. Symbols are means of three experiments. 6. Insecticidal Activity and Symptomatology One microliter of the compound in acetone TCP which are known as the noncompetitive was topically applied on the notum of 3-4 blockers of the nAChRs from the Torpedo days old female housefly (Musca domestica). electric organ and vertebrate muscle-8 gave Administration of imidacloprid was carried out the same effect. These ligands also strongly by intrathoracic injection of 0.22sl in 25% inhibited [8H]cc-BGTbinding to the honeybee dimethyl sulfoxide solution in water. Symp- nAChR (Fig. 1, Table 1). The same results toms of intoxication were observed and mor- were shown on the binding of both radio- talities were recorded after 24hr at 27+2C ligands using fresh membranes from the and 60% relative humidity. housefly heads. The IC50 values of PCP and 1-nicotine for [8H]a-BGT binding were 7.05+ RESULTSAND DISCUSSION 1.13, uM (n=3) and 3.30+0.62M (n=2), re- 1. Effect of Various Ligands on the Binding of spectively, and the IC5o values of PCP and [3H]PCP and [8H]a-BGT to the Honeybee 1-nicotine for [3H]PCP binding were 0.0562+ nAChR 0.00971uM (n=3) and 38.5+2.11uM (n=2), Eldefrawi et al. 7 indicated that [3H]Hi2- respectively. HTX or [8H]PCP binding to the housefly In case of the honeybee head nAChR, ligands nAChR was not increased by the presence of that had a high affinity to the [8H]a-BGT any vertebrate cholinergic ligand. The same binding site were PCP (1), TCP (2), lobeline was observed with honeybee nAChR.