Pharmacological Characteristics of Insect Nicotinic Acetyicholine Receptor with Its Ion Channel and the Comparison of the Effect of Nicotinoids and Neonicotinoids

J. Pesticide Sci. 20, 57-64 (1995)

Original Article

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 vertebrate 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 acetamiprid bound selectively to the ACh recognition site. In houseflies, nicotine and imidacloprid 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 containing 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. Further- (6), trimethaphane (7), dimethylphenylpipera- more, the authors found that [3H]PCP binding zinium (DMPP; 9), d-tubocurarine (11), car- was strongly inhibited by nicotine. PCP and bachol (12), cytisine (13), l- and d-nicotine (14, 60 日本農薬学会誌第2o巻第1号平成7年2月

15), anabasine (16), a-BGT (17), imidacloprid DMPP (9), carbachol (12), cytisine (13), a- (18), 6-C1-PMNI (19), acetamiprid (20) and BGT (17), imidacloprid (18), 6-Cl-PMNI (19), nitenpyram (22). Ligands that had low acetamiprid (20), NMTHT (21) and MTENI affinity were chlorpromazine (3), ketamine (4), (23) (Table 1). mecamylamine (5), coniine (8), nereistoxin (10), In case of Torpedo nAChR, using [3H]a-BGT NMTHT (21) and MTENI (23). Ligands that and [3H]PCP as the probes, ligands can be had higher affinity to the [3H]PCP binding site properly classified into agonists, competitive were PCP (1), TCP (2), chlorpromazine (3), blockers and noncompetitive blockers. How- ketamine (4), mecamylamine (5), coniine (8), ever, as indicated above, this system cannot be nereistoxin (10), d-tubocurarine (11), l- and d- applied to the insect nAChR. Thus, the nicotine (14, 15), anabasine (16), and niten- ligands are classified into the following three pyram (22), while ligands that had lower types: affinity were lobeline (6), trimethaphan (7), 1) Those having higher affinity to the [3H]a-BGT binding site than to the [3H]PCP Table 1 Effect of various ligands on the binding binding site: lobeline (6), trimethaphan (7), of [3H]PCP and [3H]cc-BGT to the membranes DMPP (9), carbachol (12), cytisine (13), a-BGT from the honeybee heads. (17), imidacloprid (18), 6-Cl-PMNI (19) and acetamiprid (20). 2) Those having higher affinity to the [3H]PCP binding site than to [3H]a-BGT binding site: chlorpromazine (3), ketamine (4), mecamylamine (5), coniine (8) and nereistoxin (10). 3) Those having high affinity to both binding sites: PCP (1), TCP (2), d-tubocurarine (11), l- and d-nicotine (14, 15), anabasine (16) and nitenpyram (22). PCP has been recognized to bind to the allosteric site located in the ion channel of the nAChR in Torpedo electric organ and vertebrate muscle (based on binding study and electrophysiological studyl-3) and in insect central nervous system (based on electrophysiological study5). Therefore, further studies were undertaken on the unique prop- erty of the insect nAChR.

2. Effect of PCP on the Saturation Isotherm of [3H]a-BGT Binding to the Honeybee nAChR (Table 2) Scatchard analysis of saturation isotherms on the [3H]a-BGT binding with or without PCP and nicotine, showed the convergence of

a) Dimethylphenylpiperazinium. three lines on the x axis (KD values in the b) Pretreated with 10 a a-BGT for 30 min be- presence of PCP and nicotine were affected as fore the binding reaction was initiated by compared with the control, but Bmaxvalues in addition of [3H]PCP. the presence of PCP and nicotine were un- Values are means + S. D, of more than three affected as compared with the control), sug- experiments. Values in the parenthesis are in- gesting that PCP and nicotine interact with hibitory percent at indicated concentrations of the [3H]a-BGT binding site, which is the ACh tested ligands. recognition site on the insect nAChR.89 Journal of Pesticide Science 20 (1) February 1995 61

Table 2 Effect of l-nicotine and PCP on KD and Bmax values of [3H]a-BGT binding to the membranes from the honeybee heads.

Values are means + S. D. of three experiments.

3. The Saturation Isotherm of [3H]PCP Bind- Fig. 2 Scatchard plots of [3H]PCP binding to ing to the Honeybee nAChR the membranes from the honeybee heads in the Previous study on the Torpedo nAChR sug- absence and the presence of 10 mat carbachol. gested that it had one binding site for [3H]- Symbols are means of three or four experiments. PCP. 3 However in honeybee nAChR, a convex curve (Fig. 2) and a Hill coefficient lower than 1.00 were obtained for [3H]PCP Table 3 KD and Bmax values of [3H]PCP binding to the membranes from the honeybee heads. binding, suggesting that there are two binding sites of high and low affinities for [3H]PCP (Table 3). Also, from the displacement curve of PCP against [3H]a-BGT binding (Fig. 1A), the ratio between concentrations of PCP at 1000 and 90% binding was calculated to be 660-fold, Hill coefficient (n)-0.8020.043. Values are suggesting the presence of at least two binding means + S. D. of four experiments. sites for PCP.

4. Characterization of [3H]PCP Binding Sites eliminated the low affinity binding (Fig. 2, in the Honeybee nAChR Table 4). It suggested that possibly only the [3H]PCP binding in the presence of 10mM low affinity site bound carbachol or cytisine. carbachol or 1 mM cytisine, which selectively In addition, the KD value of the low affinity binds to the receptors [3H]a-BGT binding site, site (1.7uM) was close to the K, value for PCP

Table 4 Effect of various ligands on KD and Bmax values of [3H]PCP binding to the membranes from the honeybee heads.

a) Hill coefficients. Values are means + S. D. of three experiments. Scatchard analysis of saturation isotherms on the binding of [3H]PCP in the presence of 10mar carbachol with or without coniine, mecamylamine, nereistoxin and 1- nicotine showed the covergence of lines to a point on the x axis. 62 日本農薬学会誌第20巻第1号平成7年2月

not only with the ACh recognition site, but also with the high affinity site of [3H]PCP binding in the honeybee nAChR (Fig. 3, Table 4).

5. I nsecticidal A ctivity and Symptoms of Toxicity (Table 5) PCP or TCP were as effective as nicotine in terms of insecticidal activity by topical application to houseflies and in potency in inhibiting [3H]PCP and [3H]a-BGT binding to the honeybee nAChR (Table 1). However, the Fig. 3 Scatchard plots of [3H]PCP binding to symptoms of toxicity by PCP or TCP were the membranes from the honeybee heads in the quite different from that of nicotine. Nicotine presence of 10mM carbachol, and 10mM car- induced convulsions with leg tremor and bachol plus 1LM l-nicotine. motion of wings, followed by paralysis at lower Symbols are means of three experiments. doses, but immediate paralysis at higher doses. On the other hand, PCP and TCP induced anesthetic effect similar to that of (2.01M) for [3H]a-BGT binding (Tables 1 and ether, and houseflies returned to normal at 3). These results suggested that the low lower doses within 5-6 hr. PCP is known as affinity binding site of [3H]PCP may be the a general anesthetic and hallucinogen to men ACh recognition site on the insect nAChR. and thus it is not surprising that PCP and Furthermore, after inhibiting the low af- TCP may also function as anesthetics in the finity [3H]PCP binding site with 10mM insect. Nereistoxin, which is known as a carbachol binding, mecamylamine, coniine or competitive blocker of the insect nAChR, 1Q nereistoxin (known to bind to the allosteric suppressed movement of houseflies as seen site in the ion channel of the vertebrate with cartap on the cockroach20 but showed leg nAChR8,18, 18 could still bind to the high tremor at higher doses (>2pg/female). affinity [3H]PCP binding site in the honeybee Nereistoxin had higher affinity to the [3H]PCP nAChR (Table 4). Thus, it is plausible that binding site than [3H]a-BGT binding site of the high affinity site of [8H]PCP binding may the honeybee nAChR (Table 1). These results correspond to the allosteric site in the ion suggest that nereistoxin interacts both with the channel of the nAChR in the insect brain. It ACh recognition site, as a partial agonist, and is interesting to note that nicotine interacts with the allosteric site located in the receptors

Table 5 Insecticidal activity and symptomatology of toxicity of various ligands against adult housefly (Musca domestics L.).

a) By injection. LD50 values are means S. D, of three experiments. Journal of Pesticide Science 20 (1) February 1995 63 ion channel as a noncompetitive blocker in the brate nAChR, imidacloprid, 6-Cl-PMNI and insect. This is similar to its action on verte- acetamiprid were weak agonists as shown by brate nAChR. 3, 6,2 binding assay, 3 and imidacloprid was also an The application of imidacloprid to houseflies agonist on the cockroach ganglia as shown by by injection induced immediate convulsion electrophysiological assays., with leg tremor and wing motion, followed by Nitenpyram, which is regarded as one of paralysis. The symptoms were similar to neonicotinoids based on its chemical structure, those by nicotine and on cockroach. 20 Imi- had binding properties that were different from dacloprid has been shown to act as an agonist those of imidacloprid, 6-Cl-PMNI and acet- on the Torpedo nAChR using binding assay3 amiprid (Table 1). In Torpedo, it was a very and on the cockroach ganglia nAChR using weak agonist and also a weak ion channel electrophysiological assay. 10 It is interesting blocker. 3 In honeybee brain, it was higher to note that the intoxication symptoms pro- in inhibition of [3H]PCP binding than in induced by imidacloprid corresponded to what hibition of [3H]a-BGT binding. This is con- is expected from the radioligand binding and trary to the action of other neonicotinoids. electrophysiological studies. NMTHT and MTENI had low affinity for both the ACh recognition and ion channel sites in 6. Differences in the Effects of Nicotinoids and the insect nAChR (Table 1), although they Neonicotinoids were weak agonists as well as weak ion channel Schematic representation of the interaction blockers in the Torpedo nAChR. 3 of nicotinoids and neonicotinoids with the In conclusion, the pharmacological charac- insect nAChR and its ion channel is shown in teristics of the insect brain nAChR are dif- Fig. 4. The possible sites of action of nico- ferent from those of vertebrate skeletal muscle tinoids, as well as PCP, are both the ACh and Torpedo electric organ nAChRs. The recognition and the allosteric sites, whereas allosteric site in the nAChR ion channel can be imidacloprid, 6-Cl-PMNI and acetamiprid, a possible target site for certain compounds. which are regarded as neonicotinoids, had far Nicotine is regarded as a typical agonist in higher affinity for the ACh recognition site than vertebrates, but has almost equal affinities for the allosteric site (Table 1). On the verte- both the ACh recognition and allosteric sites

Fig. 4 Schematic representation of the interaction of nicotinoids and neonicotinoids with the nicotinic acetylcholine receptor and its ion channel from the honeybee heads. 64 日本農薬学会誌第20巻第1号平成7年2月 in the insect nAChR. Such effects should be 107, 220 (1980) considered in the interpretation of toxicity 18) W. A. Varanda, Y. Aracava, S. M. Sherby, W. G. VanMeter, M. E. Eldefrawi & E. X. symptoms and neurophysiological results. Albuquerque: Mol. Phaymacol. 28, 128 (1985) REFERENCES 19) M. Sakai: Botyu-Kagaku 32, 21 (1967) 20) S. Sone, K. Nagata, S. Tsuboi & T. Shono: J. 1) R. S. Aronstam, A. T. Eldefrawi, I. N. Pessah, Pesticide Sci. 19, 69 (1994) J. W. Daly, E. X. Albuquerque & M. E. 21) A. T. Eldefrawi, N. M. Bakry, M. E. Eldefrawi, Eldefrawi: J. Biol. Chem. 256, 2843 (1981) M. -C. Tsai & E. X. Albuquerque: Mol. 2) M. E. Eldefrawi, A. T. Eldefrawi, R. S. Phaymacol. 17, 172 (1980) Aronstam, M. A. Maleque, J. E. Warnick & E. X. Albuquerque: Proc. Natl. Acad. See. 要約 USA 77, 7458 (1980) 3) M. Tomizawa, H. Otsuka, T. Miyamoto & I. 昆虫ニコチン性アセチルコリン受容体/イオン Yamamoto: J. Pesticide Sci. 20, 49 (1995) チャンネル複合体の薬理的性質とこれらに対す 4) D. B. Sattelle & J. A. David: Neurosci. Lett. 43, 37 (1983) るニコチノイドおよびネオニコチノイドの作用 5) D. B. Sattelle, B. Hue, M. Pelhate, S. M. Sherby, の相違 A. T. Eldefrawi & M. E. Eldefrawi: J. Insect 冨澤元博, 大塚博子, 宮本徹 Physiol. 31, 917 (1985) Mohyee E. ELDEFRAWI, 山本出 6) J. A. David, P. J. Crowley, S. G. Hall, M. Battersby & D. B. Sattelle: J. Insect Physiol. ミツバチ頭部膜画分を用い, [3H]α-ブンガロトキシ 30, 191 (1984) ン(α-BGT)および[3H]フェンサイクリジン(PCP) 7) A. T. Eldefrawi, N. Shaker & M. E. Eldefrawi: "Neuropharmacology of Insects," (Ciba Founda- をプローブとしたラジオレセプターアッセイにより各種リガンドの作用を検討した結果, 昆虫ニコチン性アセチ tion Symposium 88), ed. by D. Evered, M. OConnor & J. Whelan, Pitman, London, pp. ルコリンレセプター(nAChR)/イオンチャンネル複合 137-152, 1982 体の薬理的性質はシビレエイのそれと大ぎく異なるこ 8) D. B. Sattelle, I. D. Harrow, B. Hue, M. Pelhate, とを認めた. すなわち昆虫では供試したコリン作動性ア J. I. Gepner & L. M. Hall: J. Exp. Biol. 107, ゴニストは[3H]PCP結合を増加させず, また脊椎動物 473 (1983) 9) S. C. R. Lummis & D. B. Sattelle: Comp. でイオンチャンネル内のアロステリック部位にのみ結合 Biochem. Physiol. 80C, 75 (1985) するPCPが[3H]α-BGT結合をも阻害した. [3H]α- 10) D. Bai, S. C. R. Lummis, W. Leicht, H. Breer & BGT結合の飽和実験の結果は, ニコチン, PCPが α- D. B. Sattelle: Pestic. Sci. 33, 197 (1991) BGTと同様アセチルコリン(ACh)認識部位に結合す 11) M. Tomizawa & I. Yamamoto: J. Pesticide Sci. 17, 231 (1992) ることを示したが, さらにニコチンは[3H]α-BGT結 12) M.-Y. Liu & J. E. Casida: Pestic. Biochem. 合のみならず[3H]PCP結合をも強く阻害した. [3H]- Physiol. 46, 40 (1993) PCPはシビレエイでは単一部位に結合したが, 昆虫で 13) M. Tomizawa & I. Yamamoto: J. Pesticide Sci. 18, 91 (1993) は高親和性部位と低親和性部位の二つの結合部位が存在 14) I. Yamamoto & M. Tomizawa: "Pesticides/En- し, 前者はイオンチャンネル内のアロステリック部位, vironment: Molecular Biological Approaches," 後者はACh認識部位に相当することを推定した. ニコ ed. by T. Mitsui, F. Matsumura & I. Yamaguchi, チン, アナバシン, ニテンピラムはACh認識部位とア Pesticide Science Society of Japan, Tokyo, pp. ロステリック部位の双方に結合する一方, イミダクロプ 67-83, 1993 15) A. Kalir, H. Edery, Z. Pelah, D. Balderman & リド, 6-C1-PMNI, アセトアミプリドはACh認識部 G. Porath: J. Med. Chem. 12, 473 (1969) 位に選択的に結合した. イエバエに対してPCPはニコ 16) S. M. Sherby, A. T. Eldefrawi, J. A. David, チンと同程度の殺虫力を示したが, 中毒症状は異なり, D. B. Satelle & M. E. Eldefrawi: Arch. Insect. ニコチンやイミダクロプリドは興奮作用を示したが, Biochem. Physiol. 3, 431 (1986) 17) P. J. Munson & D. Rodbard: Anal. Biochem. PCPは麻酔作用を示した.