Actions of quinolizidine alkaloids on Periplaneta americana nicotinic acetylcholine receptors

BACKGROUND: Botanical insecticides do not play a major role as crop protectants, but they are beneficial in some applications. The authors investigated the actions of naturally occurring alkaloids on insect nicotinic acetylcholine (ACh) receptors (nAChRs) by evaluating their abilities to inhibit specific binding of [³H]imidacloprid (IMI) to nerve‐cord membranes from Periplaneta americana L. Two alkaloids were also tested for their actions on nAChRs expressed by cockroach neurons using patch‐clamp electrophysiology. RESULTS: Four natural quinolizidine alkaloids (matrine, sophocarpine, cytisine and aloperine) exhibited more than 50% inhibition of [³H]IMI binding at 10 µM , although other compounds were found to have no or low inhibitory activity. The rank order of potency based on concentration–inhibition curves was cytisine > sophocarpine ≥ aloperine ≥ matrine. Patch‐clamp analysis indicated that sophocarpine and aloperine were not agonists of nAChRs expressed in P. americana neurons, yet, at 10 µM , aloperine, but not sophocarpine, suppressed ACh‐induced inward currents significantly. CONCLUSION: Three of the four natural alkaloids tested possess structural moieties that are necessary for interaction with P. americana nAChRs. Aloperine, which possesses a unique structure and showed a distinctive dose–response curve, was found to act as an antagonist. Appropriate modifications of these alkaloids might result in novel insecticidal nAChR ligands. Copyright © 2008 Society of Chemical Industry     

Nicotinic acetylcholine receptor chimeras of rat α7 and Drosophila SAD reveal species-specific agonist binding regions

Species-specific agonist binding regions of nicotinic acetylcholine receptors (nAChR) were examined. Imidacloprid and physostigmine (Phy) selectively activated insect nAChR composed of Drosophila second alpha-like subunit (SAD) and chick β2, in contrast to rat α7 nAChR. The Phy-activated currents were α-bungarotoxin (α-BGT) sensitive, suggesting activation at the agonist binding loop C. Several SAD-α7 chimeras were constructed, by switching agonist binding regions, and expressed in oocytes. Though none of the chimeras was activated by a range of nicotinic agonists, [¹²⁵I]α-BGT binding revealed homomeric assembly of all chimeric cDNAs. Phy differentially displaced [¹²⁵I]α-BGT from the nAChR chimeras, suggesting that the β subunit is not involved in Phy binding, and that Phy targets the insect agonist binding loop C.     

Desnitroimidacloprid and Nicotine Binding Site in Rat Recombinant α4β2 Neuronal Nicotinic Acetylcholine Receptor

Desnitroimidacloprid (desnitro-IMI) is proposed to be a bioactivation product of imidacloprid and to bind at the same site as [³H]nicotine in the nicotinic acetylcholine receptor (nAChR) of mouse brain membranes. The α4β2 nAChR subtype accounts for >90% of the binding sites for nicotine in rat brain. This study further characterizes the binding site for [³]desnitro-IMI and [³H]nicotine in rat recombinant α4β2 nAChR using receptor expressed in Sf9 insect cells so that the assays involved no other receptor subtypes or interference from metabolic activation and detoxification systems. The two radioligands gave the same B_max of 7.5 pmol/mg protein and apparent K_d values of 3.3 nM for nicotine and 8.9 nM for desnitro-IMI by Scatchard analysis at 22°C. However, at 4°C, the observed apparent association rate is slower and dissociation rate is faster for [³H]desnitro-IMI than for [³H]nicotine and due to the rapid rate of dissociation of [³H]desnitro-IMI the K_d calculated from the determined association and dissociation rates more closely approximates 1.0 for both ligands. Eight cholinergic agents and nine nicotinoids are equipotent in displacing [³H]desnitro-IMI and [³H]nicotine, with IC₅₀ values (nM) of 0.5 for epibatidine, 1 for cytisine, 4–6 for nicotine and desnitro-IMI, 15 for acetylcholine, and 155 for imidacloprid, with an overall correlation for inhibitor potencies of _r² = 0.99 (n = 17). This correlation of binding site properties extends to [³H]nicotine in the recombinant α4β2 receptor and rat brain membranes (r² = 0.99, n = 12). Thus, desnitro-IMI and nicotine bind with high affinity to the same site in rat recombinant α4β2 neuronal nAChR. This recombinant receptor can be generated in sufficient quantities for high-throughput target site screening and structural analysis of the binding site.     

GABA receptors of insects susceptible and resistant to cyclodiene insecticides

Toxicity tests revealed up to 40-fold resistance to a number of cyclodiene insecticides in a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica L.). Using [³⁵S] TBPS as a probe for convulsant sites in insects, saturable specific binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of[³⁵S] TBPS binding data to CSMA and CYW membranes failed to provide evidence for significant differences between the two strains in either the affinity (K_d) or density (B_max) of saturable binding sites. For several polychlorocycloalkane insecticides, the ligand displacement profile of [³⁵S] TBPS binding was almost identical for the CSMA and CYW houseflies. Therefore, using [³⁵S] TBPS as a probe for convulsant sites, a 40-fold resistance to cyclodienes in the CYW housefly strain cannot be accounted for only in terms of alterations in TBPS binding sites.     

Toxicity and selective mechanism of imazamox by weedy rice and imidazolinone-resistant rice in China

Weedy rice (Oryza spp.) is a notorious weed in direct-seeding paddy fields. Because it has anatomical and physiological traits similar to those of cultivated rice, no selective herbicide is effective in controlling weedy rice growing among conventional rice cultivars. Imidazolinone (IMI)-resistant rice lines JD372 and JJ818 have been planted with imazamox to control weedy rice in Jiangsu and Shanghai, China. Whole-plant dose–response analysis showed that imazamox exhibited high efficacy against three populations of weedy rice. The ED₉₀ of weedy rice populations FN-5, GY-8, and HY-3 were 46.87, 61.43, and 52.17 g a.i. ha⁻¹, respectively, close to the recommended field dose (50 g a.i. ha⁻¹) of imazamox. Conversely, the ED₁₀ values of JD372 and JJ818 were slightly lower than 50 g a.i. ha⁻¹. These findings indicate that imazamox can control weedy rice production in JD372 and JJ818 fields. The acetolactate synthase (ALS) sensitivity of JD372 in vitro was 1714.89-fold lower to imazamox than was that of FN-5. ALS gene sequencing revealed a Ser653Asn point mutation—a common mutation that confers resistance to IMI herbicides in JD372. In addition, higher ALS expression levels in JD372 were found at 24 and 72 h after imazamox treatment. ALS overexpression might partially compensate for the ALS activity of JD372 that was suppressed by imazamox.     

Response of Arabidopsis thaliana to 22 ALS inhibitors: baseline toxicity and cross-resistance of csr1-1 and csr1-2 resistant mutants

The baseline toxicity of 22 acetolactate synthase (ALS)-inhibiting herbicides and the cross-resistance patterns of chlorsulfuron- and imazapyr-resistant (R) lines on these 22 ALS-inhibiting herbicides were investigated using the model species Arabidopsis thaliana. The 22 herbicides consisted of 18 sulfonylureas (SU), three imidazolinones (IMI) and one triazolopyrimidine (TP). The ED₅₀ values (doses of herbicides required to reduce dry matter by 50%) of the post-emergence-treated Col and Ler susceptible (S) lines ranged from 22 to 4822 mg ha⁻¹ and from 17 to 3143 mg ha⁻¹ respectively. The csr1-1 chlorsulfuron-resistant line (substitution of Pro₁₉₇ to Ser) conferred a high resistance to the only TP tested as well as to nine SU herbicides (R:S ratio ≥30), a low resistance to two SU herbicides (R:S≥5 and <30) and little or no resistance to the three IMI and seven other SU herbicides (R:S <5). This result contradicts the expectation that an ALS mutation selected by an SU herbicide confers high cross-resistance to other SU herbicides. We found that the efficacy of specific ALS inhibitors was different for different species and therefore could not be predicted from our results with A. thaliana; however, the cross-resistance patterns in A. thaliana were highly correlated with cross-resistance patterns in unrelated species with the same resistance mutation. These results have implications for resistance management.     

Binding Sites for Ca2+-Channel Effectors and Ryanodine in Periplaneta americana—Possible Targets for New Insecticides

The calcium channel and the ‘calcium release channel’ of muscle membrane of the cockroach Periplaneta americana have been characterized. Biological assays with calcium channel blockers and ryanodine on different insects and acari revealed pronounced insecticidal effects with ryanodine, but not with calcium channel blockers, at concentrations between 0·1 and 300 μg ml⁻¹. Skeletal muscle membranes derived either from the tubular network or from the sarcoplasmatic reticulum of P. americana were characterized with respect to the binding of the dihydropyridine (DHP) [³H]isradipine (PN 200-110), the phenyl-alkylamine [³H]verapamil and the alkaloid [³H]ryanodine. Preliminary binding studies with the benzothiazepine [³H]diltiazem suggest a low-affinity binding site with a IC₅₀ value of 3·3 μM . All binding sites tested were sensitive to treatment with proteinase K. Optimal conditions for binding of the radioligand ryanodine revealed the highest specific binding at pH 8 and at calcium chloride concentrations between 100 and 500 μM . EGTA at 10 μM abolished 95% of the ryanodine binding. Binding studies with calcium channel binding sites revealed a pronounced effect of low Ca²⁺ concentrations on specific isradipine binding, whereas verapamil and diltiazem binding were only reduced by the presence of 200 μM EGTA. With respect to high Ca²⁺ concentrations, specific binding of diltiazem, isradipine and verapamil was reduced by 73, 40 and 20%, respectively, at 5 mM Ca²⁺. Radioligand binding experiments showed high-affinity binding sites for ryanodine and isradipine. K_D values of 0·95 nM (B_max=550 fmol mg⁻¹ protein) and 0·75 nM (B_max=213 fmol mg⁻¹ protein) were determined respectively. A lower-affinity binding site was identified in binding studies with verapamil (K_D=7·4 nM and B_max=27 fmol mg⁻¹ protein). [³H]isradipine displacement studies with several dihydropyridines revealed the following ranking of affinity: nitrendipine>isradipine>Bay K8664≪nicardipine. Displacement of [³H]verapamil binding by effectors of the phenylalkylamine binding site showed that bepridil and S(-)verapamil had the highest affinities of the compounds tested followed by (±)verapamil, nor-methylverapamil and R(+)verapamil.     

Heterocyclic Insecticides Acting at the GABA-Gated Chloride Channel: 5-Alkyl-2-arylpyrimidines and -1,3-thiazines

5-tert-Butyl-2-(4-ethynylphenyl)pyrimidine and the corresponding 2,5-disubstituted-4H-1,3-thiazine block the GABA-gated chloride channel at c.20and c.200 nm , respectively, measured as 50% inhibition of the binding of 1-(4-ethynylphenyl)-4-[³H]propyl-2,6,7-trioxabicyclo[2.2.2]octane (4′-ethynyl-4-n-[³H]propylbicycloorthobenzoate; [³H]EBOB) in house fly and mouse brain membranes, and they are also toxic to topically-treated flies with LD₅₀ values of 6–27 μg g⁻¹ alone and 2–6 μg g⁻¹ with piperonyl butoxide (PB) as synergist. In the pyrimidine series, the general pattern of effectiveness of substituents in the 5-position is tert-butyl>isopropyl≈cyclohexyl≈cyclopropyl>methyl, phenyl and 3- and 4-fluorophenyl, and in the 2-position is 4-ethynylphenyl≪4-bromophenyl. These planar pyrimidines and nearly-planar 4H-1,3-thiazines with 2-ethynylphenyl or 2-bromophenyl and 5-tert-butyl or 5-isopropyl substituents are more effective than the corresponding 6H-1,3-thiazine, 6-oxo-1,3-thiazines and 4,6-dioxo-1,3-thiazine examined, but they are less active than the analogous conformationally flexible trans-1,3-dioxanes and -1,3-dithianes. The heterocyclic moiety confers a region of high electron density and positions the 2- and 5-substituents in a linear or parallel relationship for optimal affinity at the receptor. Two observations indicate that the new pyrimidines and thiazines probably act as chloride channel blockers. First, the poisoning signs are identical to those of EBOB in both mice and house flies. Second, each of the pyrimidines, thiazines and dioxanes falls on the same correlation line for inhibition of [³H]EBOB binding and toxicity to house flies (with PB) as that obtained earlier for EBOB analogs, dithianes and polychlorocycloalkanes, suggesting that they all act at the same or closely coupled binding sites in the GABA-gated chloride channel.     

House fly head GABA-gated chloride channel: [3 H] α-Endosulfan binding in relation to polychlorocycloalkane insecticide action

This study attempts to use [³H] α-endosulfan to examine directly the binding site(s) for cyclodienes, lindane and toxaphene (collectively referred to as the polychlorocycloalkane or PCCA insecticides) in the 4-aminobutyric acid (GABA)-gated chloride channel. [³H] α-Endosulfan was prepared by reduction of hexachloronorbornenedicarboxylic anhydride with sodium borotritide, then coupling the labelled alcohol with thionyl chloride followed by HPLC purification (35 Ci mmol^?1, > 99% radiochemical purity). This new candidate radioligand readily partitions into lipid membranes and undergoes indiscriminate adsorption to surfaces resulting in high levels of non-specific binding. This makes it very difficult to differentiate the small portion of specific binding at the site relevant to toxic action. This problem was partially circumvented by incubating [³H] α-endosulfan (0.1 nM) with house-fly head membranes (0.2 mg protein) for 70 min at 22°C giving 23 (±4)% specific binding (40 fmol mg^?1 protein) determined as the difference between the radioligand alone and on preincubation for 15 min with unlabelled α-endosulfan (final concentration 100 nM). This procedure is not appropriate for determination of saturation isotherms and standard binding kinetics. However, the effectiveness of 16 PCCAs (also at 100 nM final concentration) in blocking the specific binding of [³H] α-endosulfan is generally consistent with their relative potencies as inhibitors of 4-[³H] propyl-1-(4-ethynylphenyl)-2,6,7-trioxabicyclo[2.2.2] octane ([³H]EBOB) binding suggesting that the binding site for both [³H]α-endosulfan and the PCCAs is part of the GABA-gated chloride channel. Insecticidal channel blockers of other types (e.g. picrotoxinin, trioxabicyclooctanes, a dithiane, and phenylpyrazoles) and GABA are poor inhibitors of [³H] α-endosulfan binding relative to their potencies as inhibitors of [³H] EBOB binding. It therefore appears that the PCCAs compete directly for the [³H] α-endosulfan site, whereas the other channel blockers bind with different inhibition kinetics or at a site more closely coupled to the EBOB than the α-endosulfan binding domain.     

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C_1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C₃ length and the 4-alkyl groups of C₄ length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC₅₀ = 45.2 nM ), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC₅₀ = 62.2 nM ). Their receptor selectivities (rat IC₅₀/housefly IC₅₀) were 52 and 0.038, respectively. The insecticidal activity (LD₅₀) of 20 active analogues was well correlated with their potency (IC₅₀) in inhibiting [³H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABA_A receptors. © 1999 Society of Chemical Industry     

Insecticidal activity and nicotinic acetylcholine receptor binding of dinotefuran and its analogues in the housefly, Musca domestica

The insecticidal activity of dinotefuran and 23 related compounds against the housefly, Musca domestica (L) was measured by injection with metabolic inhibitors. Dinotefuran was less active than imidacloprid and clothianidin by a factor of 10 in molar concentrations. Their binding activities to the fly-head membrane preparation were measured by using [125I]alpha-bungarotoxin ([125I]alpha-BGTX) and [3H]imidacloprid ([3H]IMI) as radioligands. The activity of some selected compounds measured with [3H]IMI was 10(4)-fold higher than that measured with [125I]alpha-BGTX. With [3H]IMI as a radioligand, dinotefuran was 13-fold less active than imidacloprid. The inhibitory effect of dinotefuran on the binding of [3H]IMI to the membrane preparation was in a competitive manner. Quantitative analysis of the insecticidal activity of the test compounds with the binding activity measured with [3H]IMI showed that the higher the binding activity, the higher was the insecticidal activity.     

Insecticidal 3-benzamido-N-phenylbenzamides specifically bind with high affinity to a novel allosteric site in housefly GABA receptors

γ-Aminobutyric acid (GABA) receptors (GABARs) are an important target for existing insecticides such as fiproles. These insecticides act as noncompetitive antagonists (channel blockers) for insect GABARs by binding to a site within the intrinsic channel of the GABAR. Recently, a novel class of insecticides, 3-benzamido-N-phenylbenzamides (BPBs), was shown to inhibit GABARs by binding to a site distinct from the site for fiproles. We examined the binding site of BPBs in the adult housefly by means of radioligand-binding and electrophysiological experiments. 3-Benzamido-N-(2,6-dimethyl-4-perfluoroisopropylphenyl)-2-fluorobenzamide (BPB 1) (the N-demethyl BPB) was a partial, but potent, inhibitor of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (GABA channel blocker) binding to housefly head membranes, whereas the 3-(N-methyl)benzamido congener (the N-methyl BPB) had low or little activity. A total of 15 BPB analogs were tested for their abilities to inhibit [³H]BPB 1 binding to the head membranes. The N-demethyl analogs, known to be highly effective insecticides, potently inhibited the [³H]BPB 1 binding, but the N-methyl analogs did not even though they, too, are considered highly effective. [³H]BPB 1 equally bound to the head membranes from wild-type and dieldrin-resistant (rdl mutant) houseflies. GABA allosterically inhibited [³H]BPB 1 binding. By contrast, channel blocker-type antagonists enhanced [³H]BPB 1 binding to housefly head membranes by increasing the affinity of BPB 1. Antiparasitic macrolides, such as ivermectin B_1a, were potent inhibitors of [³H]BPB 1 binding. BPB 1 inhibited GABA-induced currents in housefly GABARs expressed in Xenopus oocytes, whereas it failed to inhibit l-glutamate-induced currents in inhibitory l-glutamate receptors. Overall, these findings indicate that BPBs act at a novel allosteric site that is different from the site for channel blocker-type antagonists and that is probably overlapped with the site for macrolides in insect GABARs.     

Effects of temperature and concentration of the accelerators ethoxylated alcohols,diethyl suberate and tributyl phosphate on the mobility of [14C]2,4‐dichlorophenoxy butyric acid in plant cuticles

Intrinsic activities of monodisperse ethoxylated dodecanols (MEDs), diethyl suberate (DESU) and tributyl phosphate (TBP) were investigated using Stephanotis floribunda leaf cuticular membranes (CMs) and [¹⁴C]2,4‐dichlorophenoxy butyric acid (2,4‐DB) as a model solute. When sorbed in cuticular membranes, MEDs, DESU and TBP increase solute mobility and are called accelerators for this reason. With MEDs, dose‐effect curves (log mobility vs accelerator concentration) were linear but, with DESU and TBP, curves convex to the x axes were obtained that approached a maximum at 90 and 150 g kg⁻¹, respectively. Accelerators increased the mobility of 2,4‐DB in the CMs by 9‐ to 48‐fold, and effects were larger at lower temperatures (range 15–30 °C). Activation energy for diffusion of 2,4‐DB was 105 kJ mol⁻¹, decreasing with increasing accelerator concentrations to 26 kJ mol⁻¹ with DESU at 90 g kg⁻¹ and 64 kJ mol⁻¹ with TBP at 150 g kg⁻¹. Thus, the intrinsic activity of DESU was much higher than that of TBP, which implies that, for a given effect, less DESU than TBP would be needed. MEDs were also very effective accelerators, lowering activation energies to 36 kJ mol⁻¹. Data are discussed in relation to increasing rates of foliar penetration of active ingredients at low temperatures. © 2001 Society of Chemical Industry     

Effect of Formulation and Application Method on the Efficacy of Aerial and Submerged Conidia of Metarhizium flavoviride for Locust and Grasshopper Control

A study was carried out to investigate the relative infectivity of aerial and submerged conidia of Metarhizium flavoviride to Schistocerca gregaria and Zonocerus variegatus. The effect of formulation and application method on initial infectivity and field persistence of these conidia was investigated. Strain IMI 330189 was highly virulent to S. gregaria but showed relatively low virulence to Z. variegatus. Direct contact with conidia from the initial spray application resulted in 100% mortality of S. gregaria for all formulation and application combinations. The mean survival time of infected locusts was significantly shorter for treatments using a knapsack sprayer containing submerged conidia in water plus 10 ml litre⁻¹ ‘Codacide’™ (seven days), than treatments with aerial conidia in oil using ULV techniques (8.9 days) or submerged conidia in modified (water plus adjuvants) ULV (MULV) (nine days) or in water-based (VLV) applications (9·3 days). Both aerial and submerged conidia persisted long enough in the environment to effect significant mortality via secondary pick-up of spray residue from vegeta-tion. Persistence was greatest in the ULV and MULV treatments, where the oil component of the formulations provided greater protection of the conidia from environmental stresses. The consequences of secondary pick-up of conidia from the different treatments on total mortality from a single application were examined using a simple host–pathogen model. This predicted that the ULV treatment would be much more effective than the other treatments under conditions where direct contact with the spray was limited. The results of these investigations are discussed in the context of development of optimum spray strategies for control of locusts and grasshoppers, and other pests, under different environmental conditions.     

Investigating nicotinic acetylcholine receptor expression in neonicotinoid resistant Myzus persicae FRC

The peach–potato aphid Myzus persicae is a pest of many commercial crops due to its polyphagous nature of feeding and has a well-documented history of acquiring resistance to insecticides. In 2009 a strain (M. persicae FRC) emerged in southern France with a point mutation (R81T) at the nicotinic acetylcholine receptor (nAChR), the target site for neonicotinoids such as imidacloprid. This point mutation was associated with the loss of the high affinity imidacloprid binding site (pM Kd), with the single remaining binding site (low nM Kd) highly overexpressed compared to laboratory controls (Bass et al., 2011 [1]). Here we report that after 2 years of continuous selection in the glass house environment with neonicotinoids, the total level of IMD-sensitive nAChRs (low nM Kd) in M. persicae FRC is now comparable to laboratory controls (pM and low nM Kd). Interestingly, despite this large reduction in IMD-sensitive nAChRs, this was not associated with any significant alteration in NNIC-lethality. Additionally, sustained absence of neonicotinoid-selection did not alter nAChR protein levels. We suggest that alterations in nAChR protein expression level described in the original characterisation of the field-isolated M. persicae FRC is unlikely to have been a direct consequence of the R81T mutation. Rather, we speculate that nAChR expression in aphids is likely influenced by as yet unknown conditions in the natural field environment that are absent in the laboratory setting.     

Protective effect of dimercaptosuccinic acid on methylmercury and mercuric chloride inhibition of rat brain muscarinic acetylcholine receptors

The reactivation of the rat brain muscarinic acetylcholine receptor (mACh-R) binding with dimercaptosuccinic acid (DMSA) after in vitro and in vivo inhibition by mercuric chloride (HgCl₂) and methylmercuric chloride (MeHg) was investigated. Receptor binding was estimated by the potent and specific antagonist l-[³H]quinuclidinyl benzilate ([³H]QNB). Rat brain synaptosomal membranes were exposed to HgCl₂ and MeHg. At 1 × 10^?4M. HgCl₂ caused complete inhibition of the [³H]QNB binding. The inhibition of [³H]QNB binding by HgCl₂ was still higher than 50% at 1 × 10^?8M. MeHg caused less inhibition of [³H]QNB binding than HgCl₂. The inhibited receptors showed a significant degree of reactivation when treated with DMSA. The recovery was almost complete after MeHg inhibition or with the lower HgCl₂ concentrations. Generally, the reactivation was dependent on the concentration of DMSA. When rats injected with either early or delayed doses of DMSA following administration with five consecutive daily doses (8 mg/kg body wt, Gavage method) of MeHg or HgCl₂, the inhibition of [³H]QNB binding was less than untreated ones. The early treatment with DMSA decreased the inhibition of [³H]QNB binding due to MeHg or HgCl₂ intoxication. However, DMSA was more effective in reducing HgCl₂ inhibition than MeHg either in vitro or in vivo treatment. The ability of DMSA to reactivate the mACh-R after inhibition with the mercurials emphasizes the involvement of essential sulfhydryl groups in [³H]QNB binding sites, and also shows that these sulfhydryl groups are the primary target for the MeHg and HgCl₂ inhibition of the rat brain muscarinic receptors.     

Novel mode of action of spinosad: Receptor binding studies demonstrating lack of interaction with known insecticidal target sites

The ability of spinosyn A to either enhance or displace binding to selected insecticidally-relevant receptors was investigated using a number of radioligands including, [³H]imidacloprid and [³H]ivermectin in tissues from the ventral nerve cord (VNC) membranes of the American cockroach, Periplaneta americana and head membranes from the housefly, Musca domestica. In these insect neural tissues, spinosyn A does not appear to alter the binding of a number of radioligands suggesting that spinosyn A does not interact directly with a variety of known receptors, including nicotinic or γ-aminobutyric acid (GABA)-based insecticidal target sites. However, available data are consistent with spinosyn A interacting with a site distinct from currently known insecticidal target sites, thus supporting a novel insecticidal mechanism of action for the spinosyns.     

Interaction of dinotefuran and its analogues with nicotinic acetylcholine receptors of cockroach nerve cords

To investigate the action of dinotefuran (MTI-446, 1-methyl-2-nitro-3-(tetrahydro-3-furylmethyl)guanidine), a recently developed insecticide, on insect nicotinic acetylcholine receptors (nAChRs), we determined the potencies of the compound and 15 analogues in inhibiting the specific binding of [3H]epibatidine (EPI), a nAChR agonist, and [3H]alpha-bungarotoxin (alpha-BGT), a competitive nAChR antagonist, to the nerve cord membranes of American cockroaches (Periplaneta americana). Racemic dinotefuran inhibited [3H]EPI binding with an IC50 of 890 nM and [3H]alpha-BGT binding with an IC50 of 36.1 microM. Scatchard analysis indicated that the dinotefuran inhibition of [3H]EPI binding was a competitive one. Slight structural modification caused a drastic reduction in potency; only four analogues were found to be equipotent to or more potent than dinotefuran. Chloropyridinyl and chlorothiazolyl neonicotinoid insecticides displayed two or three orders of magnitude higher potency than dinotefuran. There was a good correlation between the IC50 values of tested compounds obtained with [3H]EPI and those obtained with [3H]alpha-BGT. A better correlation was observed between 3-h knockdown activities (KD50) against German cockroaches (Blattella germanica) and IC50 values obtained from [3H]EPI assays than between 24-h lethal activities (LD50) and IC50 values. While the results indicate that dinotefuran and its analogues interact with the ACh-binding site in cockroach nAChRs, it remains to be elucidated why they displayed lower potencies than those expected based on their insecticidal activities.     

Effect of SS-toxin,a metabolite of <Emphasis Type="Italic">Stemphylium solani</Emphasis>, on H<Superscript>+</Superscript>-ATPase activity and standard redox system in plasma membranes from seedlings leaves of garlic (<Emphasis Type="Italic">Allium sativum</Emphasis>)

Leaf blight of garlic (Allium sativum) is a severe disease in garlic-growing regions. SS-toxin is a newly described non-proteinaceous toxin produced by the phytopathogenic fungus, Stemphylium solani, the cause of garlic leaf blight. In this study, the effects of SS-toxin on the H⁺-ATPase activity and standard redox activity in the plasma membranes isolated by aqueous polymer two-phase partitioning from garlic seedling leaves were studied in vitro. The H⁺-ATPase activity, NADH oxidation rate and Fe(CN)₆³⁻ reduction rate of the plasma membranes isolated from susceptible and resistant cultivars were all inhibited in a dose-dependent manner. Our results suggest that, under in vitro conditions, the plasma membrane H⁺-ATPase and standard redox system can be both the cellular targets of SS-toxin.