Interaction of formamidines with octopamine-, dopamine-and 5-hydroxytryptamine-sensitive adenylate cyclase in the nerve cord of Periplaneta americana

The interaction of chlordimeform, N¹-demethylchlordimeform [N²-(4-chloro-o-tolyl)-N¹-methylformamidine], BTS-27271 [N¹-methyl-N²-(2,4-xylyl)formamidine, the 2,4-xylyl analogue of N¹-demethylformamidine], and amitraz with octopamine-, dopamine-, and 5-hydroxytryptamine-mediated enhancement of cyclic-AMP production was investigated in nerve cord homogenates of the American cockroach Periplaneta americana. N¹-Demethylchlordimeform, BTS-27271 and amitraz simulated the actions of the monoamines in enhancing cyclic-AMP production, whereas chlordimeform showed no such agonism. Additivity studies indicated that these formamidine derivatives were interacting with dopamine- and 5-hydroxytryptamine-sensitive sites, in addition to their known interaction with octopamine-sensitive adenylate cyclase. N¹-Demethylchlordimeform (Ki= 0.2μM) and amitraz (Ki=0.5μM) inhibited the dopamine-mediated response, but only N¹-demethylchlordimeform (Ki=2μM) effected appreciable inhibition of the octopamine-response. Pharmacological characterisation of the formamidineinduced elevation of cyclic-AMP production indicated that the agonistic effects of formamidines are expressed primarily through the octopamine-sensitive adenylate cyclase, rather than the dopamine- or 5-hydroxytryptamine-sensitive sites. The results are discussed in the light of the proposal that the behavioural changes, associated with formamidine poisoning, reflect the cumulative effects of octopamine-agonism, and antagonism of the dopamine- and 5-hydroxytryptaminemediated processes.     

The action of formamidines on octopamine receptors in the locust

The actions of a range of formamidines have been investigated biochemically and physiologically on octopamine receptors in the locust Schistocerca gregaria. All the formamidines tested [chlordimeform (CDM), demethylchlordimeform (DCDM), amitraz, and UK 16353] mimicked the action of octopamine by (1) increasing the amplitude and relaxation rate of slow motorneurone tension in the extensor-tibiae muscle of the hind leg and (2) changing the levels of cyclic AMP in this muscle. UK 16353 was most effective in changing these parameters, followed by DCDM then amitraz and CDM. The formamidine-induced increase in cyclic AMP levels was reduced or completely blocked by phentolamine, an antagonist of insect octopamine receptors. The time course for the increase in cyclic AMP was followed for 30 min by incubating muscles in 10^?5M DCDM. The increase was potentiated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. In the presence of this compound, the response peaked within 5 min, before declining to a lower plateau after 12 min. The response to 10^?5M CDM was lower and the maximum increase occurred after 7 min, then rapidly declined. Both formamidines increased cyclic AMP in a dose-dependent way with a threshold of between 5 × 10^?7 and 10^?8M. The results are discused in terms of the relationship between the biochemical and physiological actions of the formamidines in this preparation.     

Dihydrooxadiazines: Octopaminergic system as a potential site of insecticidal action

Dihydrooxadiazines are structural analogs of octopamine and were compared with octopamine for their ability to compete with [³H]dihydroergocryptine ([³H]DHE) for binding sites on DHE-sensitive receptors, to stimulate adenylate cyclase activity in nervous system homogenates of Periplaneta americana L., and to modulate the action of the peptide proctolin on the oviducal muscles of Locusta migratoria L. [³H]DHE binding was inhibited by low concentrations (μM range) of octopamine, phentolamine, N-demethylchlordimeform (DCDM) and several dihydrooxadiazines. The tested dihydrooxadiazines acted as aminergic agonists in stimulating cyclic AMP production in cockroach nervous system homogenates and did not show additive effects with octopamine, whereas additivity was observed with 5-hydroxytryptamine. The relative potency of octopaminergic antagonists, including mianserin, cyproheptadine, phentolamine, and gramine, to block octopamine-mediated elevation of cyclic AMP production was similar to the rank-order potency of the same antagonists to inhibit dihydrooxadiazine-mediated elevation of cyclic AMP production. Octopamine, 2-(4-bromophenyl)-5,6-dihydro-4H-1,3,4-oxadiazine (4-Br-PDHO), and 8-Br-cyclic AMP caused increased phosphorylation of proteins that are phosphorylated by exogenously added cyclic AMP-dependent protein kinase. These results indicate that the dihydrooxadiazine-induced rise in cyclic AMP levels in homogenates of the cockroach nervous system results directly in activation of an endogenous cyclic AMP-dependent protein kinase. 4-Br-PDHO behaved similarly to octopamine in modulating the action of proctolin-induced contractions in locust oviducal muscles. These observations suggest that dihydrooxadiazines act as octopamine agonists and have an octopaminergic action in modulating the action of proctolin. Thus, it is proposed that dihydrooxadiazines exert at least part of their insecticidal and miticidal actions through interaction with the octopaminergic system.     

The actions of formamidine pesticides on identified central neurones of Helix aspersa

Membrane potential and voltage clamp measurements of the responses to octopamine, dopamine, O-acetylcholine, and the formamidine derivatives chlordimeform (CDM) and N¹-demethylchlordimeform (DCDM) have been made on identified central neurones of Helix aspersa. DCDM and CDM were agonists on both the inhibitory and excitatory octopamine receptors on these neurones but were less potent than octopamine. The direct inhibitory effect of DCDM was antagonised by both phentolamine and cyproheptadine. DCDM was also found to antagonise the effects of octopamine, dopamine and O-acetylcholine on these neurones. The pA₂ values for DCDM against dopamine and O-acetylcholine were 5.2 (±0.1) and 5.35 (±0.15), respectively. The pA₂ value=log[DR-(1/I)], where I is the concentration of the antagonist, and DR=dose ratio =A2/A1; A1 is the dose of agonist required to produce 50% of the maximum response, and A2 is the dose of agonist needed to produce the same degree of response in the presence of the antagonist. It is concluded that DCDM and CDM act specifically on the octopamine receptors of H. aspersa central neurones, both as agonists and antagonists. The antagonist effect of DCDM against dopamine receptors is probably non-specific.     

Formamidine structure and detachment of the cattle tick Boophilus microplus

Evaluation of the effectiveness of topically applied chlordimeform, 14 other formamidines, and 5 sulfur-containing related nonformamidine compounds in causing female Boophilus microplus ticks to detach from mice enabled activity to be related to structure. Five compounds were inactive and 15, including 2 sulfur-containing nonformamidines and 1 sulfur-containing formamidine were active at doses ranging from 0.0005 to 2.0 μg/tick. The most active compounds were the N-monomethyl formamidines, BTS-27271 [N′-(2,4-dimethylphenyl)-N-methylformamidine], and C-8520 (demethylchlordimeform). Among the analogs of chlordimeform tested, those with alkyl substitutions at the amino nitrogen decreased in effectiveness in the order, monomethyl (demethylchlordimeform), monoethyl, mono-n-butyl, mono-i-butyl, mono-i-propyl, dimethyl (chlordimeform), and di-n-propyl. Inactive compounds resulted from the replacement of the chlorine at ring position 4 of the aryl moiety of chlordimeform by bromine or hydrogen or by the conversion of the NCH amidino moiety to the NHCSN < thiourea moiety.Detachment due to chlordimeform was antagonized by piperonyl butoxide but that due to its N-monomethyl analog, a known metabolite of chlordimeform in ticks, was synergized by the same compound. These effects on the detaching response parallel those reported elsewhere concerning synergism and antagonism of toxic responses of B. microplus to formamidines.BTS-27271, which was the most effective formamidine in causing detachment after topical application to ticks was moderately effective when injected into mice but its potency relative to chlordimeform was considerably reduced; when sprayed onto cattle BTS-27271 was somewhat more effective in depressing percentage survival of ticks of all stages than chlordimeform.     

Toxicity of chlordimeform and amitraz to the egyptian cotton leafworm (Spodoptera littoralis) and the tobacco budworm (Heliothis virescens)

The relative toxicity (μg a.i. g^?1 body wt) of the formamidine insecticide chlordimeform (CDM) and the triazapentadiene insecticide amitraz was examined in two species of noctuid moth Spodoptera littoralis and Heliothis virescens. When applied topically, there was an unexpected and marked difference in the toxicity of CDM base and its hydrochloride to adults of both species, the salt being appreciably more toxic. For H. virescens at least, this difference in toxicity could not be explained by differences in penetration. This trend was reversed for larval instars of S. littoralis; while there was relatively little difference in the toxicity of the base to adult and larval stages, the salt was at least 1000-fold more toxic to adults than to larvae. N¹-Demethylchlordimeform (DCDM) was the only metabolite of CDM to show biological activity against either species, but was much less toxic than the parent compound. Amitraz was far less toxic than either CDM or DCDM; like the CDM salt, it appeared to be more toxic to adult than larval stages of S. littoralis. Application of piperonyl butoxide significantly increased the toxicity of the CDM salt, DCDM and amitraz to adult H. virescens, the synergist being particularly effective with DCDM and amitraz. In contrast, piperonyl butoxide had no significant effect on the toxicity of DCDM, and slightly antagonised the toxicity of DCDM to fourth-instar larvae of S. littoralis.     

Octopamine-like action of formamidines on hormone release in the locust, Locusta migratoria

Application of the formamidine pesticides chlordimeform (CDM) and desmethylchlordimeform (DCDM) induced the release of hyperlipemic hormone from the isolated corpus cardiacum (CC) of the locust, Locusta migratoria. Pretreatment of locusts with reserpine (10 μg/locust) had no effect on formamidine-induced release of the hyperlipemic hormone. The action of CDM and DCDM on isolated CC was blocked by the α-adrenergic-receptor blocker, phenoxybenzamine, but not by the β-adrenergic blocker, propranolol. These formamidines also potentiated the release of the hyperlipemic hormone induced by electrical stimulation of nervus corpus cardiacum II (NCC II). The evidence presented in this paper indicates that, in locusts, CDM and DCDM, or their metabolites, act on postsynaptic aminergic (octopamine) receptors in the glandular lobe of the CC, inducing the release of hyperlipemic hormone.     

Biochemical identification of putative GABA/benzodiazepine receptors in house fly thorax muscles

[³H]Flunitrazepam ([³H]Flu) was used to identify benzodiazepine binding sites in house fly thorax muscle membranes using a filter assay. [³H]Flu bound to a finite number of sites in a concentration- and time-dependent manner, reaching equilibrium in 10 min. Scatchard plots of the binding indicated a high-affinity site at 0.2 pmol/mg protein (K_d 24.3 nM) and a low-affinity site at 8.2 pmol/mg protein (K_d994nM). Binding of [³H]Flu to the high-affinity binding site was inhibited by several benzodiazepine analogs, with Flu, diazepam, and Ro 5-4864 being more potent than β-CCE, Ro 5-3027, and Ro 5-2180. Clonazepam was least potent in inhibiting [³H]Flu binding. Thus, the drug specificity of these insect muscle benzodiazepine binding sites was quite different from both the mammalian central and peripheral benzodiazepine receptor sites, though closer to the peripheral ones. GABA (γ-aminobutyric acid) and its agonists enhanced the specific binding of [³H]Flu in a dose-dependent manner, and this effect was inhibited with the GABA antagonist bicuculline. The effect was biphasic since at high GABA concentrations this stimulation was reduced. The data suggest that house fly muscles have benzodiazepine receptors, which are coupled allosterically to GABA receptors, analogous to the GABA/benzodiazepine receptors of vertebrates, but with some differences in their drug specificities.     

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.     

Effects of diazepam and chlordimeform analogs on the German and the American cockroaches

Twenty-one diazepam- and chlordimeform (CDM)-related compounds were synthesized by mimicking some parts of the 1,4-benzodiazepine tranquilizing drugs, and were tested for their insecticidal activity against the German cockroach. Some of these compounds showed knockdown effects and some were insecticidal. Against the German cockroach the most toxic CDM analog was N-propargyl CDM (compound 6), and that with a potent knockdown potency was compound 13 which has a structural resemblance to diazepam. Ligand-receptor binding assay was carried out, using [³H]diazepam as a ligand to examine the relation between CDM-related compounds and the 1,4-benzodiazepines. The [³H]diazepam binding to a specific site in the American cockroach brain was inhibited by the insecticidal compounds. Among these compounds a correlation exists between their inhibitory potency on specific [³H]diazepam binding and their insecticidal activity, suggesting a possible significance of such an interaction with the diazepam binding site for the toxicity of these compounds against cockroaches.     

Molecular cloning and pharmacological characterisation of a tyramine receptor from the rice stem borer,Chilo suppressalis (Walker)

BACKGROUND: Tyramine (TA) and octopamine (OA) are considered to be the invertebrate counterparts of the vertebrate adrenergic transmitters. Because these two phenolamines are the only biogenic amines whose physiological significance is presumably restricted to invertebrates, the attention of pharmacologists has been focused on the corresponding receptors, which are believed to represent promising targets for novel insecticides. For example, the formamidine pesticides, such as chlordimeform and amitraz, have been shown to activate OA receptors. RESULTS: A full‐length cDNA (designated CsTyR1) from the rice stem borer, Chilo suppressalis (Walker), has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction (RACE‐PCR). The mRNA of CsTyR1 is present in various tissues, including hemocytes, fat body, midgut, Malpighian tubules, nerve cord and epidermis, and it is found predominantly in the larval nerve cord with 16–80‐fold enrichment compared with other tissues. The authors generated a HEK 293 cell line stably expressing CsTyR1 in order to examine functional and pharmacological properties of this receptor. Both TA and OA at 0.01–100 µM can reduce forskolin‐stimulated intracellular cAMP levels in a dose‐dependent manner (TA, EC₅₀ = 369 nM ; OA, EC₅₀ = 978 nM ). In agonist assays, activation of CsTyR1 by clonidine and amitraz but not by naphazoline and chlordimeform can also significantly inhibit forskolin‐stimulated cAMP production. The inhibitory effect of TA at 10 µM is eliminated by coincubation with yohimbine, phentolamine or chlorpromazine (each 10 µM ). CONCLUSION: This study represents a comprehensive molecular and pharmacological characterisation of a tyramine receptor in the rice stem borer. Copyright © 2012 Society of Chemical Industry     

Presence of Muscarinic Acetylcholine Receptors in the Cattle Tick Boophilus microplus and in Epithelial Tissue Culture Cells of Chironomus tentans

A muscarinic acetylcholine receptor (mAChR) has been demonstrated and partially characterized in larvae of the cattle tick Boophilus microplus. Its properties are compared with mAChR from an epithelial cell line from the dipteran insect Chironomus tentans. Competition studies with cholinergic ligands of different specificity revealed the muscarinic nature of the cholinergic receptors investigated in both species. In homogenates from tick larvae, specific binding sites for [³H]quinuclidinyl benzilate (QNB) with high affinity (1·2±(0·13) nM ; B_max 22·5 pmol mg protein⁻¹) were detected that do not bind nicotinic compounds specifically. The estimated IC₅₀ values for nicotine, imidacloprid and α-bungarotoxin were all in the mM range. Additionally, with tick larvae, high-affinity nicotinic binding sites were detected with [³H]nicotine which could be displaced by high concentrations of imidacloprid or QNB. The estimated IC₅₀ values for nicotine, α-bungarotoxin, imidacloprid and QNB were 43(±8) nM , 0·8(±0·2) μM , 2·8(±0·6) μM and 78(±1·9) μM , respectively. With homogenates of the non-neuronal insect cell line from C. tentans, only high-affinity binding sites for [³H]QNB were found. Muscarinic antagonists selectively displaced [³H]quinuclidinyl benzilate (QNB) binding to tick larvae homogenates. The mAChR of B. microplus preferred pirenzepine (IC₅₀ 2·13(±1·02) μM ) among different subtype-specific mAChR antagonists (4-DAMP had IC₅₀ 49·9(±9·13) μM and methoctramine had IC₅₀ 121(±14·2) μM ) indicating a type of binding site similar to the vertebrate M1 mAChR subtype. The tick muscarinic receptor seems to be a G-protein-coupled receptor, as concluded from the 4·8-fold reduction in receptor affinity for binding of the muscarinic agonist oxotremorine M upon treatment with the non-hydrolysable GTP-analogue γ-S-GTP. Binding data for the agonists oxotremorine M (IC₅₀ 71·3(±19·6) μM ) and carbachol (IC₅₀ 253(±87·1) μM ) parallel the biological efficacy of these compounds, in that, while oxotremorine M showed some activity against ticks, carbachol was ineffective.     

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.     

Pyrethroid action on the nicotinic acetylcholine receptor/channel

The interactions of natural pyrethrins and nine pyrethroids with the nicotinic acetylcholine (ACh) receptor/channel complex of Torpedo electric organ membranes were studied. None caused significant reduction in [³H]ACh binding to the receptor sites, but all inhibited [³H]perhydrohistrionicotoxin ([³H]H₁₂-HTX) binding to the channel sites in presence of carbamylcholine. Allethrin inhibited [³H]H₁₂-HTX binding noncompetitively, but [³H]imipramine binding competitively, suggesting that allethrin binds to the receptor's channel sites that bind imipramine. The pyrethroids were divided into two types according to their actions: type I, which included pyrethrins, allethrin, bioallethrin, resmethrin, and tetramethrin, was more potent in inhibiting [³H]H₁₂-HTX binding and acted more rapidly (i.e., in <30 sec). Type II, which included permethrin, fluvalinate, cypermethrin and fenvalerate, was less potent and their potency increased slowly with time. Also, inhibition of the initial rate of [³H]H₁₂-HTX binding by type I compounds increased greatly by the presence of the agonist carbamylcholine, but this was not so with type II compounds. The receptor-regulated ⁴⁵Ca²⁺ flux into Torpedo microsacs was inhibited by pyrethrins and pyrethroids, suggesting that their action on this receptor function is inhibitory. There was very poor correlation between the potencies of pyrethrins and pyrethroids in inhibiting [³H]H₁₂-HTX binding and their toxicities to house flies, mosquitoes, and the American cockroach. However, the high affinities that several pyrethroids have for this nicotinic ACh receptor suggest that pyrethroids may have a synaptic site of action in addition to their well known effects on the axonal channels.     

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.     

[3H]Muscimol binding to a putative GABA receptor in honey bee brain and its interaction with avermectin B1a

A putative GABA receptor was identified in honey bee brain by virtue of its specific binding of [³H]muscimol and its drug specificity. [³H]Muscimol bound with two affinities (K_d1 of 3 nM and K_d2 of 144 nM), comparable to its affinities for binding to mammalian brain. The high-affinity binding was most sensitive to GABA agonists with the following decreasing order of potencies: muscimol>GABA>imidazole acetic acid>DL-GABOB>Zβ-guanidine propionic acid. However, it was insensitive to the antagonist bicuculline, which is potent on [³H]muscimol binding to the mammalian GABA_A receptor. It was also insensitive to baclofen, which is a potent agonist of mammalian GABA_B receptor, as well as to picrotoxinin, pentobarbital, flunitrazepam, and ethyl-β-carboxylate, which bind to allosteric sites in mammalian GABA receptor. The low-affinity [³H]muscimol binding was inhibited with GABA agonists with the following decreasing order of potencies: imidazole acetic acid = β-guanidine propionic acid>dl-GABOB. The two muscimol binding affinities may represent binding to two sites on the same GABA receptor or to two kinds of GABA receptor. The most potent inhibitor of the high-affinity [³H]muscimol binding to honey bee brain was avermectin B_1a (AVM), whose IC₅₀ was 0.01 nM. AVM also inhibited the low-affinity [³H]muscimol binding with an IC₅₀ of 2 μM.     

Effects of insecticides and GABAergic agents on a house fly [35S]t-butylbicyclophosphorothionate binding site

Specific binding of [³⁵S]t-butylbicyclophosphorothionate ([³⁵S]TBPS) to a house fly thorax-plus-abdomen membrane preparation at 20°C is characterized by apparent K_d and B_max values of 0.21 μM and 2.5 pmol/mg protein, respectively, an association half-time of 13 min at 2 nM, and a biphasic dissociation curve showing half-times of 15 and 35 min. Specific binding is reduced at 37°C apparently due to instability of the receptor-ligand complex and at 0°C as the result of very slow association. [³⁵S]TBPS binding is diminished by detergents, stimulated by GABA at low ligand concentration, and inhibited by picrotoxinin and certain barbiturates, benzodiazepines, bicyclophosphorus compounds, and polychlorocycloalkane insecticides. The potency of TBPS and three related phosphorothionates in displacing [³⁵S]TBPS parallels their toxicity on injection into house flies; the corresponding bicyclophosphates are less active in both assays. Cyclodienes of low toxicity are generally poor inhibitors of radioligand binding. α-Endosulfan and syn-12-hydroxyendrin are more potent than their β and anti isomers, respectively, both as inhibitors of TBPS binding and as toxicants. Analysis of Scatchard plots indicates that picrotoxinin and heptachlor epoxide are non-competitive inhibitors of [³⁵S]TBPS binding. The [³⁵S]TBPS binding site of the house fly membrane preparation differs from that extensively studied in mammalian brain with respect to their responses to many insecticides and GABAergic agents.     

DDT and pyrethroids: Receptor binding and mode of action in the house fly

The mode of action of DDT and pyrethroids was investigated in the house fly, Musca domestica L, using drug:receptor binding techniques. Both in vivo and in vitro binding studies demonstrated the existence of membrane receptors which bind specifically to [¹⁴C]DDT and [¹⁴C]cis-permethrin. The receptors show properties to be expected of a critical target site of these insecticides. These include negative temperature correlation with binding, relatively nonsensitivity to DDE, and sensitivity to Ca²⁺. The receptor sites are readily saturated at 45–90 nM [¹⁴C]DDT and have an apparent disassociation constant (K_d) of 12.2 nM. The maximum number of binding sites was estimated to be 17 pmol DDT/mg membrane protein (0.34 pmol/house fly head). Competition studies showed DDT, cis-permethrin, and cypermethrin bind to the same receptor but not at precisely the same site. The addition of Ca²⁺ to the incubation buffer significantly inhibited the binding of both [¹⁴C]DDT and [¹⁴C]cis-permethrin, suggesting the receptor binding is Ca²⁺ sensitive and may have a role in ion conductance.     

Effects of chlordimeform and lindane on monoamine levels in the central nervous system of the american cockroach, Periplaneta americana L

The effects of chlordimeform and lindane on levels of 5-hydroxytryptamine, tryptophan, and N-acetyl dopamine were studied in the cerebral ganglia of the american cockroach, Periplaneta americana. The effects of chlordimeform on nerve cord levels of 5-hydroxytryptamine, tryptophan, dopamine, and octopamine, and the effect of lindane on cerebral ganglia levels of dopamine were also investigated in this species. Topical applications of chlordimeform deplete 5-hydroxytryptamine and tryptophan from the cerebral ganglia whereas levels of n-acetyl dopamine are elevated. The effect of chlordimeform on these compounds is dose-dependent. Similar chlordimeform-induced effects are observed in the nerve cord, and octopamine levels are also depleted in this tissue following treatment with chlordimeform. A biphasic response to chlordimeform is observed in the nerve cord for dopamine levels with a 40% decrease evident after 2 hr and a 30% increase apparent after 6 hr. In contrast to chlordimeform, lindane does not affect 5-hydroxytryptamine and tryptophan levels in the cebral ganglion but low doses of this insecticide effect increases in brain levels of dopamine and n-acetyl dopamine.