Pharmacology of insect GABA receptors

A GABA-operated Cl^– channel that is bicuculline-insensitive is abundant in the nervous tissue of cockroach, in housefly head preparations and thorax/abdomen preparations, and in similar preparations from several insect species. Bicuculline-insensitive GABA-operated Cl^– channels, which are rare in vertebrates, possess sites of action of benzodiazepines, steroids and insecticides that are pharmacologically-distinct from corresponding sites on vertebrate GABA_A receptors. The pharmacological profile of the benzodiazepine-binding site linked to an insect CNS GABA-operated Cl^– channel resembles more closely that of vertebrate peripheral benzodiazepine-binding sites. Six pregnane steroids and certain polychlorocycloalkane insecticides, which are active att-butylbicy-clophosphorothionate (TBPS)-binding sites, also differ in their effectiveness on vertebrate and insect GABA receptors. Radioligand binding and physiological studies indicate that in insects there may be subtypes of the GABA receptor. Molecular biology offers experimental approaches to understanding the basis of this diversity.Special issue dedicated to Dr. Eugene Roberts     

5-amino-1-(2,6-dichloro-4-trifluoromethyl-phenyl)-3-[3H3]-methylsulfanyl-1H-pyrazole-4-carbonitrile (CTOM): synthesis and characterization of a novel and selective insect GABA receptor radioligand

Pyrazole 2a is a novel, potent ligand for insect GABA receptors obtained from housefly head membrane preparations (K(i)=8 nM). It is 500-fold selective against the mammalian receptor (mouse brain preparations). Its specifically tritiated version (2b) was synthesized by reduction of disulfide 10 with NaBH(4) followed by alkylation with [3H(3)]-CH(3)I.     

Comparative studies on cholinergic activities in the central nervous system ofLocusta migratoria

Summary Comparative studies on biochemical and pharmacological aspects of cholinergic transmission in the cerebral ganglion of locust and the mouse cerebral cortex were performed. The results point to common features and significant differences in the cholinergic pharmacology of insect and vertebrate nervous system: ACh content as well as AChE and ChAT activities reflect a highly cholinergic innervation of the locust cerebral ganglion. While the nicotinic ACh-receptor type predominates in locust nervous system, the muscarinic type is the main receptor type in mouse brain.The kinetic data for the high affinity choline uptake of locust synaptosome preparations corroborate the cholinergic properties of nerve endings from locust head ganglion.Abbreviations ACh acetylcholine - ChAT cholineacetyltransferase - AChE acetylcholinesterase - QNB quinuclidihylbenzilate - -BTX -bungarotoxin The author is indebted to Prof. Lueken for support and encouragement during the course of this work. I also gratefully acknowledge the excellent technical assistance of Mrs. M. Düwel. This work was financially supported by the Deutsche Forschungsgemeinschaft (Br 712/2-2).     

Structure-activity relationships of seco-prezizaane and picrotoxane/picrodendrane terpenoids by Quasar receptor-surface modeling

The seco-prezizaane-type sesquiterpenes pseudoanisatin and parviflorolide from Illicium are noncompetitive antagonists at housefly (Musca domestica) gamma-aminobutyric acid (GABA) receptors. They show selectivity toward the insect receptor and thus represent new leads toward selective insecticides. Based on the binding data for 13 seco-prezizaane terpenoids and 17 picrotoxane and picrodendrane-type terpenoids to housefly and rat GABA receptors, a QSAR study was conducted by quasi-atomistic receptor-surface modeling (Quasar). The resulting models provide insight into the structural basis of selectivity and properties of the binding sites at GABA receptor-coupled chloride channels of insects and mammals.     

Antigenic differences between a proteolipid and a proteodetergent from Torpedo electroplax having similar cholinergic binding properties

The nicotinic acetylcholine receptor from Torpedo marmorata was extracted and purified from the electroplax membranes by using both aqueous detergent (proteodetergent) or chloroform-methanol (proteolipid). When studied with a highly sensitive radioimmunoassay, it was found that both proteins do not cross-react immunologically against an antireceptor antiserum prepared with the proteodetergent. Treatment with organic solvents of the electroplax membranes, as well as of the proteodetergent receptor purified by affinity chromatography, impaired the radioimmunoassay. This suggests that the antigenicity has been affected by the change in solvent polarity, even though both proteins have similar binding properties for cholinergic ligands. These findings do not allow a simple immunological comparison between the cholinergic proteodetergent and the proteolipid as previously stated in the literature.     

A C-terminal aldehyde analog of the insect kinins inhibits diuresis in the housefly

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides in flies. A C-terminal aldehyde insect kinin analog, Fmoc-RFFPWG-H (R-LK-CHO), demonstrates stimulation of Malpighian tubule fluid secretion in crickets, but shows inhibition of both in vitro and in vivo diuresis in the housefly. R-LK-CHO reduced the total amount of urine voided over 3 h from flies injected with 1 microL of distilled water by almost 50%. The analog not only inhibits stimulation of housefly fluid secretion by the native kinin Musdo-K, but also by thapsigargin, a SERCA inhibitor, and by ionomycin, a calcium ionophore. The activity of R-LK-CHO is selective, however, as related C-terminal aldehyde analogs do not demonstrate an inhibitory response on housefly fluid secretion. The selective inhibitory activity of R-LK-CHO on housefly tubules represents an important lead in the development of environmentally friendly insect management agents based on the insect kinins.     

Binding of octopamine to membranes from the white-eyed and wild-type housefly heads

In attempting to develop an octopamine (OA) receptor preparation with ready access to large amounts of tissue, we examined the binding of OA to membranes from the heads of white and red houseflies (Musca domestica L.). Binding was dependent on the presence of L-ascorbic acid in the medium. However, equilibrium was reached only over 24–36 h at 4°C and reversal of binding was also slow and incomplete. Scatchard analysis revealed at least two binding sites in the white-eyed housefly. A high-affinity site (K_d = 13.9 nM and B_max = 3.9 pmol/mg protein) was present, but the majority of the binding had low affinity (K_d = 1130 nM and B_max = 165 pmol/mg protein). Scatchard analysis revealed a low affinity in the red-eyed housefly (K_d = 240 nM and B_max = 12 pmol/mg protein). Catecholamines were the best competitors for OA binding followed by phenolamines such as OA and synephrine. 5-Hydroxytryptamine was less effective. Phentolamine and mianserin, which are good antagonists of the ability of OA to stimulate adenylate cyclase in housefly head membranes, and formamidine and imidazolines, which are potent partial agonists of this adenylate cyclase, were poor competitors of OA binding. The slow kinetics, low affinity, large amount, and unconventional pharmacological profile of this binding is not congruent with it being a neuroreceptor. When the brain was dissected free from the head, less than 10% of the total specific binding of OA was found in the brain membrane fraction. This suggests that most of the binding of OA may be to cuticular sites that possibly are associated with the metabolism of catecholamines in cuticular synthesis. Thus, binding studies made with labeled catecholamines and phenolamines on insect tissues containing significant cuticular elements should be interpreted with caution.     

Nitromethylene actions on in situ and expressed insect nicotinic acetylcholine receptors

Single channel recordings from dissociated housefly (Musca domestica) neurons show that a novel type of nitromethylene insecticide, 2(nitro-methylene)tetrahydro-1,3-thiazine (NMTHT) gates a channel, the conductance and open time histogram of which resemble those obtained when acetylcholine is the agonist. Injection into Xenopus oocytes of a locust (Schistocerca gregaria) alpha-subunit mRNA results in the expression of functional nicotinic receptors sensitive to NMTHT. Control oocytes injected with distilled water are insensitive to the same concentration of this compound. Thus NMTHT exhibits agonist actions at both in situ and expressed insect nicotinic receptors, and one site of action of this compound is on an insect nicotinic receptor alpha-subunit.     

Neuroprotective and Neurological Properties of <Emphasis Type="Italic">Melissa officinalis</Emphasis>

Melissa officinalis has traditionally been used due to its effects on nervous system. Both methanolic and aqueous extracts were tested for protective effects on the PC12 cell line, free radical scavenging properties and neurological activities (inhibition of MAO-A and acetylcholinesterase enzymes and affinity to the GABA_A-benzodiazepine receptor). The results suggest that the plant has a significant (P < 0.05) protective effect on hydrogen peroxide induced toxicity in PC12 cells. The radical scavenging properties were also investigated in cells and in cell free systems, where this plant was shown to be a good free radical scavenger. The MAO-A bioassay was also performed to detect possible antidepressant activities demonstrating that both extracts inhibited this enzyme, which has a key role in neurotransmitters metabolism. However, no activity was detected in the acetylcholinesterase and GABA assays. In general, the methanolic extract was more effective than the aqueous.     

Characterization of the channel properties of a neuronal acetylcholine receptor reconstituted into planar lipid bilayers

An alpha-toxin-binding membrane protein, isolated from the head and thoracic ganglia of the locus (Locusta migratoria), was reconstituted into planar lipid bilayers. Cholinergic agonists such as acetylcholine, carbamylcholine, and suberyldicholine induced fluctuations of single channels, which suggests that the protein represents a functional cholinergic receptor channel. The antagonist d-tubocurarine blocked the activation of the channels, whereas hexamethonium had only a weak effect; similar properties have been described for nicotinic insect receptors in situ. The channel was selectively permeable to monovalent cations but was impermeable to anions. The conductance of the channel (75 pS in 100 mM NaCl) was independent of the type of agonist used to activate the receptor. Kinetic analysis of the channel gating revealed that, at high agonist concentrations (50 microM carbamylcholine), more than one closed state exists and that multiple gating events, bursting as well as fast flickering, appeared. At very high agonist concentrations (500 microM carbamylcholine), desensitization was observed. Channel kinetics were dependent on the transmembrane potential. Comparing the conductance, the kinetics, and the pharmacology of nicotinic acetylcholine receptor from insect ganglia and fish electroplax reconstituted into bilayers revealed obvious similarities but also significant differences.     

入侵植物香丝草水浸提液对蚕豆和玉米根尖染色体行为的影响

以采自农田中自然生长的植物群落中的香丝草为供体,以典型的双子叶植物蚕豆和典型的单子叶植物玉米的幼苗为受体,运用根尖微核试验和染色体畸变试验,研究了香丝草的根、茎、叶和幼果4种器官水浸提液对受体的遗传毒性。结果表明:(1)在香丝草不同器官水浸提液作用下,蚕豆和玉米根尖细胞的有丝分裂各时期均受到明显影响,细胞中出现了微核、染色体桥、染色体断片、染色体环、染色体粘连及染色体滞后等多种染色体畸变。(2)香丝草各器官水浸提液对蚕豆幼苗根尖细胞分裂的抑制作用明显大于玉米。(3)香丝草各器官水浸提液对蚕豆和玉米幼苗根尖的染色体畸变诱导存在显著的浓度效应,即水浸提液浓度越高,受体的微核率和畸变率越高,相应的有丝分裂指数越低,水浸提液的诱导作用与浓度呈正相关关系,但不是简单的加和作用。(4)香丝草各器官水浸提液均具有较强的遗传毒性,但整体化感效应表现为叶＞幼果＞茎＞根,即叶片产生的化感作用最强。因此,香丝草分泌的化感物质可能通过对受体植物生长点的细胞有丝分裂和细胞形态产生影响,造成受体植物染色体的多种畸变和不可逆的遗传损伤,从而成功入侵新的栖息地。     

Affinity to the Nicotinic Acetylcholine Receptor and Insecticidal Activity of Chiral Imidacloprid Derivatives with a Methylated Imidazolidine Ring

Four imidacloprid derivatives with an asymmetrically methylated imidazolidine ring were synthesized. Their affinity to the nicotinic acetylcholine receptor of housefly Musca domestica and insecticidal activity against the housefly were measured. The compound with a 5R-methylated imidazolidine ring demonstrated intrinsic activity comparable to that of the unsubstituted compound. Most of the compounds were synergized by oxygenase inhibitors.     

Cordeauxia edulis and Rhododendron tomentosum extracts disturb orientation and feeding behavior of Hylobius abietis and Phyllodecta laticollis

The use of plant‐based compounds to control insect pests is an alternative to the use of synthetic pesticides. We evaluated the repellent and antifeedant effects of Cordeauxia edulis Hemsley (Fabaceae) and Rhododendron tomentosum Harmaja (Ericaceae) extracts against Hylobius abietis L. (Coleoptera: Curculionidae) and Phyllodecta laticollis Suffrian (Coleoptera: Chrysomelidae). Repellent properties were evaluated by monitoring responses of adult insects toward the odor from extracts or extraction solvents (controls) in a Y‐tube olfactometer, and choice or no‐choice feeding tests were conducted by applying extracts or extraction solvents on stems of Scots pine [Pinus sylvestris L. (Pinaceae)] and European aspen [Populus tremula L. (Salicaceae)], host plants of H. abietis and P. laticollis, respectively. Extracts of R. tomentosum repelled adults of both insect species effectively. However, extracts of C. edulis did not repel H. abietis although its ethyl acetate extract showed repellence against the adults of P. laticollis. Feeding by H. abietis was significantly reduced by a methanol extract of C. edulis, and methanol and hexane extracts of R. tomentosum. Feeding by the adults and larvae of P. laticollis was significantly reduced by extracts from both plant species. Concomitant with less feeding, larval growth was retarded by ethyl acetate extracts of both plant species. Gas chromatography‐mass spectrometry analyses of the volatile components of the extracts showed that extracts from both plant species were mixtures of various terpene and non‐terpene compounds, which showed quantitative and/or qualitative variations between plant species and extraction solvents. This experiment showed that extracts from both plant species effectively manipulated the orientation and/or feeding behavior of the two beetle species. Hence, they may be considered as potential alternatives to synthetic chemical pesticides.     

Inhibition of the onset of vitellogenesis by abnormal retention of mature eggs in the stick insect,Carausius morosus

Mature eggs stored in the lateral oviducts are unusual in Carausius morosus. When egg laying is experimentally prevented, ovarian production is inhibited by the accumulation of mature eggs within the lateral oviducts. Such storage affects only the very small terminal oocytes and not the vitellogenic follicles, which attain to term and are ovulated. The fact that unilateral retention of eggs affects only the corresponding ovary strongly suggests that the mature eggs themselves inhibit ovarian production in the stick insect without the involvement of an endocrine organ. It is likely that this mode of action, different from that suggested for the housefly or Rhodnius prolixus, is correlated with the independence of ovarian development from the corpus allatum hormone in the stick insect.     

Photoaffinity labeling of insect nicotinic acetylcholine receptors with a novel [(3)H]azidoneonicotinoid

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel in the insect CNS and a target for major insecticides. Here we use photoaffinity labeling to approach the functional architecture of insect nAChRs. Two candidate 5-azido-6-chloropyridin-3-yl photoaffinity probes are evaluated for their receptor potencies: azidoneonicotinoid (AzNN) with an acyclic nitroguanidine moiety; azidodehydrothiacloprid. Compared to their non-azido parents, both probes are of decreased potencies at Drosophila (fruit fly) and Musca (housefly) receptors but AzNN retains full potency at the Myzus (aphid) receptor. [(3)H]AzNN was therefore radiosynthesized at high specific activity (84 Ci/mmol) as a novel photoaffinity probe. [(3)H]AzNN binds to a single high-affinity site in Myzus that is competitively inhibited by imidacloprid and nicotine and further characterized as to its pharmacological profile with various nicotinic ligands. [(3)H]AzNN photoaffinity labeling of Myzus and Homalodisca (leafhopper) detects a single radiolabeled peak in each case displaceable with imidacloprid and nicotine and with molecular masses corresponding to approximately 45 and approximately 56 kDa, respectively. The photoaffinity-labeled receptor in both Drosophila and Musca has imidacloprid- and nicotine-sensitive profiles and migrates at approximately 66 kDa. These photoaffinity-labeled polypeptides are considered to be the insecticide-binding subunits of native insect nAChRs.     

Conditions for balanced polymorphism in the presence of differential delay in developmental time

The effect of differential development on the genetic properties of a population is studied. A specific model applicable to a wide variety of insect populations is used and the conditions under which the population is polymorphic when differential delay in developmental time is present are investigated. It is found that polymorphism is present for a very wide range of selection. The effect of the delay is to introduce frequency dependent factors and thus to maintain polymorphism even in the absence of heterosis. The results are applied to experimental data on the moth species Gonodontis bidentata and on the Canberra strain of the housefly, Musca domestica.     

Hydrophobin (HFBI): A potential fusion partner for one-step purification of recombinant proteins from insect cells

Hydrophobins play an important role in binding and assembly of fungal surface structures as well as in medium-air interactions. These, hydrophobic properties provide interesting possibilities when purification of macromolecules is concerned. In aqueous micellar two-phase systems, based on surfactants, the water soluble hydrophobins are concentrated inside micellar structures and, thus, distributed to defined aqueous phases. This, one-step purification is attractive particularly when large-scale production of recombinant proteins is concerned. In the present study the hydrophobin HFBI of Trichoderma reesei was expressed as an N-terminal fusion with chicken avidin in baculovirus infected insect cells. The intracellular distribution of the recombinant fusion construct was analyzed by confocal microscopy and the protein subsequently purified from cytoplasmic extracts in an aqueous micellar two-phase system by using a non-ionic surfactant. The results show that hydrophobin and an avidin fusion thereof were efficiently expressed in insect cells and that these hydrophobic proteins could be efficiently purified from these cells in one-step by adopting an aqueous micellar two-phase system.     

Expression of the housefly acetylcholinesterase in a bioreactor and its potential application in the detection of pesticide residues

Acetylcholinesterase is a key enzyme of the animal nerve system. The enzyme is the primary target of organophosphorous (OP) and carbamate (CB) insecticides. The insect AChE is being extensively used in development of new insecticides or in vitro selection of the new designed insecticides, and in pharmacological and toxicological field. Rapid assays using AChE-based methods have been proposed as an efficient and rapid method for the detection of pesticides, especially in many Asian markets. In this study, the acetylcholinesterase gene was cloned from housefly (Musca domestica) susceptible to organophosphate (OP) and carbamate (CB) insecticides, and expressed in baculovirus-insect cells system using a bioreactor with oxygen supplementation. The recombinant housefly AChE was purified using ammonium sulfate precipitation and procainamide affinity chromatography, and approximately 0.42 mg of the purified AChE with high biological activity (118.9 U/mg) was obtained from 100 ml of culture solution. The purified AChE was highly sensitive to OP and CBs insecticides. In conclusion, an efficient expression and purification system has been developed for large-scale production of recombinant housefly AChE. The recombinant enzyme is potential to be used for the detection of pesticide residues.     

Chemical profiling of polyphenols in extracts from bark of Acacia nilotica (Linn.) and their efficacy against Spodoptera litura (Fab.)

Six different crude extracts from bark powder of an important medicinal plant, Acacia nilotica (L.) were investigated for their toxicity against Spodoptera litura (Fab.). Total phenolic content in six crude extracts was measured. Ultra-High Performance Liquid Chromatography (UHPLC) was also performed for qualitative analysis of six crude extracts which revealed the presence of 11 phenolic compounds. Bioassays conducted with all crude extracts revealed maximum adverse effect with acetone extract. Duration of larval period and total development period was found to be prolonged. Various aberrations were observed in pupae and adults in the form of wrinkled pupae, blackened pupae, bulged out head part of pupae, partially emerged adults and adults with crumpled wings. All nutritional indices viz. RGR, RCR, ECI, ECD and AD decreased with treatment. The findings indicated that A. nilotica had considerable potential in the management of this serious insect pest.