The biochemical mode of action of some modern anthelmintics

The possible biochemical modes of action of levamisole, morantel, benzimidazoles and salicylanilides are discussed. It is concluded that the modes of action of none of these anthelmintics is properly understood, but it seems likely that levamisole and pyrantel act on the nematode nervous system, benzimidazoles affect cellular integrity and the uncoupling properties of salicylanilides could enable them to act at several sites.     

The activation of nicotinic acetylcholine receptors in the nematode parasite Ascaris suum by the application of levamisole to the cytoplasmic surface of muscle membrane

The patch-clamp technique was used to examine activation by levamisole of nicotinic acetylcholine receptors in muscle vesicle preparations from Ascaris suum. Previous experiments have shown that levamisole, when applied at concentrations greater than 30 μM to the extracellular surface of the membrane, activated channels which showed characteristics of a voltage-sensitive open channel-block and desensitisation. In this study initial experiments were performed where channels were activated with levamisole (2 μM ) applied in the patch-pipette. Isolated inside-out patches were used. In addition, levamisole (30-926 μM ) was added to the cytoplasmic membrane surface via the bath solution. In eight out of nine experiments, addition of levamisole to the bath solution resulted in an increase in channel activity, a voltage-sensitive open channel-block and desensitisation. The open channel-block occurred at hyperpolarised potentials, an observation consistent with levamisole (a cationic substance) blocking the channel from the extracellular surface. Thus it was concluded that levamisole crossed from the cytoplasmic side of the membrane, via the lipid phase, to the extracellular surface of the patch. In the presence of high cytoplasmic concentrations of levamisole, open channel-block was not observed at depolarised potentials, suggesting channel asymmetry.     

Anthelmintic Actions of the Cyclic Depsipeptide PF1022A and its Electrophysiological Effects on Muscle Cells of Ascaris suum

The cyclic depsipeptide PF1022A, given orally to mice, showed very good anthelmintic activity against Heligmosomoides polygyrus and Heterakis spumosa at 50 mg kg⁻¹. In vitro, PF1022A was very active against Trichinella spiralis and had good activity against Nippostrongylus brasiliensis at 1 μg ml⁻¹. An 18-membered enniatin analogue, JES 1798, showed good activity only against N. brasiliensis at 10 μg ml⁻¹. The optical antipode of PF1022A had poor activity even at 100 μg ml⁻¹. The effects of PF1022A on the membrane potential and input conductance of somatic muscle of Ascaris suum were examined using a two-microelectrode current-clamp technique. PF1022A did not antagonize the effects of the selective nicotinic agonist levamisole. PF1022A and an analogue, JES 1798, but not the PF1022A antipode, produced a small time-dependent increase in input conductance associated with no potential change. The increase in input conductance did not occur in the Cl⁻-free bathing solution, suggesting that the increase in input conductance was mediated by Cl⁻ ions. The addition of high concentrations of Ca²⁺ to the preparation after the addition of PF1022A did not lead to production of Ca²⁺-activated Cl⁻ channels, suggesting that its mode of action was not that of a Ca²⁺ ionophore. The mechanism by which the cyclic depsipeptide might increase the Cl⁻ conductance is discussed.     

Phenylpyrazoles,a new class of pesticide: Effects on neuromuscular transmission and acetylcholine responses

Phenylpyrazoles constitute a newly developed class of chemicals with insecticidal and herbicidal properties. In acute toxicity experiments in rats, near-lethal doses of phenylpyrazoles produce neurotoxic symptoms with an unknown mode of action. We have investigated the effects of two phenylpyrazoles, SLA4722 and SLA4685, in the frog neuromuscular junction and in the clonal muscle cell line BC3H-1. In the frog neuromuscular junction SLA4722 at concentrations ? 10^?5 M caused a pronounced increase in the frequency of miniature endplate potentials (MEPPs). SLA4685 caused a similar increase but was less potent. In the clonal muscle cell line BC3H-1, SLA4685 produced a concentration-dependent block of the acetylcholine (ACh) response (IC₅₀ = 2.4(±0.3) × 10^?5 M). At 10^?6 M a slight potentiating effect of SLA4685 on the ACh response was observed. SLA4722 was less potent in this preparation. The increased transmitter release might, at least partly, be responsible for the excitatory effects of the phenylpyrazoles in intact animals.     

Investigation of the Mode of Action of Nematode Neuropeptides

The neuropeptide AF2 has a complex set of actions on the dorsal muscle strip of Ascaris suum, including a potentiation of the acetylcholine-stimulated muscle contraction. Caffeine at 100 μm and 5 mm inhibited this potentiation, as did 100 μm theophylline in two out of six studies. The cyclic-AMP-potentiating compounds IBMX, dibutyryl cAMP and forskolin had no effect on the AF2-induced potentiation of the acetylcholine-stimulated muscle contraction. These preliminary data suggest that the potentiating action of AF2 is not mediated by a cAMP pathway.     

Insect background sodium channel as a new target for scorpion alpha toxin

The effects of an anti-insect scorpion alpha toxin, LqhαIT, isolated from the venom of the Israeli scorpion Leiurus quinquestriatus hebraeus was studied on isolated adult dorsal unpaired median (DUM) neurones isolated from the cockroach Periplaneta americana terminal abdominal ganglion. Using the cell-attached patch-clamp configuration, a new type of sodium channel, called background sodium channel (bNa), was recently characterized. At −50 mV, the channel activity was observed as unclustered brief single openings. For hyperpolarized steady-state holding potential (−100 mV) the patches contained large unitary current steps, appearing generally in bursts. The open probability (Po) calculated at −50 mV was low (0.008 (± 0.004), n = 5) and displayed a typical bell-shaped voltage dependence. LqhαIT (10⁻⁸ M ) altered the bNa activity in a time-dependent manner. At −50 mV the channel activity appeared in bursts. Po calculated at −50 mV was about 20 times greater than Po calculated in controls and also showed bell-shaped voltage dependence. At 10⁻⁷ M , LqhαIT induced longer silent periods interrupted by bursts of increased channel activity. Whole-cell recordings revealed that 10⁻⁷ M LqhαIT transformed regular beating DUM neurone pacemaker activity into a rhythmic bursting. In this paper we demonstrate, for the first time, that bNa is a new target for anti-insect scorpion toxin. © 1999 Society of Chemical Industry     

Effects of abamectin on intracellular potassium ion activity and membrane potential in dipteran skeletal muscle

The effects of abamectin (AVMB₁) on intracellular potassium ion activity (aK_i) and resting membrane potential (E_m) of the skeletal muscle cells of final instar larvae of Phormia terraenovae (Diptera) were investigated using K⁺-selective micro-electrodes. Bathing the preparation in 10^? M AVMB₁(+ 1 ml litre^?1 dimethylsulfoxide) for 60 min caused a significant (31%) decrease in aK_i, whilst E_m depolarized on average by 19 mV (nearly 50% of the original control value). The difference E_K-E_m increased by 9 mV, although E_K (potassium equilibrium potential) remained more negative than E_m. These results could be due to a cationic effect of AVMB₁ possibly involving an increase in K⁺ and Na⁺ conductances of the muscle plasma membrane.     

Action of pyrethroids on a nerve-muscle preparation of the clawed frog,Xenopus laevis

The effects of a range of pyrethroids on end-plate potentials and muscle action potentials were studied in the pectoralis nerve-muscle preparation of the clawed frog, Xenopus laevis. The noncyano pyrethroids allethrin, cismethrin, bioresmethrin, and IR-cisphenothrin caused moderate presynaptic repetitive activity only, resulting in the occurrence of multiple end-plate potentials (epps). Trains of repetitive muscle action potentials without presynaptic repetitive activity were observed after the α-ethynyl pyrethroid S-5655 and after the α-cyano pyrethroids cypermethrin, deltamethrin, FCR 1272, and FCR 2769. An intermediate group of pyrethroids consisting of the non-cyano compounds 1R-permethrin, des-cyano-deltamethrin, NAK 1901 and NAK 1963, and the α-cyano pyrethroids cyphenothrin and fenvalerate caused both types of effect. The insecticidally inactive S-enantiomers of permethrin had no effect on the nerve-muscle preparation. Trains of repetitive action potentials in pyrethroid-treated muscle fibers were followed by a depolarizing afterpotential which in general decayed more rapidly for the non-cyano pyrethroids than for the α-cyano pyrethroids. The rate of decay of the depolarizing afterpotential decreased gradually as the temperature was lowered, whereas the pre- and postsynaptic repetitive activity remained largely unaffected over a large temperature range. It is concluded that in muscle membrane like in nerve membrane the pyrethroid-induced repetitive activity is due to a prolongation of the sodium current and that a clear distinction between non-cyano pyrethroids on the one hand and α-cyano compounds on the other cannot be made on the basis of the present results.     

Effects of metaldehyde and acetaldehyde on specific membrane currents in neurones of the pond snail Lymnaea stagnalis

Bath application of metaldehyde or acetaldehyde led to tonic depolarization and bursting activity in identified neurones in the isolated central nervous system of Lymnaea stagnalis. This activity was sometimes accompanied by paroxysmal depolarizing shifts in membrane potential. Those neurones which possessed type 2 action potentials showed marked spike broadening in response to metaldehyde, but little increase in action potential duration in the presence of acetaldehyde. Under voltage clamp, metaldehyde caused a reduction in the delayed rectifier and A-currents, both of which are outward potassium currents. Acetaldehyde also reduced the delayed rectifier but had no effect on the A-current. In type 2 neurones, voltage-dependent calcium current appeared to increase in the presence of metaldehyde, but was decreased by acetaldehyde. The action of metaldehyde and acetaldehyde on firing activity, as well as their differential effects on action potential duration, may be partly explained by alterations in these specific membrane currents.     

拟环纹豹蛛神经细胞的急性分离及其电压门控钠通道的膜片钳研究

对拟环纹豹蛛雌蛛中枢神经细胞进行急性分离,并利用全细胞膜片钳技术对神经细胞的电压门控钠通道基本电生理学特性进行了研究,同时测定了氯氰菊酯及其不同浓度对Na 通道门控过程的影响。结果表明,拟环纹豹蛛头胸部神经团在消化酶和培养液混合作用下能急性分离出单个神经细胞,在4～6h内适合用于膜片钳研究。在全细胞电压钳条件下,神经细胞Na 通道电流(INa)在-60mV左右激活, 10mV左右达峰值,对河豚毒素敏感。用氯氰菊酯处理后,Na 通道的激活电压向负电位方向移动约10mV,为-70mV左右,峰值提前在 5mV左右出现。INa先增大然后迅速减小。用不同浓度的氯氰菊酯处理神经细胞后,INa在各电压下的差异不大,但达到最大INa所需时间随氯氰菊酯浓度的增加而缩短。     

The toxicity of phosphine,methyl bromide, 1,1,1-trichloroethane and carbon dioxide alone and as mixtures to the pupae of red flour beetle,Tribolium castaneum herbst

The toxicity of phosphine, methyl bromide, 1,1,1-trichloroethane (methyl chloroform) and carbon dioxide and mixtures of phosphine + methyl bromide, methyl bromide + methyl chloroform, phosphine + carbon dioxide, and methyl bromide + carbon dioxide to one- to two-day-old pupae of Tribolium castaneum Herbst was studied. Joint action ratios estimated at LD₅₀ and LD₉₀ for a 24-h exposure indicated no enhancement of toxicity for mixtures of phosphine and methyl bromide, or methyl chloroform and methyl bromide on the pupae. Carbon dioxide up to 40% in air enhanced the toxic action of phosphine as well as of methyl bromide. Higher levels of carbon dioxide, however, failed to improve the toxicity of phosphine or methyl bromide proportionately. Carbon dioxide used alone produced a maximum of 11% mortality of the pupae exposed to 10–70% levels for 24 h. The order of toxicity of the fumigants both at LD₅₀ and LD₉₀ was phosphine > methyl bromide > methyl chloroform.     

Characterization of the germination and emergence response to temperature and soil moisture of Avena fatua and A. sterilis

Seeds of Avena fatua L. and )A. sterilis L. were germinated under a wide range of temperatures (5–30°C) and osmotic potentials (?25 to ?1400 KPa) in order to characterize their responses to these two environmental factors. Although both species behaved similarly at moderate temperatures, different responses were observed at the two extremes. )A. sterilis germinated and emerged in a higher proportion than A. fatua at temperatures below 10°C but the opposite was true at temperatures above 20°C. Although the rates of these two processes were similar in both species up to 18°C, above this temperature the germination and emergence of )A. sterilis was considerably delayed in comparison with that of A. fatua. The effect of decreasing osmotic potentials in reducing the germination was more pronounced in A. sterilis than in A. fatua. However, no differences were observed in the emergence responses of either species. The adaptative advantages of these characteristics and their relationship with the geographic distribution of the two species is discussed.     

A patch—clamp study of the action of a nitromethylene heterocycle insecticide on cockroach neurones growing in vitro

The mode of action of a nitromethylene heterocycle (NMH) insecticide was studied by patch–clamp techniques using cockroach embryonic cultures as an experimental model. Under whole-cell recordings, this compound elicited inward currents resembling those induced by O-acetylcholine (ACh). The reversal potentials for both ACh and the NMH were similar, suggesting that the inward currents induced by both were carried by the same species of ion. Pharmacological investigations of NMH-induced responses revealed that the insecticidal action of this compound is exerted through agonistic action at the nicotinic acetylcholine receptor. Single-channel studies were also performed to study the interaction of NMH with the nicotinic-receptor-coupled ion channel.     

Pharmacological differentiation of responses to dopamine,octopamine and noradrenaline recorded from a cockroach motoneurone

Recordings have been made from the soma of a cockroach common inhibitory motoneurone. Locally applied dopamine, octopamine or noradrenaline all depolarised the neurone and produced a modest increase in membrane conductance. Under voltage-clamp, currents evoked by these amines showed considerable voltage dependence. The current-voltage relationships for dopamine and octopamine responses were similar, but differed from that for noradrenaline. The unusual voltage-dependent characteristics of these responses suggest that these compounds may serve a modulatory function in the insect CNS. The effects of a range of pharmacological antagonists indicate that dopamine acts upon a class of receptors distinct from those mediating responses to octopamine, noradrenaline and acetylcholine (ACh). Dopamine currents were abolished by SCH 23390 or cis-(Z)-flupenthixol, which did not depress responses to the other agonists. Octopamine and noradrenaline currents were selectively blocked by metoclopramide and ergometrine respectively, while β-bungarotoxin and gallamine preferentially suppressed ACh responses. At the present time the ionic basis of dopamine responses is unclear. Dopamine-evoked currents were totally abolished by application of Cd2²⁺ or verapamil or when the preparation was bathed in Ca²⁺ free external solutions. Changes in the external concentrations of Na⁺, K⁺ or Cl^?, however, also had some effect upon dopamine currents.     

Comparison of symptomatic and neurophysiological activities of enantiomers of the insecticide 3-phenoxybenzyl 1-(4-ethoxyphenyl)-2,2-dichlorocyclopropane-1-carboxylate

The insecticidal activities of optical isomers of 3-phenoxybenzyl 1-(4-ethoxyphenyl)-2,2-dichlorocyclopropanecarboxylate and related compounds were measured with American cockroaches and their knockdown activities were evaluated with house flies. The activities of the S(?)-isomer of the dichlorocyclopropanecarboxylate were higher than those of the R(+)-isomer. The effects of the compounds on the inward membrane currents induced by a stepdepolarizing pulse in crayfish axonal membranes were examined under voltage clamp conditions by the sucrose gap method. The compounds induced a tail current upon step repolarization of the membrane. The tail current decayed to zero in each record, but developed with time after the start of application of the compound until a steady level was reached. The rate of decay of the tail current observed in axonal membranes treated with the S(?)-isomer was slower than with the R(+)-isomer. The rates of development of the tail current induced by the two isomers were not very different.     

Recent studies on the effects of DDT and pyrethroid insecticides on nervous activity in cockroaches

A summary is given of the results of three sets of electrophysiological experiments on the American cockroach (Periplaneta americana). The effects of DDT and S-bioallethrin [bioallethrin (S)-cyclopentenyl isomer] on single giant axons were studied using the voltage-clamp technique. Whereas both molecules induced long tails of inward (sodium ion) current, the voltage and time-dependency of these tails differed. With DDT, the tail was proportional to the activation of the peak current and decreased with the duration of the pulse, suggesting that the molecules were bound to open sodium channels and delayed their closing. With S-bioallethrin, the voltage dependency of the tail was different from that of the peak current, and the tail current increased exponentially with the duration of the depolarisation, suggesting that the pyrethroid insecticide modified resting (or silent) sodium channels into slowly activating channels. Modified action potentials, mimicking those produced by the two molecules, were computed on the basis of these results. Deltamethrin, one of the most potent pyrethroid insecticides, was applied topically on a leg mechanoreceptor and was found to have little effect on the local ‘receptor potential’ but to inhibit action potential production. The effects of topical applications of deltamethrin on the dorsal part of the abdomen, on nervous activity in the abdominal connectives, were studied under different experimental conditions. The results suggest that the insecticide molecules diffused rapidly through the cuticle, were concentrated in the haemolymph, and eventually reached the central nervous system, where they inhibited nerve activity.     

Determination of unchanged residues of a pyrethroid insecticide,PYR-VU-TO2, in sheep internal organ tissues

During subacute toxicological testing of PYR-VU-TO2, a novel cypermethrin-like pyrethroid with a high (150:1) cis: trans ratio, its distribution in skeletal muscle and internal organ tissues of two groups of experimental sheep was investigated after oral administration of two different doses (1/10 and 1/20 of the LD₁₀). Residual levels of PYR-VU-TO2 were also observed in sheep blood after administration of a single dose of the compound. Homogenized internal organ tissues and blood samples were extracted in light petroleum distillate+acetone for 15 min. After filtration, the extract was cleaned up on a ‘Florisil’^R and sodium sulfate layer. The pyrethroid elution was processed with acetone+light petroleum distillate (2+98 by volume). Recoveries ranged from 80.7 to 93.5%. Unchanged PYR-VU-TO2 residues were determined on a 1.5-m stainless steel packed column under isothermal conditions with an electron-capture detector. The results were evaluated by the calibration curve method. The coefficients of variation were generally lower than 5% and determination limit for PYR-VU-TO2 was 20 μg kg^?1. Mean unchanged PYR-VU-TO2 residues lay within a wide range. After six weeks of treatment the following order of tissue residue levels could be stated: heart>lung>muscle>spleen>kidney>liver>brain, and spleen>muscle>lung>heart>liver>kidney>brain for experimental groups I and II, respectively. Only small differences between the residues in the tissues of males and females were found in the first group (daily PYR-VU-TO2 dose of 50 mg kg^?1 live weight). The experimental females of the second group (daily PYR-VU-TO2 dose of 200 mg kg^?1 body weight) revealed higher levels (other than spleen) than did the males. Maximum PYR-VU-TO2 residues in sheep blood were found 24 h after administration of a single dose. Confirmation of the results was carried out by determination of 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid methyl ester by capillary GC.     

Mode of action of nereistoxin on the neuromuscular transmission in the frog

The effects of nereistoxin on the neuromuscular transmission of the frog have been studied by means of intracellular microelectrode and voltage clamp techniques. Nereistoxin blocked the neuromuscular transmission without causing depolarization of end-plate membranes. A small end-plate potential was elicited upon nerve stimulation. The frequency and amplitude of miniature end-plate potentials decreased after exposure to nereistoxin, and the quantum content of the end-plate potential tended to decrease also. Chemical sensitivity of the end-plate membrane as examined by iontophoretic application of acetylcholine was markedly reduced. Both sodium and potassium components of the end-plate current were suppressed almost equally by application of nereistoxin. It is concluded that inhibition of the mechanisms whereby the end-plate membrane conductances to sodium and potassium ions are increased is the major action of nereistoxin responsible for neuromuscular blockade.     

Membrane partitioning of organophosphorus and organochlorine insecticides and its implications for mechanisms of toxicity

Partition coefficients of organophosphorus and organochlorine insecticides were determined in several types of membrane. Insecticide partitioning varied among the membranes under study, depending on temperature, cholesterol content and on the physico-chemical profiles of membrane components and the insecticides themselves. The maximal partitions for DDT, lindane, parathion and malathion in egg phosphatidylcholine bilayers over the temperature range from 10 to 37°C (at which the lipid is in the liquid-crystalline state) were about 260000, 2000, 1000 and 120, respectively. Incorporation of 50 mol% cholesterol in egg phosphatidylcholine bilayers dramatically decreased the partition and almost abolished the temperature effect. First-order phase transitions of dimyristoyl-, dipalmitoyl- and distearoylphosphatidylcholines (DMPC, DPPC and DSPC) were accompanied by a sharp increase in insecticide partition. Furthermore, the insecticides under study were more easily accommodated in bilayers of short-aliphatic-chain lipids, since higher values of partition were obtained in DMPC bilayers. Partition values in native membranes depended considerably on the membrane type and composition and were higher in sarcoplasmic reticulum (SR) and mitochondria than in brain microsomes, myelin and erythrocytes. Identical results were obtained in related liposomes of total extracted lipids, although the absolute partitions showed decreased values. In general, the incorporation of insecticides correlated reasonably with the cholesterol content of the membranes. The order of partitioning of the above insecticides did not run parallel with their toxicity to mammals and, both in model and in native membranes, followed the sequence: DDT ? lindane > parathion > malathion.     

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