Action of avermectin B1a on the leg muscles and the nervous system of the American cockroach

The action of avermectin was studied in the leg muscle and the central nervous system of the American cockroach, Periplanata americana L. Avermectin at a low concentration (10^?7M) causes a failure of the leg muscles to respond to external stimuli within 30 min without affecting the magnitude of contraction. Avermectin was found to stimulate Cl^? uptake by the leg muscles within 4 min at 10^?7M. The threshold concentration to cause such stimulation was on the order of 10^?8M. This stimulatory action could be antagonized by picrotoxinin (10^?4M) and to a lesser extent by bicuculline methiodide (10^?4M). The phenomenon is observable under both Na⁺-free and K⁺-free conditions. It was concluded that the action of avermectin is to open the chloride channel on the plasma membrane. This action of avermectin does not seem to be mediated through GABA, GABA receptors, diazepine receptors, or picrotoxinin receptor in this insect species, and therefore suggests that avermectin directly attacks the chloride channel proper both in the central nervous and the neuromuscular systems.     

Mechanism of neuromuscular block by chlordimeform

The mechanism of action of chlordimeform on the frog sciatic nerve-sartorius muscle preparation has been studied by means of microelectrodes. Chlordimeform (0.1 mM) suppresses the amplitude of spontaneous miniature end-plate potentials without much changing their frequency. At 1 mM, it completely blocks the miniature end-plate potentials. The resting membrane potential is not affected. The end-plate potential evoked by nerve stimulation is also suppressed, while the action potential from the nerve terminal is not impaired. The sensitivity of the end-plate membrane to the transmitter substance as examined by iontophoretic applications of acetylcholine is effectively decreased by chlordimeform. The quantal content of transmitter release is not affected. It is concluded that the block of neuromuscular transmission by chlordimeform is due primarily to a depression of the end-plate sensitivity to the transmitter.     

Potassium uptake in atrazine-treated roots of sensitive and resistan plants

The action of atrazine and its biodegradation products on the membrane transport of potassium in roots was evaluated in both sensitive and resistant plants. Excised roots of maize and oat showed inhibition of potassium uptake efficiency in the presence of 1.4 × 10^?4M atrazine and 1.4 × 10^?4M deethylated atrazine. Other biodegradation products such as 2-chloro-4-amino-6-ethylamino-1,3,5-triazine,2-chloro-4,6-,bisamino-1,3,5-triazine, and 2-chloro-4-amino-1,3,5-triazine showed no inhibitory effect on the K⁺ uptake capacity. Two maize hybrids showing different uptake efficiency were inhibited differently by atrazine. We suggest that atrazine and deethylated atrazine inhibited the K⁺ transport interacting directly with the plant cell membranes without discerning between resistant and sensitive plants.     

Effects of chlordimeform on vascular smooth muscle

The effects of chlordimeform on agonist-induced contractions of rabbit aortic strips and on calcium flux in the deadventitiated strips have been studied. Chlordimeform antagonizes contractions induced by several vasoactive agents, with the order of antagonism being potassium . histamine > serotonin > norepinephrine, and decreases the rate of contraction of strips exposed to each of the four agonists. Muscles contracted by each of the agonists are rapidly relaxed by application of chlordimeform, although in the case of norepinephrine the relaxation only proceeds to approximately one-half of the maximal tension. The rate of washout of ⁴⁵Ca from the media-intimal layer of the rabbit aorta was increased by chlordimeform, but the norepinephrine-induced decrease in the rate of washout of ⁴⁵Ca was not affected by chlordimeform. Chlordimeform did not affect ⁴⁵Ca uptake by the media-intimal strips. The results of this study indicate that chlordimeform probably interferes with loosely bound calcium to a greater extent than it does with more tightly bound stores.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

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.     

Differential inhibition of potassium ion absorption by difenzoquat in wild oat and cereals

Over a concentration range of 5.0 × 10^?6?7.5 × 10^?4M, the selective herbicide difenzoquat (1,2-dimethyl-3,5-diphenyl-1H-pyrazolium) caused more pronounced inhibition of potassium ion (K⁺) absorption by excised seedling roots of susceptible wild oat (Avena fatua L.) compared to those of tolerant barley (Hordeum vulgare L. cv. Bonanza) or wheat (Triticum aestivum L. cv. Neepawa). At 2.5 × 10^?5M difenzoquat, the relative inhibition of K⁺ (⁸⁶Rb) absorption by wild oat root segments inceased from 30% with a 10-min uptake period to 75% with an uptake period of 90 min, whereas no inhibition at all was evident for wheat root segments even after a 90-min exposure to the herbicide. An ion efflux compartmental analysis procedure demonstrated that difenzoquat did not affect the passive permeability properties of the plasma membrane of wild oat root cells. The experimental findings indicated that difenzoquat interfered directly with the process of active ion transport across the plasma membrane of root cells.     

Studies on the action of chlordimeform in cockroaches

The effects of chlordimeform [N-(2-methyl-4-chlorophenyl)-N′,N′-dimethylformamidine] on amine regulatory mechanisms in insects were studied. Chlordimeform inhibits monoamine oxidase (MAO) from cockroach heads in vitro, and the MAO substrates serotonin and norepinephrine accumulate in poisoned insects in vivo. Chlordimeform synergizes the toxicity of tryptamine, another MAO substrate. The significance of these findings in relation to the mode of action of chlordimeform is discussed.     

Modification of frescon action in Lymnaea stagnalis smooth and cross-striated muscles by agents that alter potential-dependent or receptor-operated calcium permeability

The calcium entry blockers sodium nitroprusside (10^?2M) and sodium pentobarbital (5 × 10^?3M) had different effects on high-potassium-induced and Frescon-induced contractures in a smooth and a cross-striated muscle of the snail Lymnaea stagnalis. This suggests that Frescon causes an increase in sarcolemmal calcium permeability by a mechanism which is different from that utilized by high extracellular potassium.     

Trifluralin effects on carrot callus tissue

The effect of trifluralin on the growth, oxygen uptake, and adenosine phosphates level of carrot (Daucus carota L.) callus tissue was determined over a period of 18 days after subculture. The herbicide (10^?4 and 10^?5M) reduced fresh weight gain significantly; the reduction was less with lower trifluralin concentrations. Dry weight accumulation was not inhibited until after Day 6 of the test period and thereafter was reduced by all concentrations tested. Oxygen consumption was inhibited by trifluralin (10^?4 and 10^?5M) throughout the test period. Concentrations of 10^?6.10^?7, and 10^?8M produced variable effects. The response profile of O₂ consumption in the presence of dinitrophenol was different from that of trifluralin. Analysis of adenosine phosphates level gave no clear response trend. Energy charge values of 0.7 to 0.85 were obtained for untreated tissue. Trifluralin had no effect on energy charge until Day 9 (10^?4M) and after Day 15 (all concentrations).     

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.     

DDT inhibition of Ca-ATPase of the peripheral nerves of the American lobster

A Ca-ATPase highly sensitive to DDT has been found in peripheral nerves of lobster, Homarus americanus. The observed I₅₀ for this Ca-ATPase toward DDT is on the order of 10^?9M and has a low temperature quotien. The ATPase seems to work over a wide range of ATP concentrations. It is stimulated by Ca²⁺ (optimum 0.1 mM) and shows sensitivity to Na⁺ (optimum 20 mM) and K⁺ (optimum 20 mM) ions. The fact that it is highly sensitive to ruthenium red (I₅₀ = 10 μM) suggests that the enzyme is a Ca-ATPase and not a Mg-ATPase. Furthermore the enzyme is not a CaMg-ATPase, since the presence of Mg²⁺ along with Ca²⁺ ion is not required for its activity. DDT is found to inhibit the process of Ca²⁺ binding in the axonic membrane only in the presence of ATP. The evidence suggests the important role of the Ca-ATPase in regulating Ca²⁺ concentrations in the membrane. The possible significance of DDT inhibition of the ATPase is discussed.     

Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase,diacylglycerol kinase,and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10^?2–10^?6M) and diazoxon (10^?2–10^?4M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10^?2M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10^?2–10^?3M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.     

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.     

Actions of formamidines,local anesthetics,octopamine and related compounds upon the electrical activity of neurohaemal organs of the stick insect (Carausius morosus) and sense organs of fly larvae (Musca domestica; Calliphora erythrocephala)

The pesticides, chlordimeform and amitraz, and their metabolites, demethylchlordimeform, N¹-(2,4-dimethylphenyl)-N-methylformamidine, and 2,4-dimethylformanilide, are effective at concentrations as low as 3 μM in raising the firing rate of endogenously active neurosecretory fibres in the isolated neurohaemal organs of Carausius morosus. Molecules such as bunamidine and cetrimide, with cationic detergent properties, produced sporadic bursting which did not elevate the overall firing rate to any great extent. Indeed, bunamidine could induce complete block of action potentials at concentrations as low as 30 μM. The local anesthetics, procaine, lidocaine, and benzocaine, do not induce block of activity at least up to a level of 1 mM. They have no effect at concentrations lower than 100 μM. Between 100 μM and 1 mM lidocaine and benzocaine produce a small increase in firing rate. Procaine produced a pronounced increase in the frequency of firing. The phenolic amines, octopamine, synephrine, and tyramine, markedly increased electrical activity. The catecholamines, dopamine, noradrenaline, and adrenaline, by contrast, only produced a weak excitation. Neither α- nor β-adrenergic blocking agents were effective in antagonizing the electrical activity induced by chlordimeform or phenolic amines until relatively high concentrations of about 1 mM were used. Chlordimeform was able to induce high-frequency bursts from sense organs associated with the epidermis and body-wall musculature in larvae of Musca domestica and Calliphora erythocephala. Octopamine did not induce any similar bursting activity in these sense organs. These results are discussed in relationship to current views on the mode of action of the N-aryl amidines. It is concluded that the excitatory effects of these compounds upon neurohaemal organs and sense organs are more likely to result from a direct action upon voltage sensitive channels of the nerve membranes, rather than by an effect mediated by interactions with octopamine receptors.     

Interaction of formamidine pesticides with insect neural octopamine receptors: Effects on radioligand binding and cyclic AMP production

The formamidines chlordimeform, demethylchlordimeform (DCDM), amitraz and BTS-2727l were tested for their respective abilities to inhibit [³H]mianserin binding in membrane preparations of cockroach brain and, in the same tissue, to stimulate octopamine-sensitive receptors coupled to adenylate cyclase. Analysis of [³H]mianserin binding indicated negative cooperativity between two binding sites with respective K_d and B_max values of 3.82 mM and 0.886 pmol (mg protein)^?1 for a high-affinity site and 218 nM and 13.56pmol (mg protein)^?1 for a low-affinity site. DCDM, BTS-27271 and amitraz inhibit [³H]mianserin binding with IC₅₀ values similar to those obtained with octopamine and the μ-adrenergic antagonist, phentolamine, whereas chlordimeform is a poor competitor for the binding sites. Similarly DCDM, BTS-27271 and amitraz elevate cyclic AMP production in brain membrane preparations in a dose-dependent manner with K_a values of 0.32 uM, 1.5 uM and 3.4 uM respectively, whereas chlordimeform was again without effect. The formamidine-mediated responses were fully additive with the evaluation of cyclic AMP obtained using the maximal concentration of dopamine but not with octopamine-mediated increases; thus the formamidine effects appear to be expressed through partial agonism of octopamine receptors that are coupled to adenylate cyclase.     

Pyrethroid insecticides: Potent,stereospecific enhancers of mouse brain sodium channel activation

Deltamethrin and NRDC 157, pyrethroid insecticides that produce different poisoning syndromes in mammals, enhanced veratridine-dependent, sodium channel-mediated ²²Na⁺ uptake in mouse brain synaptosomes. Concentrations producing half-maximal enhancement were 2.5 × 10^?8M (deltamethrin) and 2.2 × 10^?7M (NRDC 157). This effect was stereospecific: The nontoxic 1S enantiomers had no significant effect on veratridine-dependent activation. At high deltamethrin concentrations, enhancement was maximal at 5 × 10^?5?1 × 10^?4M veratridine. Pyrethroid enhancement was completely blocked by 5 × 10^?6M tetrodotoxin, and neither pyrethroid affected ²²Na⁺ uptake in the absence of veratridine at concentrations up to 1 × 10^?5M. The relative potencies of deltamethrin and NRDC 157 in the synaptosomal sodium channel assay agree well with their relative acute toxicities to mice when administered by intracerebral injection. These findings demonstrate that pyrethroids exemplifying both characteristic poisoning syndromes are potent, stereospecific modifiers of sodium channel function in mammalian brain.     

Effects of herbicidal carbamates on mitochondria and chloroplasts

At concentrations near 2 × 10^?4M, barban, chlorpropham, and phenmedipham are inhibitors of the electron transfer in potato and mung bean mitochondria. The inhibition seems to be localized in the flavoprotein region. It affects preferentially the exogenous NADH dehydrogenation, in potato mitochondria (I₅₀, 10^?4M). Succinate dehydrogenation is less inhibited. At noninhibiting concentrations, the studied carbamates cannot uncouple the oxidative phosphorylations. Photosynthesis is completely inhibited by 2.10^?7M phenmedipham, 5 × 10^?5M barban, and 2 × 10^?4M chlorpropham. The inhibition takes place at the PS II level. Moreover, barban and chlorpropham are uncouplers of the photophosphorylations for concentrations between 5 × 10^?5 and 5 × 10^?4M. The effects observed on mitochondrial respiration can also be found on respiration of Acer cultured cells. The effects on isolated chloroplast photosynthesis are also observed for slightly higher concentrations on cultured Chlorella and on pea and oat leaf fragments.     

Degradation of ioxynil to CO2 in soil

Degradation of ioxynil (4-hydroxy-3,5-diiodobenzonitrile) to CO₂ was detected in a clay loam, high organic matter content soil. The majority of radioactivity was recovered as ¹⁴CO₂ from both ring-labeled and cyano-labeled ioxynil; however, ¹⁴CO₂ was always released from cyano-labeled ioxynil at a much faster initial rate. No ¹⁴CO₂ was released in treated sterile soil, either aerobically or anaerobically. Production of ¹⁴CO₂ from cyanolabeled and ring-labeled ioxynil was greatly inhibited by HgCl₂ (10^?5M), and p-chloromercuribenzoate (5 × 10^?5M), but slightly inhibited by ferricyanide (10^?4M). No ¹⁴CO₂ was evolved from ring-labeled ioxynil under anaerobic conditions. These observations indicated that the degradation of ioxynil to CO₂ in soil was a microbial action and was oxygen dependent. This is consistent with the known mechanism of oxygenases in degrading benzene rings. Anaerobically, a small amount of ¹⁴CO₂ was released from cyano-labeled ioxynil. Thin-layer chromatographic analyses of the culture supernatant revealed that 3,5-diiodo-4-hydroxybenzamide and 3,5-diiodo-4-hydroxybenzoic acid were intermediate metabolites.     

Activation of an O-ethyl S-n-propyl phosphorothiolate,TIA-230, in the central nerve of Spodoptera larvae

TIA-230, O-[1-(4-chlorophenyl)-4-pyrazolyl] O-ethyl S-propyl phosphorothiolate, showed strong insecticidal activity against Spodoptera larvae, in spite of its weak in vitro anti-AChE activity. Head AChE of Spodoptera was, however, inhibited with the progress of TIA-230 intoxication. When the isolated central nerve cord was incubated with TIA-230, AChE in the tissue was strongly inhibited even by concentration (10^?5M) lower than in vitro I₅₀ against AChE (10^?4M). The frequency of spontaneous firing of the nerve cord was increased by treatment of TIA-230 at low concentrations (10^?6–10^?5M) after a latent time of several minutes. The firing was increased by fenitroxon, but without the latent time. The length of the latent time agreed well with the time necessary for rising the inhibition of nerve cord AChE by TIA-230. AChE inhibition of TIA-230 in the nerve cord was reduced by the treatment of piperonyl butoxide, an inhibitor of mixed-function oxidases. From these results, TIA-230 was regarded as being activated oxidatively in the nerve cord to inhibit AChE. Profenofos was also activated in the nerve cord. It was concluded, therefore, that O-ethyl S-n-propyl phosphorothiolate insecticides were activated in the central nerve of the insect.