Corrigendum: Pharmacological effects of monoterpene carveol on the neuromuscular system of nematodes and mammals.

[This corrects the article DOI: 10.3389/fphar.2024.1326779.].

Design of Novel TRPA1 Agonists Based on Structure of Natural Vasodilator Carvacrol-In Vitro and In Silico Studies.

Considering the escalating global prevalence and the huge therapeutic demand for the treatment of hypertension, there is a persistent need to identify novel target sites for vasodilator action. This study aimed to investigate the role of TRPA1 channels in carvacrol-induced vasodilation and to design novel compounds based on carvacrol structure with improved activities. In an isolated tissue bath experiment, it was shown that 1 µM of the selective TRPA1 antagonist A967079 significantly (p < 0.001) reduced vasodilation induced by 3 mM of carvacrol. A reliable 3D-QSAR model with good statistical parameters was created (R2 = 0.83; Q2 = 0.59 and Rpred2 = 0.84) using 29 TRPA1 agonists. Obtained results from this model were used for the design of novel TRPA1 activators, and to predict their activity against TRPA1. Predicted pEC50 activities of these molecules range between 4.996 to 5.235 compared to experimental pEC50 of 4.77 for carvacrol. Molecular docking studies showed that designed molecules interact with similar amino acid residues of the TRPA1 channel as carvacrol, with eight compounds showing lower binding energies. In conclusion, carvacrol-induced vasodilation is partly mediated by the activation of TRPA1 channels. Combining different in silico approaches pointed out that the molecule D27 (2-[2-(hydroxymethyl)-4-methylphenyl]acetamide) is the best candidate for further synthesis and experimental evaluation in in vitro conditions.

Pharmacological effects of monoterpene carveol on the neuromuscular system of nematodes and mammals.

The control of parasitic nematode infections relies mostly on anthelmintics. The potential pharmacotherapeutic application of phytochemicals, in order to overcome parasite resistance and enhance the effect of existing drugs, is becoming increasingly important. The antinematodal effects of carveol was tested on the free-living nematode Caenorhabditis elegans and the neuromuscular preparation of the parasitic nematode Ascaris suum. Carveol caused spastic paralysis in C. elegans. In A. suum carveol potentiated contractions induced by acetylcholine (ACh) and this effect was confirmed with two-electrode voltage-clamp electrophysiology on the A. suum nicotinic ACh receptor expressed in Xenopus oocytes. However, potentiating effect of carveol on ACh-induced contractions was partially sensitive to atropine, indicates a dominant nicotine effect but also the involvement of some muscarinic structures. The effects of carveol on the neuromuscular system of mammals are also specific. In micromolar concentrations, carveol acts as a non-competitive ACh antagonist on ileum contractions. Unlike atropine, it does not change the EC50 of ACh, but reduces the amplitude of contractions. Carveol caused an increase in Electrical Field Stimulation-evoked contractions of the isolated rat diaphragm, but at higher concentrations it caused an inhibition. Also, carveol neutralized the mecamylamine-induced tetanic fade, indicating a possibly different pre- and post-synaptic action at the neuromuscular junction.

Action of Carvacrol on Parascaris sp. and Antagonistic Effect on Nicotinic Acetylcholine Receptors.

Parascaris sp. is the only ascarid parasitic nematode in equids and one of the most threatening infectious organisms in horses. Only a limited number of compounds are available for treatment of horse helminthiasis, and Parascaris sp. worms have developed resistance to the three major anthelmintic families. In order to overcome the appearance of resistance, there is an urgent need for new therapeutic strategies. The active ingredients of herbal essential oils are potentially effective antiparasitic drugs. Carvacrol is one of the principal chemicals of essential oil from Origanum, Thymus, Coridothymus, Thymbra, Satureja and Lippia herbs. However, the antiparasitic mode of action of carvacrol is poorly understood. Here, the objective of the work was to characterize the activity of carvacrol on Parascaris sp. nicotinic acetylcholine receptor (nAChR) function both in vivo with the use of worm neuromuscular flap preparations and in vitro with two-electrode voltage-clamp electrophysiology on nAChRs expressed in Xenopus oocytes. We developed a neuromuscular contraction assay for Parascaris body flaps and obtained acetylcholine concentration-dependent contraction responses. Strikingly, we observed that 300 µM carvacrol fully and irreversibly abolished Parascaris sp. muscle contractions elicited by acetylcholine. Similarly, carvacrol antagonized acetylcholine-induced currents from both the nicotine-sensitive AChR and the morantel-sensitive AChR subtypes. Thus, we show for the first time that body muscle flap preparation is a tractable approach to investigating the pharmacology of Parascaris sp. neuromuscular system. Our results suggest an intriguing mode of action for carvacrol, being a potent antagonist of muscle nAChRs of Parascaris sp. worms, which may account for its antiparasitic potency.

Interaction of agonists of a different subtype of the nAChR and carvacrol with GABA in Ascaris suum somatic muscle contractions.

Resistance of parasitic nematodes to anthelmintic drugs is a growing problem in human and veterinary medicine. The molecular mechanisms by which nematodes become resistant are different, but certainly one of the possible processes involves changing the drug binding site on the specific receptor. The significance of changes in individual subtypes of nicotinic acetylcholine receptors (nAChRs) for the development of resistance has not been clarified in detail. This study investigates the interaction of antinematodal drugs, agonist of different types of nAChRs and carvacrol with gamma aminobutyric acid (GABA) on the contractions of parasitic nematode A. suum. In our study, GABA (3 µM) produced significant increase of contractile EC50 value for pyrantel, and nonsignificant for bephenium and morantel, from 8.44 to 28.11 nM, 0.62 to 0.96 µM, and 3.72 to 5.69 nM, respectively. On the other hand, the maximal contractile effect (R max) did not change in the presence of GABA. However, when A. summ muscle flaps were incubated with GABA 3 µM and carvacrol 100 µM, the EC50 value of pyrantel, bephenium, and morantel was increased significantly to 44.62 nM, 1.40 µM, and nonsignificantly to 7.94 nM, respectively. Furthermore, R max decreased by 70, 60, and 65%. Presented results indicate that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional, due to the potential development of resistance.

Potential modulating effect of the Ascaris suum nicotinic acetylcholine receptor (nAChR) by compounds GSK575594A, diazepam and flumazenil discovered by structure-based virtual screening approach.

Parasitic infections are a widespread health problem and research of novel anthelmintic compounds is of the utmost importance. In this study we performed a virtual screening campaign by coupling ligand-based pharmacophore, homology modeling and molecular docking. The virtual screening campaign was conducted using a joined pool of the Drugbank database and a library of purchasable compounds in order to identify drug like compounds with similar pharmacological activity. Our aim was to identify compounds with a potential antihelmintic modulatory effect on nicotinic acetylcholine receptors (nAChR). We derived a 3D pharmacophore model based on the chemical features of known Ascaris suum nAChR modulators. To evaluate the in silico predictions, we tested selected hit compounds in contraction assays using somatic muscle flaps of the Ascaris suum neuromuscular tissue. We tested the modulatory effects of GSK575594A, diazepam and flumazenil hit compounds on nematode contractions induced by acetyl choline (ACh). The compound GSK575594A (3 µM) increased the Emax by 21 % with the EC50 dose ratio of 0.96. Diazepam (100 µM) decreased the Emax by 15 % (1.11 g to 0.95 g) with the EC50 ratio of 1.42 (shifted to the left from 11.25 to 7.93). Flumazenil decreased the EC50 value (from 11.22 µM to 4.88 µM) value showing dose ratio of 2.30, and increased the Emax by 4 % (from 1.54 g to 1.59 g). The observed biological activity was rationalized by molecular docking calculations. Docking scores were calculated against several binding sites within the Ascaris suum homology model. We constructed the homology model using the ACR-16 subunit sequence. The compound GSK575594A showed strong affinity for the intersubunit allosteric binding site within the nAChR transmembrane domain. The binding modes of diazepam and flumazenil suggest that these compounds have a comparable affinity for orthosteric and allosteric nAChR binding sites. The selected hit compounds displayed potential for further optimization as lead compounds. Therefore, such compounds may be useful in neutralizing the growing resistance of parasites to drugs, either alone or in combination with existing conventional anthelmintics.

Carvacrol acts as a potent selective antagonist of different types of nicotinic acetylcholine receptors and enhances the effect of monepantel in the parasitic nematode Ascaris suum.

The neuromuscular system of parasitic nematodes has proven to be an efficient pharmacological target for antihelmintics. Some of the most frequently used antiparasitic drugs are agonists or antagonists of nicotinic acetylcholine receptors (nAChRs). The antinematodal mechanism of action of carvacrol involves the inhibition of parasite muscle contraction. We have examined the interaction of carvacrol with antinematodal drugs that are agonists of different subtypes of nAChRs and monepantel, which is a non-competitive antagonist of this receptor in A. suum. Additionally, we investigated the effect of carvacrol on the muscle type of nAChRs in the mammalian host. As orthosteric agonists of nAChR, pyrantel, morantel and befinijum lead to dose-dependent contractions of the neuromuscular preparation of Ascaris suum. Carvacrol 100 µM decreased the Emax of pyrantel, morantel and bephenium by 29%, 39% and 12 %, 39 % and 12 % respectively. The EC50 ratio was 3.43, 2.95 and 2.47 for pyrantel, morantel and bephinium, respectively. Carvacrol 300 u µM reduces the Emax of pyrantel, morantel and bephenium by 71%, 80% and 75 %, 80 % and 75 % respectively. The EC50 ratio for pyrantel, morantel and bephenium was 3.88, 3.19 and 4.83 respectively. Furthermore, carvacrol enhances the inhibitory effect of monepantel on A. suum contractions, which may have an effective clinical application. On the other hand, tested concentrations of carvacrol did not significantly affect the EFS-induced contractions of the rat diaphragm, indicating a lack of interaction with the postsynaptic nAChR at the muscle end plate in mammals, but the highest concentration (300 µM) caused a clear tetanic fade. Carvacrol exhibited a time and dose-dependent effect on the Rota-rod performances of rats with a high value of the ED50 (421.6 mg/kg). In our research, carvacrol dominantly exhibited characteristics of a non-competitive antagonist of nAChR in A. suum, and enhances the inhibitory effect of monepantel. The combination of monepantel and carvacrol may be clinically very effective, and the carvacrol molecule itself can be used as a promising platform for the development of new anthelmintic drugs.

Menthol acts as a positive allosteric modulator on nematode levamisole sensitive nicotinic acetylcholine receptors.

The ongoing and widespread emergence of resistance to the existing anti-nematodal pharmacopeia has made it imperative to develop new anthelminthic agents. Historically, plants have been important sources of therapeutic compounds and offer an alternative to synthetic drugs. Monoterpenoids are phytochemicals that have been shown to produce acute toxic effects in insects and nematodes. Previous studies have shown nicotinic acetylcholine receptors (nAChRs) to be possible targets for naturally occurring plant metabolites such as carvacrol and carveol. In this study we examined the effects of monoterpenoid compounds on a levamisole sensitive nAChR from Oesophagostomum dentatum and a nicotine sensitive nAChR from Ascaris suum. We expressed the receptors in Xenopus laevis oocytes and used two-electrode voltage-clamp to characterize the effect of various compounds on these cys-loop receptors. At 100 µM the majority of these compounds acted as antagonists. Interestingly, further experiments revealed that both 0.1 µM and 10 µM menthol potentiated acetylcholine and levamisole responses in the levamisole sensitive receptor but not the nicotine sensitive receptor. We also investigated the effects of 0.1 µM menthol on the contractility of A. suum somatic muscle strips. Menthol produced significant potentiation of peak contractions at each concentration of acetylcholine. The positive allosteric modulatory effects of menthol in both in vivo and in vitro experiments suggests menthol as a promising candidate for combination therapy with cholinergic anthelmintics.

Monepantel is a non-competitive antagonist of nicotinic acetylcholine receptors from Ascaris suum and Oesophagostomum dentatum.

Zolvix® is a recently introduced anthelmintic drench containing monepantel as the active ingredient. Monepantel is a positive allosteric modulator of DEG-3/DES-2 type nicotinic acetylcholine receptors (nAChRs) in several nematode species. The drug has been reported to produce hypercontraction of Caenorhabditis elegans and Haemonchus contortus somatic muscle. We investigated the effects of monepantel on nAChRs from Ascaris suum and Oesophagostomum dentatum heterologously expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp electrophysiology, we studied the effects of monepantel on a nicotine preferring homomeric nAChR subtype from A. suum comprising of ACR-16; a pyrantel/tribendimidine preferring heteromeric subtype from O. dentatum comprising UNC-29, UNC-38 and UNC-63 subunits; and a levamisole preferring subtype (O. dentatum) comprising UNC-29, UNC-38, UNC-63 and ACR-8 subunits. For each subtype tested, monepantel applied in isolation produced no measurable currents thereby ruling out an agonist action. When monepantel was continuously applied, it reduced the amplitude of acetylcholine induced currents in a concentration-dependent manner. In all three subtypes, monepantel acted as a non-competitive antagonist on the expressed receptors. ACR-16 from A. suum was particularly sensitive to monepantel inhibition (IC50 values: 1.6 ±â€¯3.1 nM and 0.2 ±â€¯2.3 µM). We also investigated the effects of monepantel on muscle flaps isolated from adult A. suum. The drug did not significantly increase baseline tension when applied on its own. As with acetylcholine induced currents in the heterologously expressed receptors, contractions induced by acetylcholine were antagonized by monepantel. Further investigation revealed that the inhibition was a mixture of competitive and non-competitive antagonism. Our findings suggest that monepantel is active on multiple nAChR subtypes.

Two opposite dose-dependent effects of diazinon on the motor activity of the rat ileum.

Acute poisoning with OPs may lead to a range of neurological effects, which cannot be explained by AChE inhibition alone. Several OPs interact directly with cholinergic receptors in mammals, but such data does not exist for invertebrates. The aim of current study was to investigate the direct and indirect effects of diazinon on the contractions of rat ileum and to compare those effects on the nervemuscle preparation of the Ascaris suum. In the presence of increasing concentrations of diazinon (3, 10 and 30nM), EFS-induced ileal contractions were increased significantly. In the same preparation, diazinon 3nM, significantly increased contractions induced by EFS, but did not affect the contractions caused by 5MFI. Contrarily, 1µM of diazinon significantly and reversibly inhibited the EFS-induced ileal contractions. Diazinon exhibited competitive and non-competitive inhibitions of 5MFI induced contractions. The control EC50 of 5MFI was 2.48µM with Rmax=1.88g. In the presence of diazinon, EC50 was 12.45µM, while Rmax was reduced to 0.43g. After washing, the EC50 and Rmax values were again closer to the control level (3.80µM and 1.04g). Diazinon 1µM did not inhibit Ascaris suum contractions caused by ACh, but it increased the Rmax. Diazinon in our study exhibits two opposite effects on the motor activity of the ileum. In low nanomolar concentrations the dominat is its effect on AChE and the stimulation of contractions. Furthermore, in concentrations that approach micromolar values diazinon has a direct inhibitory effect on muscarinic receptors. The direct inhibitory effect of diazinon on A. suum contractions was not found.

DIAGNOSIS AND THERAPY OF LIVER FLUKE (FASCIOLOIDES MAGNA) INFECTION IN FALLOW DEER (DAMA DAMA) IN SERBIA.

Giant liver fluke ( Fascioloides magna ) infection is an important health problem of cervids in southeastern Europe. We measured the prevalence and intensity of infection with F. magna in a fenced area near the Danube River in the South Backa District of Serbia. Parasitologic, pathomorphologic, and histopathologic examinations were conducted from November 2007 to February 2008, beginning with a population of 127 adult fallow deer ( Dama dama ). After a positive diagnosis, therapy with triclabendazole-medicated corn was applied. Deer were treated at four baiting stations, using medicated feed providing triclabendazole at an estimated dose of 10-14 mg/kg of body weight per deer. Treatment lasted for 7 d in early February 2008 and an additional 7 d 2 wk later. For the complete success of pharmacotherapy it was necessary to prevent any contact of deer with the snail intermediate host ( Galba truncatula ). Intervention in the habitat, removing grass and low vegetation, and draining ponds reduces the possibility of contact. Six months after the treatment, livers of hunted deer were reddish, with fibrous tracks; pigmentation and cysts in the parenchyma were surrounded by a fibrous capsule and their fecal samples contained no eggs of F. magna . Over the following years, livers of hunted deer were negative, and the last control cull in March 2015 confirmed complete absence of infection. We reconfirmed the presence of giant liver flukes in fallow deer in Serbia, apparently the result of natural spread across the Danube from Hungary and Croatia. We also report that the treatment of deer with triclabendazole-medicated corn is an effective method for administration of therapeutic doses of drug in semicaptive deer. Interventions in the environment are necessary to prevent recontact of deer with habitats used by the snail intermediate host, and enable the success of the therapy.

Pharmacological and morphological characteristics of the muscular system of the giant liver fluke (Fascioloides magna - Bassi 1875).

Motility is required for feeding, reproduction and maintenance of the fluke in the host's liver. According to that, the neuromuscular system can be an attractive drugable target for chemotherapy. Musculature of the Fascioloides magna is organized into three layers, an outer circular layer, beneath this layer the longitudinal layer, and third, the oblique, or diagonal layer underlies the longitudinal layer. In our study, the administration of atropine or caffeine did not cause classic muscle contractions of F. magna muscle strips. However, the Electrical Field Stimulation (EFS) induced stable and repeatable contractions, which enabled us to examine their sensitivity to the various substances. Acetylcholine (ACh) (300 µM and 1 mM), caused only a slight relaxation, without affecting the amplitude of spontaneous contractions or the amplitude of contractions induced by EFS. Contrary to that, atropine (100 µM) caused a significant increase in the basal tone and an increase of EFS-induced contractions. If acetylcholine is an inhibitory neurotransmitter in trematodes, the described effects of atropine are achieved by the blockade of inhibitory neurotransmission. On the other hand, with respect to the process of excitation-contraction coupling, the plant alkaloid ryanodine (30 µM) significantly reduced the basal tone, as well as EFS-induced contractions of F. magna muscle strips. Ryanodine inhibited the potentiating effect of atropine on the basal tone and contractions caused by EFS, which indicates that the contractile effect of atropine is dependent on Ca(++) release from intracellular stores. Caffeine (500 µM) caused relaxation of fluke muscle strips and at the same time significantly enhanced the EFS-induced contractions. Both effects of caffeine can be explained by entry of extracellular Ca(++) into muscle cells. The muscle contractility of F. magna depends both on the entry of extracellular calcium, and calcium release from intracellular stores, which are under the control of RyRs. Our results also suggest that antitrematodal drugs could potentially be developed from substances with selective anti-cholinergic activity.

Interaction of carvacrol with the Ascaris suum nicotinic acetylcholine receptors and gamma-aminobutyric acid receptors, potential mechanism of antinematodal action.

Essential plant oils (or their active principles) are safe to use and a potentially attractive alternative to current antiparasitic drugs. In the present study, we tested the effects of carvacrol on the isolated tissues of Ascaris suum and investigated potential interactions with other antiparasitic drugs. We used somatic muscle flaps for contraction assays, as well as for electrophysiological investigations. Carvacrol 300 µM highly significantly inhibited contractions caused by 1, 3, 10, 30, and 100 µM of ACh (p = 0.0023, p = 0.0002, p = 0.0002, p < 0.0001, and p < 0.0001). The control EC50 for acetylcholine was 8.87 µM (log EC50 = 0.95 ± 0.26), while R max was 2.53 ± 0.24 g. The EC50 of acetylcholine in the presence of 300 µM of carvacrol was 27.71 µM (log EC50 = 1.44 ± 0.28) and the R max decreased to 1.63 ± 0.32 g. Furthermore, carvacrol highly significant potentiates inhibitory effect of GABA and piperazine on the contractions induced by ACh. However, carvacrol (100 and 300 µM), did not produce any changes in the membrane potential or conductance of the A. suum muscle cell. While, 300 µM of carvacrol showed a significant inhibitory effect on ACh-induced depolarization response. The mean control depolarization was 13.58 ± 0.66 mV and decreased in presence of carvacrol to 4.50 ± 1.02 mV (p < 0.0001). Mean control Δg was 0.168 ± 0.017 µS, while in the presence of 300 µM of carvacrol, Δg significantly decreased to 0.060 ± 0.018 ΔS (p = 0.0017). The inhibitory effect on contractions may be the explanation of the antinematodal potential of carvacrol. Moreover, inhibition of depolarizations caused by ACh and reduction of conductance changes directly points to an interaction with the nAChR in A. suum.

Derquantel and abamectin: effects and interactions on isolated tissues of Ascaris suum.

Startect(®) is a novel anthelmintic combination of derquantel and abamectin. It is hypothesized that derquantel and abamectin interact pharmacologically. We investigated the effects of derquantel, abamectin and their combination on somatic muscle nicotinic acetylcholine receptors and pharyngeal muscle glutamate gated chloride receptor channels of Ascaris suum. We used muscle-strips to test the effects of abamectin, derquantel, and abamectin+derquantel together on the contraction responses to different concentrations of acetylcholine. We found that abamectin reduced the response to acetylcholine, as did derquantel. In combination (abamectin+derquantel), inhibition of the higher acetylcholine concentration response was greater than the predicted additive effect. A two-micropipette current-clamp technique was used to study electrophysiological effects of the anthelmintics on: (1) acetylcholine responses in somatic muscle and; (2) on l-glutamate responses in pharyngeal preparations. On somatic muscle, derquantel (0.1-30µM) produced a potent (IC50 0.22, CI 0.18-0.28µM) reversible antagonism of acetylcholine depolarizations. Abamectin (0.3µM) produced a slow onset inhibition of acetylcholine depolarizations. We compared effects of abamectin and derquantel on muscle preparations pretreated for 30min with these drugs. The effect of the combination was significantly greater than the predicted additive effect of both drugs at higher acetylcholine concentrations. On the pharynx, application of derquantel produced no significant effect by itself or on responses to abamectin and l-glutamate. Abamectin increased the input conductance of the pharynx (EC50 0.42, CI 0.13-1.36µM). Our study demonstrates that abamectin and derquantel interact at nicotinic acetylcholine receptors on the somatic muscle and suggested synergism can occur.

Ivermectin effects on motor coordination and contractions of isolated rat diaphragm.

Ivermectin, the antiparasitic drug from the macrocyclic lactones class raises attention due to its high efficiency against nematodes and arthropods and very specific toxic and side effects that it may produce in host. Dominant clinical symptoms of adverse effects and toxicity of ivermectin in animals are tremor, ataxia, CNS depression and coma which often results in mortality. In our study increasing intravenous doses of ivermectin, (6 or more times higher than therapeutic dose: 1.25, 2.5, 3.75, 5.0, 6.25 and 7.5 mg/kg), caused dose-dependent disturbance of motor coordination in treated rats. The median effective dose (ED50) that was able to impair the rota-rod performance in rats treated 3 min before testing was 2.52 mg/kg. This effect weakens over time, while in the rats treated 60 min before the rota-rod test, ED50 of ivermectin was 4.21 mg/kg. Whereas, all tested doses of ivermectin did not cause any other clinical symptoms of toxicity. Ivermectin has no effect on the contractions of isolated diaphragm caused by the EFS, which effectively blocked mecamylamine (100 µM) and pancuronium (1 and 2 µM). Effect on motor coordination is the first detectable clinical symptom of ivermectin toxicity and apparently is a result of its central effects.

Central and peripheral neurotoxic effects of ivermectin in rats.

Ivermectin is considered a very safe drug; however, there are reports of toxic effects in particularly sensitive populations or due to accidental overdose. The aim of this study was (1) to further characterize the central and peripheral toxic effects of ivermectin in animals and (2) to determine possible therapeutic strategies for use in cases of ivermectin poisoning. We tested the effects of experimental doses of ivermectin previously reported to cause various intensities of CNS depression. However, in our study, ivermectin at 2.5, 5.0 and 7.5 mg/kg i.v. did not produce visible CNS depression in rats and 10 mg/kg resulted in sleepiness and staggering 10 to 40 min after application, while a dose of 15 mg/kg caused CNS depression very similar to general anesthesia. Ivermectin dose-dependently potentiates thiopentone-induced sleeping time in rats. Flumazenil (0.2 mg/kg), the benzodiazepine antagonist, did not affect the action of thiopentone; however, it significantly reduced sleeping time in rats treated with a combination of ivermectin (10 mg/kg) and thiopentone (25 mg/kg; from 189.86 ± 45.28 min to 83.13 ± 32.22 min; mean ± SD). Ivermectin causes an increase in the tonus (EC(50)=50.18 µM) and contraction amplitude (EC(50)=59.32 µM) of isolated guinea pig ileum, very similar to GABA, but without the initial relaxation period. These effects are dose-dependent and sensitive to atropine. Our results confirm the central and peripheral GABAergic properties of ivermectin in mammals and also indicate involvement of the cholinergic system in its toxicity. In addition, the results suggest that flumazenil and atropine have potential clinical roles in the treatment of ivermectin toxicity.

Effects of the muscarinic agonist, 5-methylfurmethiodide, on contraction and electrophysiology of Ascaris suum muscle.

Contraction and electrophysiological effects of 5-methylfurmethiodide (MFI), a selective muscarinic agonist in mammals, were tested on Ascaris suum muscle strips. In a contraction assay, MFI produced weak contraction and was less potent than levamisole and acetylcholine. Atropine (3microM) a non-selective muscarinic antagonist in mammalian preparations, did not affect contractions produced by MFI. Mecamylamine (3microM) a nicotinic antagonist in A. suum preparations, blocked the MFI contractions indicating that MFI had weak nicotinic agonist actions. In two-micropipette current-clamp experiments MFI, at concentrations greater than 10microM, produced concentration-dependent depolarizations and small increases in membrane conductance. The depolarizing effects were not abolished by perfusing the preparation in a calcium-free Ascaris Ringer solution to block synaptic transmission, suggesting that MFI effects were mediated by receptors on the muscle and were calcium-independent. A high concentration of mecamylamine, 30microM, only reduced the depolarizing responses by 42%, indicating that MFI also had effects on non-nicotinic receptors. Three non-nicotinic effects in the presence of 30microM mecamylamine were identified using voltage-clamp techniques: (i) MFI produced opening of mecamylamine-resistant non-selective-cation channel currents; (ii) MFI inhibited opening of voltage-activated potassium currents; and (iii) MFI increased the threshold of voltage-activated calcium currents. We suggest that a drug that is more selective for voltage-activated potassium currents, without effects on other channels like MFI, may be exploited pharmacologically as a novel anthelmintic or as an agent to potentiate the action of levamisole. In a larval migration assay we demonstrated that 4-aminopyridine (4-AP: a potassium channel blocker) potentiated the effects of levamisole but MFI did not.

Brief application of AF2 produces long lasting potentiation of nAChR responses in Ascaris suum.

Resistance of parasitic nematodes to the cholinergic anthelmintic levamisole is associated with a reduction in the proportion of time that acetylcholine receptor ion-channels are in the open state decreasing the response of nematode parasites to the drug. Here we examine electrophysiological and contractile responses to acetylcholine and the cholinergic agonist, levamisole, in Ascaris suum muscle looking for a pharmacological approach that may be developed to increase the response to cholinergic agonists. We found that short application of the FMRFamide, AF2, produced modulation (long lasting potentiation) of the peak membrane potential response to acetylcholine but not to levamisole. Since levamisole preferentially activates L-type acetylcholine receptors, we also tested the effect of nicotine (selective activator of N-type acetylcholine receptors) and bephenium (selective activator of B-type acetylcholine receptors) and found again no effect of AF2 on peak membrane potential responses. We then tested atropine on the AF2 potentiation of acetylcholine and found it to inhibit the peak potentiation suggesting that AF2 receptors interact with muscarinic receptors to produce the potentiation of acetylcholine. We saw similar atropine sensitive potentiation of acetylcholine responses in our muscle contraction experiments. The potentiation of the acetylcholine responses shows that nematode acetylcholine receptors are capable of a level of plasticity. A model involving calcium release from the sarcoplasmic reticulum, CaM Kinase, calcineurin, muscarinic receptors and AF2 receptors is proposed to explain our observations. These observations are important because they point to a pharmacological approach that may be developed to counter resistance to cholinergic anthelmintics.

Oxantel is an N-type (methyridine and nicotine) agonist not an L-type (levamisole and pyrantel) agonist: classification of cholinergic anthelmintics in Ascaris.

Three pharmacological subtypes of cholinergic receptors have been distinguished in Ascaris suum using a muscle contraction assay and classical pharmacological techniques. The receptor subtypes are: a B-subtype (sensitive to bephenium); an L-subtype (sensitive to levamisole and pyrantel); and an N-subtype (sensitive to nicotine and methyridine). Oxantel is a cholinergic anthelmintic that was first introduced for the treatment of whipworm, Trichuris, infections in children. Here, we compare the subtype selectivity of oxantel with thenium and other cholinergic anthelmintics. We used the A. suum assay to derive pA(2) values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. pA(2) values, rather than pK(B) values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The pA(2) of oxantel was 6.58+/-0.25 for paraherquamide; 5.39+/-0.28 for 2-desoxyparaherquamide; 7.01+/-0.19 for methyllycaconitine. Comparison of pA(2) values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had pA(2)s of 7.84+/-0.41 for paraherquamide; 5.52+/-0.50 for 2-desoxyparaherquamide; 6.33+/-0.19 for methyllycaconitine. Cluster analysis placed thenium between the L-subtype agonists and the B-subtype agonist. The therapeutic significance of classification of cholinergic anthelmintics is discussed. Combination of oxantel and pyrantel would have therapeutic advantages, covering N- and L-subtypes, and so increasing spectrum of action and reducing the potential for development of resistance. Our results predict that oxantel may remain effective in some nematode isolates that have become levamisole- and pyrantel-resistant.

Levamisole receptor phosphorylation: effects of kinase antagonists on membrane potential responses in Ascaris suum suggest that CaM kinase and tyrosine kinase regulate sensitivity to levamisole.

A two-micropipette current-clamp technique was used to record electrophysiological responses from the somatic muscle of Ascaris suum. Levamisole and acetylcholine were applied to the bag region of the muscle using a microperfusion system. Depolarizations produced by 10 s applications of 10 micro mol l(-1) levamisole or 20 s applications of 10 micro mol l(-1) acetylcholine were recorded. The effect on the peak membrane potential change of the kinase antagonists H-7, staurosporine, KN-93 and genistein was observed. H-7 (30 micro mol l(-1)), a non-selective antagonist of protein kinases A, C and G but which has little effect on Ca(2+)/calmodulin-dependent kinase II (CaM kinase II), did not produce a significant effect on the peak response to levamisole or acetylcholine. Staurosporine (1 micro mol l(-1)), a non-selective kinase antagonist that has effects on protein kinases A, C and G, CaM kinase and tyrosine kinase, reduced the mean peak membrane potential response to levamisole from 6.8 mV to 3.9 mV (P<0.0001) and the mean response to acetylcholine from 5.5 mV to 2.8 mV (P=0.0016). The difference between the effects of H-7 and staurosporine suggested the involvement of CaM kinase II and/or tyrosine kinase. KN-93, a selective CaM kinase II antagonist, reduced the mean peak response to levamisole from 6.2 mV to 2.7 mV (P=0.035) and the mean peak response of acetylcholine from 4.7 mV to 2.0 mV (P=0.0004). The effects indicated the involvement of CaM kinase II in the phosphorylation of levamisole and acetylcholine receptors. The effect of extracellular Ca(2+) on the response to levamisole was assessed by comparing responses to levamisole in normal and in low-Ca(2+) bathing solutions. The response to levamisole was greater in the presence of Ca(2+), an effect that may be explained by stimulation of CaM kinase II. Genistein (90 micro mol l(-1)), a selective tyrosine kinase antagonist, reduced peak membrane potential responses to levamisole from a mean of 6.4 mV to 3.3 mV (P=0.001). This effect indicated the involvement of tyrosine kinase in maintaining the receptor.