Cholinergic Anthelmintics Research Articles

Cholinergic receptors on intestine cells of Ascaris suum and activation of nAChRs by levamisole

Cholinergic agonists, like levamisole, are a major class of anthelmintic drugs that are known to act selectively on nicotinic acetylcholine receptors (nAChRs) on the somatic muscle and nerves of nematode parasites to produce their contraction and spastic paralysis. Previous studies have suggested that in addition to the nAChRs found on muscle and nerves, there are nAChRs on non-excitable tissues of nematode parasites. We looked for evidence of nAChRs expression in the cells of the intestine of the large pig nematode, Ascaris suum, using RT-PCR and RNAscope in situ hybridization and detected mRNA of nAChR subunits in the cells. These subunits include components of the putative levamisole receptor in A. suum muscle: Asu-unc-38, Asu-unc-29, Asu-unc-63 and Asu-acr-8. Relative expression of these mRNAs in A. suum intestine was quantified by qPCR. We also looked for and found expression of G protein-linked acetylcholine receptors (Asu-gar-1). We used Fluo-3 AM to detect intracellular calcium changes in response to receptor activation by acetylcholine (as a non-selective agonist) and levamisole (as an L-type nAChR agonist) to look for evidence of functioning nAChRs in the intestine. We found that both acetylcholine and levamisole elicited increases in intracellular calcium but their signal profiles in isolated intestinal tissues were different, suggesting activation of different receptor sets. The levamisole responses were blocked by mecamylamine, a nicotinic receptor antagonist in A. suum, indicating the activation of intestinal nAChRs rather than G protein-linked acetylcholine receptors (GARs) by levamisole. The detection of nAChRs in cells of the intestine, in addition to those on muscles and nerves, reveals another site of action of the cholinergic anthelmintics and a site that may contribute to the synergistic interactions of cholinergic anthelmintics with other anthelmintics that affect the intestine (Cry5B).

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.

Deciphering the molecular determinants of cholinergic anthelmintic sensitivity in nematodes: When novel functional validation approaches highlight major differences between the model Caenorhabditis elegans and parasitic species.

Cholinergic agonists such as levamisole and pyrantel are widely used as anthelmintics to treat parasitic nematode infestations. These drugs elicit spastic paralysis by activating acetylcholine receptors (AChRs) expressed in nematode body wall muscles. In the model nematode Caenorhabditis elegans, genetic screens led to the identification of five genes encoding levamisole-sensitive-AChR (L-AChR) subunits: unc-38, unc-63, unc-29, lev-1 and lev-8. These subunits form a functional L-AChR when heterologously expressed in Xenopus laevis oocytes. Here we show that the majority of parasitic species that are sensitive to levamisole lack a gene orthologous to C. elegans lev-8. This raises important questions concerning the properties of the native receptor that constitutes the target for cholinergic anthelmintics. We demonstrate that the closely related ACR-8 subunit from phylogenetically distant animal and plant parasitic nematode species functionally substitutes for LEV-8 in the C. elegans L-AChR when expressed in Xenopus oocytes. The importance of ACR-8 in parasitic nematode sensitivity to cholinergic anthelmintics is reinforced by a ‘model hopping’ approach in which we demonstrate the ability of ACR-8 from the hematophagous parasitic nematode Haemonchus contortus to fully restore levamisole sensitivity, and to confer high sensitivity to pyrantel, when expressed in the body wall muscle of C. elegans lev-8 null mutants. The critical role of acr-8 to in vivo drug sensitivity is substantiated by the successful demonstration of RNAi gene silencing for Hco-acr-8 which reduced the sensitivity of H. contortus larvae to levamisole. Intriguingly, the pyrantel sensitivity remained unchanged thus providing new evidence for distinct modes of action of these important anthelmintics in parasitic species versus C. elegans. More broadly, this highlights the limits of C. elegans as a predictive model to decipher cholinergic agonist targets from parasitic nematode species and provides key molecular insight to inform the discovery of next generation anthelmintic compounds.

Functional genomics in Brugia malayi reveal diverse muscle nAChRs and differences between cholinergic anthelmintics

Many techniques for studying functional genomics of important target sites of anthelmintics have been restricted to Caenorhabditis elegans because they have failed when applied to animal parasites. To overcome these limitations, we have focused our research on the human nematode parasite Brugia malayi, which causes elephantiasis. Here, we combine single-cell PCR, whole muscle cell patch clamp, motility phenotyping (Worminator), and dsRNA for RNAi for functional genomic studies that have revealed, in vivo, four different muscle nAChRs (M-, L-, P-, and N-). The cholinergic anthelmintics had different selectivities for these receptors. We show that motility and patch-clamp responses to levamisole and pyrantel, but not morantel or nicotine, require the unc-38 and/or unc-29 genes. Derquantel behaved as a competitive antagonist and distinguished M-nAChRs activated by morantel (Kb 13.9 nM), P-nAChRs activated by pyrantel (Kb 126 nM), and L-nAChRs activated by levamisole (Kb 0.96 µM) and bephenium. Derquantel was a noncompetitive antagonist of nicotine, revealing N-type nAChRs. The presence of four diverse nAChRs on muscle is perhaps surprising and not predicted from the C. elegans model. The diverse nAChRs represent distinguishable drug targets with different functions: Knockdown of unc-38+unc-29 (L- and/or P-receptors) inhibited motility but knockdown of acr-16+acr-26 (M- and/or N-receptors) did not.

Pharmacological profile of Ascaris suum ACR-16, a new homomeric nicotinic acetylcholine receptor widely distributed in Ascaris tissues.

SummaryBackground and PurposeControl of nematode parasite infections relies largely on anthelmintic drugs, several of which act on nicotinic ACh receptors (nAChRs), and there are concerns about the development of resistance. There is an urgent need for development of new compounds to overcome resistance and novel anthelmintic drug targets. We describe the functional expression and pharmacological characterization of a homomeric nAChR, ACR‐16, from a nematode parasite.Experimental ApproachUsing RT‐PCR, molecular cloning and two‐electrode voltage clamp electrophysiology, we localized acr‐16 mRNA in Ascaris suum (Asu) and then cloned and expressed acr‐16 cRNA in Xenopus oocytes. Sensitivity of these receptors to cholinergic anthelmintics and a range of nicotinic agonists was tested.Key ResultsAmino acid sequence comparison with vertebrate nAChR subunits revealed ACR‐16 to be most closely related to α7 receptors, but with some striking distinctions. acr‐16 mRNA was recovered from Asu somatic muscle, pharynx, ovijector, head and intestine. In electrophysiological experiments, the existing cholinergic anthelmintic agonists (morantel, levamisole, methyridine, thenium, bephenium, tribendimidine and pyrantel) did not activate Asu‐ACR‐16 (except for a small response to oxantel). Other nAChR agonists: nicotine, ACh, cytisine, 3‐bromocytisine and epibatidine, produced robust current responses which desensitized at a rate varying with the agonists. Unlike α7, Asu‐ACR‐16 was insensitive to α‐bungarotoxin and did not respond to genistein or other α7 positive allosteric modulators. Asu‐ACR‐16 had lower calcium permeability than α7 receptors.Conclusions and ImplicationsWe suggest that ACR‐16 has diverse tissue‐dependent functions in nematode parasites and is a suitable drug target for development of novel anthelmintic compounds.

Tribendimidine: mode of action and nAChR subtype selectivity in Ascaris and Oesophagostomum.

The cholinergic class of anthelmintic drugs is used for the control of parasitic nematodes. One of this class of drugs, tribendimidine (a symmetrical diamidine derivative, of amidantel), was developed in China for use in humans in the mid-1980s. It has a broader-spectrum anthelmintic action against soil-transmitted helminthiasis than other cholinergic anthelmintics, and is effective against hookworm, pinworms, roundworms, and Strongyloides and flatworm of humans. Although molecular studies on C. elegans suggest that tribendimidine is a cholinergic agonist that is selective for the same nematode muscle nAChR as levamisole, no direct electrophysiological observations in nematode parasites have been made to test this hypothesis. Also the hypothesis that levamisole and tribendimine act on the same receptor, does not explain why tribendimidine is effective against some nematode parasites when levamisole is not. Here we examine the effects of tribendimidine on the electrophysiology and contraction of Ascaris suum body muscle and show that tribendimidine produces depolarization antagonized by the nicotinic antagonist mecamylamine, and that tribendimidine is an agonist of muscle nAChRs of parasitic nematodes. Further pharmacological characterization of the nAChRs activated by tribendimidine in our Ascaris muscle contraction assay shows that tribendimidine is not selective for the same receptor subtypes as levamisole, and that tribendimidine is more selective for the B-subtype than the L-subtype of nAChR. In addition, larval migration inhibition assays with levamisole-resistant Oesophagostomum dentatum isolates show that tribendimidine is as active on a levamisole-resistant isolate as on a levamisole-sensitive isolate, suggesting that the selectivity for levamisole and tribendimidine is not the same. It is concluded that tribendimidine can activate a different population of nematode parasite nAChRs than levamisole, and is more like bephenium. The different nAChR subtype selectivity of tribendimidine may explain why the spectrum of action of tribendimidine is different to that of other cholinergic anthelmintics like levamisole.

The Conqueror Worm: recent advances with cholinergic anthelmintics and techniques excite research for better therapeutic drugs

The following account is based on a review lecture given recently at the British Society of Parasitology. We point out that nematode parasites cause very widespread infections of humans, particularly in economically underdeveloped areas where sanitation and hygiene are not adequate. In the absence of adequate clean water and effective vaccines, control and prophylaxis relies on anthelmintic drugs. Widespread use of anthelmintics to control nematode parasites of animals has given rise to the development of resistance and so there is a concern that similar problems will occur in humans if mass drug administration is continued. Recent research on the cholinergic anthelmintic drugs has renewed enthusiasm for the further development of cholinergic anthelmintics. Here we illustrate the use of three parasite nematode models, Ascaris suum, Oesophagostomum dentatum and Brugia malayi, microfluidic techniques and the Xenopus oocyte expression system for testing and examining the effects of cholinergic anthelmintics. We also show how the combination of derquantel, the selective nematode cholinergic antagonist and abamectin produce increased inhibition of the nicotinic acetylcholine receptors on the nematode body muscle. We are optimistic that new compounds and combinations of compounds can limit the effects of drug resistance, allowing anthelmintics to be continued to be used for effective treatment of human and animal helminth parasites.

Nicotinic acetylcholine receptors: expression demonstrates functional diversity in parasitic nematodes (844.4)

Nicotinic acetylcholine receptors mediate fast neurotransmission in both vertebrates and invertebrates. Selective targeting of parasite nAChR by antiparasitic drugs requires functional and/or structural differences between the receptors of the host and the parasite. Anti‐parasitic drugs like levamisole, pyrantel, tribendimidine, and derquantel (cholinergic anthelmintics) selectively target the nAChRs of nematodes. Unfortunately, regular use of anthelmintics has resulted in increasing reports of resistance in livestock parasites. Our aim has been to understand the molecular pharmacology of the nAChRs in parasitic nematodes, to inform development of better anthelmintics. We have cloned homologues of the nAChR subunit genes unc‐29, unc‐38, unc‐63 and acr‐8 from Oesophagostomum dentatum, a pig GI parasite. By employing the Xenopus laevis oocyte expression system, we demonstrated that four, pharmacologically different receptor subtypes can be reconstituted with various combinations of the four subunits. Single‐channel properties of one of the reconstituted receptor subtypes matched with one of the four subtypes recorded in vivo from O. dentatum, demonstrating the physiological relevance of the reconstituted receptor subtypes. Comparing these results with reconstitution results from other nematodes demonstrates the diverse, plastic nature of nAChRs in parasitic nematodes.Grant Funding Source: Supported by NIH R01 AI047194‐12, R21AI092185‐01 and Chateaubriand Fellowship

Investigation of acetylcholine receptor diversity in a nematode parasite leads to characterization of tribendimidine- and derquantel-sensitive nAChRs.

Nicotinic acetylcholine receptors (nAChRs) of parasitic nematodes are required for body movement and are targets of important “classical” anthelmintics like levamisole and pyrantel, as well as “novel” anthelmintics like tribendimidine and derquantel. Four biophysical subtypes of nAChR have been observed electrophysiologically in body muscle of the nematode parasite Oesophagostomum dentatum, but their molecular basis was not understood. Additionally, loss of one of these subtypes (G 35 pS) was found to be associated with levamisole resistance. In the present study, we identified and expressed in Xenopus oocytes, four O. dentatum nAChR subunit genes, Ode-unc-38, Ode-unc-63, Ode-unc-29 and Ode-acr-8, to explore the origin of the receptor diversity. When different combinations of subunits were injected in Xenopus oocytes, we reconstituted and characterized four pharmacologically different types of nAChRs with different sensitivities to the cholinergic anthelmintics. Moreover, we demonstrate that the receptor diversity may be affected by the stoichiometric arrangement of the subunits. We show, for the first time, different combinations of subunits from a parasitic nematode that make up receptors sensitive to tribendimidine and derquantel. In addition, we report that the recombinant levamisole-sensitive receptor made up of Ode-UNC-29, Ode-UNC-63, Ode-UNC-38 and Ode-ACR-8 subunits has the same single-channel conductance, 35 pS and 2.4 ms mean open-time properties, as the levamisole-AChR (G35) subtype previously identified in vivo. These data highlight the flexible arrangements of the receptor subunits and their effects on sensitivity and resistance to the cholinergic anthelmintics; pyrantel, tribendimidine and/or derquantel may still be effective on levamisole-resistant worms.

Levamisole receptors: a second awakening

Levamisole and pyrantel are old (1965) but useful anthelmintics that selectively activate nematode acetylcholine ion channel receptors; they are used to treat roundworm infections in humans and animals. Interest in their actions has surged, giving rise to new knowledge and technical advances, including an ability to reconstitute receptors that reveal more details of modes of action/resistance. We now know that the receptors are plastic and may form diverse species-dependent subtypes of receptor with different sensitivities to individual cholinergic anthelmintics. Understanding the biology of the levamisole receptors is expected to inform other studies on anthelmintics (ivermectin and emodepside) that act on ion channels.

ACR-26: A novel nicotinic receptor subunit of parasitic nematodes

Nematode nicotinic acetylcholine receptors are the targets for many effective anthelmintics, including those recently introduced into the market. We have identified a novel nicotinic receptor subunit sequence, acr-26, that is expressed in all the animal parasitic nematodes we examined from clades III, IV and V, but is not present in the genomes of Trichinella spiralis, Caenorhabditis elegans, Pristionchus pacificus and Meloidogyne spp. In Ascaris suum, ACR-26 is expressed on muscle cells isolated from the head, but not from the mid-body region. Sequence comparisons with other vertebrate and nematode subunits suggested that ACR-26 may be capable of forming a functional homomeric receptor; when acr-26 cRNA was injected into Xenopus oocytes along with Xenopus laevis ric-3 cRNA we occasionally observed the formation of acetylcholine- and nicotine-sensitive channels. The unreliable expression of ACR-26 in vitro may suggest that additional subunits or chaperones may be required for efficient formation of the functional receptors. ACR-26 may represent a novel target for the development of cholinergic anthelmintics specific for animal parasites.

Levamisole and ryanodine receptors (I): A contraction study in Ascaris suum

Cholinergic anthelmintics (like levamisole) are important drugs but resistance with reduced responses by the parasite to these compounds is a concern. There is a need to study and understand mechanisms that affect the amplitude of the responses of parasites to these drugs. In this paper, we study interactions of levamisole and ryanodine receptors on contractions of Ascaris suum body muscle flaps. In our second paper, we extend these observations to examine electrophysiological interactions of levamisole, ryanodine receptors (RyRs) and AF2. We report that the maximum force of contraction, g max , was dependent on the extracellular concentration of calcium but the levamisole EC 50 (0.8 μM) was not. The relationship between maximum force of contraction and extracellular calcium was described by the Michaelis–Menten equation with a K m of 1.8 mM. Ryanodine inhibited g max without effect on EC 50 ; ryanodine inhibited only 44% of the maximum contraction ( K i of 40 nM), revealing a ryanodine-insensitive component in the levamisole excitation–contraction pathway. Dantrolene had the same effect as ryanodine but was less potent. The neuropeptide AF2 (1 μM) decreased the levamisole EC 50 to 0.2 μM without effect on g max ; 0.1 μM ryanodine and 100 μM dantrolene, inhibited the g max of the AF2-potentiated levamisole response. High concentrations of caffeine, 30 mM, produced weak contraction of the body-flap preparation. Caffeine behaved like ryanodine in that it inhibited the maximum force of contraction, g max , without effects on the levamisole EC 50 . Thus, RyRs play a modulatory role in the levamisole excitation–contraction pathway by affecting the maximum force of contraction without an effect on levamisole EC 50 . The levamisole excitation–contraction coupling is graded and has at least two pathways: one sensitive to ryanodine and one not.

The Nicotinic Acetylcholine Receptors of the Parasitic Nematode Ascaris suum: Formation of Two Distinct Drug Targets by Varying the Relative Expression Levels of Two Subunits

Parasitic nematodes are of medical and veterinary importance, adversely affecting human health and animal welfare. Ascaris suum is a gastrointestinal parasite of pigs; in addition to its veterinary significance it is a good model of the human parasite Ascaris lumbricoides, estimated to infect ∼1.4 billion people globally. Anthelmintic drugs are essential to control nematode parasites, and nicotinic acetylcholine receptors (nAChRs) on nerve and muscle are the targets of cholinergic anthelmintics such as levamisole and pyrantel. Previous genetic analyses of nematode nAChRs have been confined to Caenorhabditis elegans, which is phylogenetically distinct from Ascaris spp. and many other important parasites. Here we report the cloning and expression of two nAChR subunit cDNAs from A. suum. The subunits are very similar in sequence to C. elegans UNC-29 and UNC-38, are expressed on muscle cells and can be expressed robustly in Xenopus oocytes to form acetylcholine-, nicotine-, levamisole- and pyrantel-sensitive channels. We also demonstrate that changing the stoichiometry of the receptor by injecting different ratios of the subunit cRNAs can reproduce two of the three pharmacological subtypes of nAChR present in A. suum muscle cells. When the ratio was 5∶1 (Asu-unc-38∶Asu-unc-29), nicotine was a full agonist and levamisole was a partial agonist, and oocytes responded to oxantel, but not pyrantel. At the reverse ratio (1∶5 Asu-unc-38∶Asu-unc-29), levamisole was a full agonist and nicotine was a partial agonist, and the oocytes responded to pyrantel, but not oxantel. These results represent the first in vitro expression of any parasitic nicotinic receptor and show that their properties are substantially different from those of C. elegans. The results also show that changing the expression level of a single receptor subunit dramatically altered the efficacy of some anthelmintic drugs. In vitro expression of these subunits may permit the development of parasite-specific screens for future anthelmintics.

Mode of action of levamisole and pyrantel, anthelmintic resistance, E153 and Q57

Here we review molecular information related to resistance to the cholinergic anthelmintics in nematodes. The amount of molecular information available varies between the nematode species, with the best understood so far being C. elegans. More information is becoming available for some other parasitic species. The cholinergic anthelmintics act on nematode nicotinic acetylcholine receptors located on somatic muscle cells. Recent findings demonstrate the presence of multiple types of the nicotinic receptors in several nematodes and the numerous genes required to form these multimeric proteins. Not only are the receptors the product of several genes but they are subject to modulation by several other proteins. Mutations altering these modulatory proteins could alter sensitivity to the cholinergic anthelmitics and thus lead to resistance. We also discuss the possibility that resistance to the cholinergic anthelmintics is not necessarily the result of a single mutation but may well be polygenic in nature. Additionally, the mutations resulting in resistance may vary between different species or between resistant isolates of the same species. A list of candidate genes to examine for SNPs is presented.

The nematode neuropeptide, AF2 (KHEYLRF‐NH2), increases voltage‐activated calcium currents in Ascaris suum muscle

Resistance to all the classes of anti-nematodal drugs like the benzimidazoles, cholinergic agonists and avermectins, has now been recorded in parasites of animals and/or humans. The development of novel anthelmintics is an urgent and imperative need. Receptors of nematode neuropeptides have been suggested to be suitable target sites for novel anthelmintic drugs. To investigate the effect of AF2 on calcium-currents in Ascaris suum somatic muscle cells we employed the two-micropipette current-clamp and voltage-clamp techniques and a brief application of AF2. Here we report the isolation of voltage-activated, transient, inward calcium currents. These currents are similar in characteristics to Caenorhabditis elegans UNC-2 type currents, non-L-type calcium currents. Following a 2-minute application of 1 microM AF2 , there was a significant long-lasting increase in the transient inward calcium current; AF2 increased the maximum current (from -84 nA to -158 nA) by shifting the threshold in the hyperpolarising direction (V (50) changed from -7.2 to -12.8 mV) and increasing the maximum conductance change from 1.91 to 2.94 microS. These studies demonstrate a mechanism by which AF2 increased the excitability of the neuromuscular system by modulating calcium currents in nematodes. A selective small molecule agonist of the AF2 receptor is predicted to increase the contraction and act synergistically with cholinergic anthelmintics and could counter resistance to these compounds.

Pharmacology of N ‐, L ‐, and B ‐subtypes of nematode nAChR resolved at the single‐channel level in Ascaris suum

Pharmacological experiments on Ascaris suum have demonstrated the presence of three (N-, L-, and B-) subtypes of cholinergic receptor mediating contraction of body wall muscle in parasitic nematodes. In the present study, these ionotropic acetylcholine (ACh) receptors (nAChRs) were activated by levamisole and bephenium under patch-clamp conditions and competitively antagonized by paraherquamide and 2-desoxoparaherquamide. A number of recordings exhibited three separate current amplitude levels, indicating the presence of small, intermediate, and large conductance subtypes of receptor. The mean conductance of the small conductance subtype, G25, was 22 +/- 1 pS; the intermediate conductance channel, G35, was 33 +/- 1 pS; and the large conductance channel, G45, was 45 +/- 1 pS. The small channel was not antagonized significantly by paraherquamide and was identified as the N-subtype. The intermediate channel was preferentially activated by levamisole rather than bephenium and antagonized by paraherquamide: the intermediate channel was identified as the L-subtype. The large conductance channel was preferentially activated by bephenium, antagonized more by 2-desoxoparaherquamde than by paraherquamide and was identified as the B-subtype. These observations reveal that the three channel subtypes have different selectivity for cholinergic anthelmintics. The different selectivity of these compounds should be considered when dealing with drug resistant infections.

Drug resistance and neurotransmitter receptors of nematodes: recent studies on the mode of action of levamisole

Here we review recent studies on the mode of action of the cholinergic anthelmintics (levamisole, pyrantel etc.). We also include material from studies on the free living nematode Caenorhabditis elegans. The initial notion that these drugs act on a single receptor population, while attractive, has proven to be an oversimplification. In both free living and parasitic nematodes there are multiple types of nicotinic acetylcholine receptor (nAChR) on the somatic musculature. Each type has different (sometimes subtly so) pharmacological properties. The implications of these findings are: (1) combinations of anthelmintic that preferentially activate a broad range of nAChR types would be predicted to be more effective; (2) in resistant isolates of parasite where a subtype has been lost, other cholinergic anthelmintics may remain effective. Not only are there multiple types of nAChR, but relatively recent research has shown these receptors can be modulated; it is possible to increase the response of a parasite to a fixed concentration of drug by altering the receptor properties (e.g. phosphorylation state). These findings offer a potential means of increasing efficacy of existing compounds as an alternative to the costly and time consuming development of new anthelmintic agents.

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 p A 2 values for the agonists: oxantel, thenium, bephenium, levamisole, pyrantel, nicotine and methyridine with the antagonists: paraherquamide, 2-desoxyparaherquamide and methyllycaconitine. p A 2 values, rather than p K B values, were determined for all agonists when it was found that Schild slopes for some agonists were significantly less than 1.0. The p A 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 p A 2 values using cluster analysis showed that oxantel was grouped with nicotine and methyridine, the N-subtype agonists. Thenium had p A 2s 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.

Anthelmintic actions on homomer-forming nicotinic acetylcholine receptor subunits: chicken α7 and ACR-16 from the nematode Caenorhabditis elegans

Two homomer-forming nicotinic acetylcholine receptor subunits with 47% identity in their amino acid sequences were employed to compare the actions of cholinergic anthelmintics and ivermectin on expressed vertebrate and nematode nicotinic receptors of known molecular composition. Voltage-clamp electrophysiology was used to study recombinant nicotinic receptors expressed in Xenopus laevis oocytes following nuclear injection of cDNA encoding either chicken α7 or Caenorhabditis elegans ACR-16 (Ce21) subunits. Butamisole, morantel and metyridine were without agonist actions on either α7 or ACR-16 nicotinic receptors in the range 10 nM–1 mM. However, butamisole (pIC 50=4.9 for both α7 and ACR-16) and morantel (pIC 50=5.6 for α7 and 5.7 for ACR-16) antagonized responses of both α7 and ACR-16 receptors to acetylcholine. Metyridine (1 mM) did not affect responses to acetylcholine of either receptor. Oxantel was without agonist actions on ACR-16, but was an acetylcholine antagonist (pIC 50=5.4). In contrast, it was found to have low efficacy agonist action (pEC 50=4.4) on α7 at concentrations in the range 10–300 μM. In agreement with a previous study, ivermectin (30 μM), an agonist of l-glutamate-gated chloride channels, enhanced the amplitude of responses to acetylcholine of α7 nicotinic receptors. However, this same concentration of ivermectin (30 μM) did not potentiate the acetylcholine-induced responses of ACR-16, but rather resulted in a slight attenuation. We conclude that oxantel and ivermectin have identified new pharmacological differences between the chicken α7 nicotinic receptor and its C. elegans homologue ACR-16.