Activation of Caenorhabditis elegans Levamisole-Sensitive and Mammalian Nicotinic Receptors by the Antiparasitic Bephenium.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels involved in neuromuscular transmission. In nematodes, muscle nAChRs are targets of antiparasitic drugs. Bephenium is an anthelmintic compound whose molecular action in the free-living nematode Caenorhabditis elegans, which is a model for anthelmintic drug discovery, is poorly known. We explored the effect of bephenium on C. elegans locomotion and applied single-channel recordings to identify its molecular target, mechanism of action, and selectivity between mammalian and C. elegans nAChRs. As in parasites, bephenium paralyzes C. elegans A mutant strain lacking the muscle levamisole-sensitive nAChR (L-AChR) shows full resistance to bephenium, indicating that this receptor is the target site. Bephenium activates L-AChR channels from larvae muscle cells in the micromolar range. Channel activity is similar to that elicited by levamisole, appearing mainly as isolated brief openings. Our analysis revealed that bephenium is an agonist of L-AChR and an open-channel blocker at higher concentrations. It also activates mammalian muscle nAChRs. Opening events are significantly briefer than those elicited by ACh and do not appear in activation episodes at a range of concentrations, indicating that it is a very weak agonist of mammalian nAChRs. Recordings in the presence of ACh showed that bephenium acts as a voltage-dependent channel blocker and a low-affinity agonist. Molecular docking into homology-modeled binding-site interfaces represent the binding mode of bephenium that explains its partial agonism. Given the great diversity of helminth nAChRs and the overlap of their pharmacological profiles, unraveling the basis of drug receptor-selectivity will be required for rational design of anthelmintic drugs.

Whole-cell patch-clamp recording of nicotinic acetylcholine receptors in adult Brugia malayi muscle.

Lymphatic filariasis is a debilitating disease caused by clade III parasites like Brugia malayi and Wuchereria bancrofti. Current recommended treatment regimen for this disease relies on albendazole, ivermectin and diethylcarbamazine, none of which targets the nicotinic acetylcholine receptors in these parasitic nematodes. Our aim therefore has been to develop adult B. malayi for electrophysiological recordings to aid in characterizing the ion channels in this parasite as anthelmintic target sites. In that regard, we recently demonstrated the amenability of adult B. malayi to patch-clamp recordings and presented results on the single-channel properties of nAChR in this nematode. We have built on this by recording whole-cell nAChR currents from adult B. malayi muscle. Acetylcholine, levamisole, pyrantel, bephenium and tribendimidine activated the receptors on B. malayi muscle, producing robust currents ranging from >200 pA to ~1.5 nA. Levamisole completely inhibited motility of the adult B. malayi within 10 min and after 60 min, motility had recovered back to control values.

Selective effect of the anthelmintic bephenium on Haemonchus contortus levamisole-sensitive acetylcholine receptors.

Acetylcholine receptors (AChRs) are pentameric ligand-gated ion channels involved in the neurotransmission of both vertebrates and invertebrates. A number of anthelmintic compounds like levamisole and pyrantel target the AChRs of nematodes producing spastic paralysis of the worms. The muscle AChRs of nematode parasites fall into three pharmacological classes that are preferentially activated by the cholinergic agonists levamisole (L-type), nicotine (N-type) and bephenium (B-type), respectively. Despite a number of studies of the B-type AChR in parasitic species, this receptor remains to be characterized at the molecular level. Recently, we have reconstituted and functionally characterized two distinct L-AChR subtypes of the gastro-intestinal parasitic nematode Haemonchus contortus in the Xenopus laevis oocyte expression system by providing the cRNAs encoding the receptor subunits and three ancillary proteins (Boulin et al. in Br J Pharmacol 164(5):1421-1432, 2011). In the present study, the effect of the bephenium drug on Hco-L-AChR1 and Hco-L-AChR2 subtypes was examined using the two-microelectrode voltage-clamp technique. We demonstrate that bephenium selectively activates the Hco-L-AChR1 subtype made of Hco-UNC-29.1, Hco-UNC-38, Hco-UNC-63, Hco-ACR-8 subunits that is more sensitive to levamisole than acetylcholine. Removing the Hco-ACR-8 subunit produced the Hco-L-AChR2 subtype that is more sensitive to pyrantel than acetylcholine and partially activated by levamisole, but which was bephenium-insensitive indicating that the bephenium-binding site involves Hco-ACR-8. Attempts were made to modify the subunit stoichiometry of the Hco-L-AChR1 subtype by injecting five fold more cRNA of individual subunits. Increased Hco-unc-29.1 cRNA produced no functional receptor. Increasing Hco-unc-63, Hco-unc-38 or Hco-acr-8 cRNAs did not affect the pharmacological characteristics of Hco-L-AChR1 but reduced the currents elicited by acetylcholine and the other agonists. Here, we provide the first description of the molecular composition and functional characteristics of any invertebrate bephenium-sensitive receptor.

Phenotypic characterization of two Ancylostoma caninum isolates with different susceptibilities to the anthelmintic pyrantel.

The anthelmintic pyrantel plays an important role in the control of gastrointestinal helminths of humans and domestic animals. Despite the demonstration of pyrantel resistance in several helminth species over the last 20 years, the resistance mechanism remains unclear. It has been hypothesized that resistance may arise as a consequence of changes to the relative proportions of subpopulations of nicotinic acetylcholine receptors (nAchRs). To test this hypothesis, we examined the responses of two isolates of the canine hookworm Ancylostoma caninum with low-level resistance (isolate NT) and high-level resistance (isolate PR) to pyrantel to nicotinic agonist drugs reported to be selective for three nAchR subtypes. We used larval motility and conformation assays and force transduction experiments with adult worms. Pyrantel and levamisole were less potent against larvae of isolate PR than larvae of isolate NT (up to an 18-fold increase in the 50% inhibitory concentration); on the other hand, bephenium was more potent against larvae of isolate PR than larvae of isolate NT (twofold) and nicotine had the same potency against larvae of both isolates. In adults, pyrantel, levamisole, and nicotine were less potent against isolate PR than isolate NT (two- to threefold), but the potency of bephenium against the two isolates was equivalent. Our data indicate a complex pattern of nAchRs in this species and suggest that the two isolates differ in their relative sensitivities to agonists targeting different nAchRs.

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.

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.

Methyridine (2-[2-methoxyethyl]-pyridine]) and levamisole activate different ACh receptor subtypes in nematode parasites: a new lead for levamisole-resistance.

1. The development of resistance to all chemotherapeutic agents increases and needs to be addressed. We are interested in resistance in parasitic nematodes to the anthelmintic levamisole. During studies on methyridine, we found that it gave us a new insight into pharmacological changes associated with levamisole resistance. Initially, electrophysiological investigation using a two-micropipette current-clamp recording technique revealed that methyridine acts as a cholinergic agonist on nematode muscle receptors (Ascaris suum). Methyridine (>30 microm) produced reversible concentration-dependent depolarizations and increases in input conductance. Mecamylamine (30 microm) and paraherquamide (0.3 microm) produced reversible antagonism of the depolarization and conductance responses to methyridine. These observations suggest that methyridine, like acetylcholine and levamisole, gates ion channels on the muscle of parasitic nematodes. 2. The antagonistic effects of dihydro-beta-erythroidine and paraherquamide on methyridine-induced contractions of A. suum muscle flaps were then examined to determine if methyridine showed subtype selectivity for N-subtype (nicotine-sensitive) or L-subtype (levamisole-sensitive) acetylcholine receptors. Dihydro-beta-erythroidine weakly antagonized the effects of methyridine (but had no effect on levamisole responses). The antagonism of methyridine (pA2, 5.9) and nicotine (pA2, 6.1) by paraherquamide was similar, but was less than the antagonism of levamisole (pA2, 7.0). The antagonist profiles suggested that methyridine has a selective action on the N-subtype rather than on the L-subtype. 3. A novel use for a larval inhibition migration assay was made using L3 larvae of Oesophagostomum dentatum. Inhibitory effects of nicotine, levamisole, pyrantel and methyridine on the migration of larvae of levamisole-sensitive (SENS) and levamisole-resistant (LEV-R) isolates were tested at different concentrations. Levamisole and pyrantel (putative L-subtype-selective agonists) concentration-response plots were displaced to the right in LEV-R isolates. Nicotine (an N-subtype-selective agonist) and methyridine produced little shift in concentration-response plots in the LEV-R isolates. Resistance dose ratios were used to calculate the relative selectivity, rhoL, for the L-type receptor (levamisole rhoL=1.0; pyrantel rhoL=0.93; methyridine rhoL=0.17; nicotine rhoL=0.06). These observations reveal an N-subtype-selective action of methyridine and suggest that levamisole resistance may be associated with a loss of the L-subtype, but not the N-subtype receptors. The pharmacology of methyridine suggests an approach for the treatment of levamisole-resistant parasites.

[Morphological changes in the mucosa of the stomach and the small intestine of rats under the action of mebendazole]. / Morfologicheskie izmeneniia slizistoi obolochki zheludka i tonkoi kishki krys pod deistviem mebendazola.

The effect of mebendazole on the gastric and intestinal mucosa was studied in 52 intact white rats. It was shown that the drug caused a significant damage to the mucosa, especially of goblet cells of villi and crypts and interfere with the mucoid secretion. The alterations after mebendazole administration were more severe than those caused by naphthamon. On the 13-15th day after the treatment the complete restoration of the damage was not seen.

Response of preadult Necator americanus to some known anthelmintics in hamsters.

Hamsters infected with laboratory-adapted preadult Necator americanus were dosed with 6 reference anthelmintics. Their efficacy was measured in terms of percentage cure of infected animals as well as percentage worm reduction following treatment. Mebendazole and pyrantel were equally effective in this system. Other anthelmintics, including anti-hookworm compound, bephenium hydroxynaphthoate, were less effective. The comparative results revealed that the N. americanus model is sensitive and reliable for identifying and characterizing new anti-parasite preparations.

The interaction between anthelmintic drugs and histamine in Ascaris suum.

1 Piperazine reduced the histamine content of Ascaris suum, yet it greatly increased the uptake of histamine from the surrounding medium, the neuromuscular structures of the nematode preferentially increasing in amount. 2 Bephenium reduced the histamine content of Ascaris and the uptake of histamine from the surrounding medium. However, the relative amount in the neuromuscular structures increased. 3 The flaccid paralysing action of piperazine may thus involve increased histamine absorption whereas the spastic paralysing action of bephenium may be independent of histamine.

Detection of activity for various anthelmintics against in vitro-produced Cooperia punctata.

The in vitro-grown parasitic stages of Cooperia punctata were used to evaluate 28 compounds with different kinds and degrees of in vivo activity. Using presumptive and confirmatory tests, it was possible to establish a group order of in vitro potency that compared favorably with an order based on established in vivo use of these compounds. The procedure lends itself to evaluating activity against a given parasitic growth stage and gives a quantitative estimate (range) of the concentration that produces 50% nematode kill. The system was most successful in detecting compounds with in vivo activity for C punctata, followed in order by compounds active against Cooperia spp, other gastrointestinal nematodes of ruminants, and other nematodes of non-bovine hosts. The procedure showed some differentiation between activity against nematodes versus that against cestodes, trematodes, and arthropods. The system permits considerable flexibility in experimental design, thus making possible the acquisition of the particular information desired. In addition to establishing lethal effects on the nematode, the procedure detected compounds with nematode-anesthetizing effects. The results indicate this in vitro system can be used with some expediency as a preliminary screening method in the search for new anthelmintic compounds.