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Neuromuscular transmission in nematodes and anthelmintic drug action
Abstract
Some anthelmintic drugs interfere selectively with nematode neuromuscular transmission. These drugs include: the nicotinic agonists, e.g. levamisole, the gamma-amino butyric acid agonist piperazine, and the avermectins which open Cl- channels. The physiology and pharmacology of neuromuscular transmission in nematodes is reviewed and the actions of antinematodal drugs which interfere with the transmission described. The results of experiments on the large porcine-intestinal nematode parasite, Ascaris suum, form the basis of the account presented but experiments on other nematodes suggest that these observations may be generalized. Results of some experiments on the small free living nematode Caenorhabditis elegans are also included.
... Indeed, many nematicides work on the nervous system. For example, levamisol is a potent cholinergic agonist [Lewis et al. 1980;Martin 1993;Rand 2007], piperazine is a GABA agonist [Martin 1993], avermectins, like ivermectin, irreversibly open glutamate-gated chloride channels [Campbell et al. 1983;Martin 1993;Wolstenholme and Rogers 2005], aldicarb is a cholinesterase inhibitor [Baron R L 1994], and pyrantel pamoate inhibits neuromuscular transmission [Jain et al. 2006]. These examples demonstrate that the nervous system offers good targets for the development of treatments. ...
... Indeed, many nematicides work on the nervous system. For example, levamisol is a potent cholinergic agonist [Lewis et al. 1980;Martin 1993;Rand 2007], piperazine is a GABA agonist [Martin 1993], avermectins, like ivermectin, irreversibly open glutamate-gated chloride channels [Campbell et al. 1983;Martin 1993;Wolstenholme and Rogers 2005], aldicarb is a cholinesterase inhibitor [Baron R L 1994], and pyrantel pamoate inhibits neuromuscular transmission [Jain et al. 2006]. These examples demonstrate that the nervous system offers good targets for the development of treatments. ...
... Indeed, many nematicides work on the nervous system. For example, levamisol is a potent cholinergic agonist [Lewis et al. 1980;Martin 1993;Rand 2007], piperazine is a GABA agonist [Martin 1993], avermectins, like ivermectin, irreversibly open glutamate-gated chloride channels [Campbell et al. 1983;Martin 1993;Wolstenholme and Rogers 2005], aldicarb is a cholinesterase inhibitor [Baron R L 1994], and pyrantel pamoate inhibits neuromuscular transmission [Jain et al. 2006]. These examples demonstrate that the nervous system offers good targets for the development of treatments. ...
Because of its complexity and intricacy, studying the nervous system is often challenging. Fortunately, the small nematode roundworm Caenorhabditis elegans is well established as a model system for basic neurobiological research. The C. elegans model is also the only organism with a supposedly complete connectome, an organism-wide map of synaptic connectivity resolved by electron microscopy, which provides some understanding of how the nervous system works as a whole. However, the number of available data-sets is small and the connectome contains errors and gaps. One example of this concerns electrical synapses. Electrical synapses are formed by gap junctions and difficult to map due to their often ambiguous morphology in electron micrographs, leading to misclassification or omission. On the other hand, chemical synapses are more easily mapped, but many aspects of their mode of operation remain elusive and their role in the C. elegans connectome is oversimplified. A comprehensive understanding of signal transduction of neurons between each other and other cells will be indispensable for a comprehensive understanding of the nervous system. In this thesis, I approach these challenges with a combination of advanced light and electron microscopy techniques. First, this thesis describes a strategy to increase synaptic specificity in connectomics. Specifically, I classify gap junctions with a high degree of confidence. To achieve this, I utilized array tomography (AT). In this thesis, AT is adapted for high-pressure freezing to optimize for structure preservation and for super-resolution light microscopy; in this manner, I aim to bridge the gap between light and electron microscopy resolutions. I call this adaptation super-resolution array tomography (srAT). The srAT approach made it possible to clearly identify and map gap junctions with high precision and accuracy. The results from this study showcased the feasibility of incorporating electrical synapses into connectomes in a systematic manner, and subsequent studies have used srAT for other models and questions. As mentioned above, the C. elegans connectomic model suffers from a shortage of datasets. For most larval stages, including the special dauer larval stage, connectome data is completely missing up to now. To obtain the first partial connectome data-set of the C. elegans dauer larva, we used focused ion-beam scanning electron microscopy (FIB-SEM). This technique offers an excellent axial resolution and is useful for acquiring large volumes for connectomics. Together with our collaborators, I acquired several data-sets which enable the analysis of dauer stage-specific “re-wiring” of the nervous system and thus offer valuable insights into connectome plasticity/variability. While chemical synapses are easy to map relative to electrical synapses, signal transduction via chemical transmitters requires a large number of different proteins and molecular processes acting in conjunction in a highly constricted space. Because of the small spatial scale of the synapse, investigating protein function requires very high resolution, which electron tomography provides. I analyzed electron tomograms of a worm-line with a mutant synaptic protein, the serine/threonine kinase SAD-1, and found remarkable alterations in several architectural features. My results confirm and re-contextualize previous findings and provide new insight into the functions of this protein at the chemical synapse. Finally, I investigated the effectiveness of our methods on “malfunctioning,” synapses, using an amyotrophic lateral sclerosis (ALS) model. In the putative synaptopathy ALS, the mechanisms of motor neuron death are mostly unknown. However, mutations in the gene FUS (Fused in Sarcoma) are one known cause of the disease. The expression of the mutated human FUS in C. elegans was recently shown to produce an ALS-like phenotype in the worms, rendering C. elegans an attractive disease model for ALS. Together with our collaboration partners, I applied both srAT and electron tomography methods to “ALS worms” and found effects on vesicle docking. These findings help to explain electrophysiological recordings that revealed a decrease in frequency of mini excitatory synaptic currents, but not amplitudes, in ALS worms compared to controls. In addition, synaptic endosomes appeared larger and contained electron-dense filaments in our tomograms. These results substantiate the idea that mutated FUS impairs vesicle docking and also offer new insights into further molecular mechanisms of disease development in FUS-dependent ALS. Furthermore, we demonstrated the broader applicability of our methods by successfully using them on cultured mouse motor neurons. Overall, using the C. elegans model and a combination of light and electron microscopy methods, this thesis helps to elucidate the structure and function of neuronal synapses, towards the aim of obtaining a comprehensive model of the nervous system.
... This result is in accordance with those found by Quijada et al. [52], who reported that condensed tannins have different modes of action against different parasite species. Considering their mode of action, anthelmintic drugs may be distinguished into nicotinic agonists, acetylcholinesterase inhibitors, GABA agonists, GluCl potentiators, calcium permeability increasers, β-tubulin binders, proton ionophores, inhibitors of malate metabolism, inhibitors of phosphoglycerate kinase and mutase, inhibitors of arachidonic acid metabolism, and stimulators of innate immunity [53,54]. Indeed, levamisole acts as a selective nicotinic agonist to induce contraction of nematode somatic muscle tissue and therefore leads to paralysis [55]. ...
... Indeed, levamisole acts as a selective nicotinic agonist to induce contraction of nematode somatic muscle tissue and therefore leads to paralysis [55]. The known target sites on nematode parasites are proteins including ion channels, enzymes, structural proteins, and molecule transporters [53,54]. Consequently, different bioactive compounds of C. mucronatum could have different targets to exert their anthelmintic activity on infective larvae. ...
Background
Soil-transmitted helminths (STH) infect more than a quarter of the world’s human population. In the absence of vaccines for most animal and human gastrointestinal nematodes (GIN), treatment of infections primarily relies on anthelmintic drugs, while resistance is a growing threat. Therefore, there is a need to find alternatives to current anthelmintic drugs, especially those with novel modes of action. The present work aimed to study the composition and anthelmintic activity of Combretum mucronatum leaf extract (CMLE) by phytochemical analysis and larval migration inhibition assays, respectively.
Methods
Combretum mucronatum leaves were defatted with petroleum ether and the residue was extracted by ethanol/water (1/1) followed by freeze-drying. The proanthocyanidins and flavonoids were characterized by thin layer chromatography (TLC) and ultra-high performance liquid chromatography (UPLC). To evaluate the inhibitory activity of this extract, larval migration assays with STH and GIN were performed. For this purpose, infective larvae of the helminths were, if necessary, exsheathed (Ancylostoma caninum, GIN) and incubated with different concentrations of CMLE.
Results
CMLE was found to be rich in flavonoids and proanthocyanidins; catechin and epicatechin were therefore quantified for standardization of the extract. Data indicate that CMLE had a significant effect on larval migration. The effect was dose-dependent and higher concentrations (1000 µg/mL) exerted significantly higher larvicidal effect (P < 0.001) compared with the negative control (1% dimethyl sulfoxide, DMSO) and lower concentrations (≤ 100 µg/ml). Infective larvae of Ascaris suum [half-maximal inhibitory concentration (IC50) = 5.5 µg/mL], Trichuris suis (IC50 = 7.4 µg/mL), and A. caninum (IC50 = 18.9 µg/mL) were more sensitive to CMLE than that of Toxocara canis (IC50 = 310.0 µg/mL), while infective larvae of Toxocara cati were largely unaffected (IC50 > 1000 µg/mL). Likewise, CMLE was active against most infective larvae of soil-transmitted ruminant GIN, except for Cooperia punctata. Trichostrongylus colubriformis was most sensitive to CMLE (IC50 = 2.1 µg/mL) followed by Cooperia oncophora (IC50 = 27.6 µg/mL), Ostertagia ostertagi (IC50 = 48.5 µg/mL), Trichostrongylus axei (IC50 = 54.7 µg/mL), Haemonchus contortus (IC50 = 145.6 µg/mL), and Cooperia curticei (IC50 = 156.6 µg/mL).
Conclusions
These results indicate that CMLE exhibits promising anthelmintic properties against infective larvae of a large variety of soil-transmitted nematodes.
Graphical Abstract
... Στην ομάδα αυτή περιλαμβάνονται μερικές από τις ασφαλέστερες και αποτελεσματικότερες αντιπαρασι τικές ουσίες (Πίνακας 2). Οι ουσίες αυτές συνδέονται με τους δίαυλους χλωρίου, διευκολύνοντας έτσι τη μα ζική εισροή των ιόντων χλωρίου, μέσω της οποίας προ καλείται υπερπόλωση των νευρώνων, με αποτέλεσμα το θάνατο του παράσιτου λόγω χαλαρής παράλυσης (Martin 1993, Shoop et al. 1995, Lynn 2003. Στις συ χνότερα χρησιμοποιούμενες μακροκυκλικές λακτόνες περιλαμβάνονται η ιβερμεκτινη, η μιλβεμυκινη, η μοξιδεκτινη και η σελαμεκτινη, που βασικά χρησιμοποι ούνται στην πρόληψη της διροφιλαριωσης του σκύλου (Lynn 2001, Bowman 2003a. ...
... Οι ουσίες αυτές, που δρουν ως νικοτινικοί αγωνι στές, προκαλοΰν σπαστική παράλυση των μυών, που οδηγεί γρήγορα στο θάνατο των νηματωδών παρασι-158 Δ. Κ. ΤΑΠΕΣ, Μ. Ε. ΜΥΛΩΝΑΚΗΣ, Η. ΠΑΠΑΔΟΠΟΥΛΟΣ, Α. Φ. ΚΟΥΤΙΝΑΣ των (Martin 1993). Από την κατηγορία αυτή, μόνο η παμοϊκή (εμβονική) πυραντέλη χρησιμοποιείται στο σκΰλο και τη γάτα (Πίνακας 2), το ανθελμινθικό φά σμα της οποίας περιλαμβάνει τα νηματώδη παράσιτα Toxocara spp., Τ. leonina, Angylostoma spp. ...
Gastrointestinal parasitic infections in dogs and cats are a common problem in the clinical setting. Ascarids, hookworms, whipworms and tapeworms should be eliminated on a regular basis, not only to avoid possible health implications on the host itself, but also for the zoonotic potential of certain helminthes (ascarids, hookworms, Echinococcus spp.). Of the highly efficacious and safe medications that eliminate gastrointestinal helminths, commercially available in Greece are benzimidazoles, macrocyclic lactones, tetrahydropyrimidines and isokinolones. Since puppies, kittens, pregnant and nursing animals have a greater risk to acquire gastrointestinal parasites, more intensive anthelmintic schedules are usually instituted. In the less than 6-month old domestic carnivores, deworming should begin at the age of 2-3 weeks and proceed at biweekly intervals through the 12th week, followed thereafter by monthly administrations of the anthelmintics up to the age of 6 months. Nursing bitches and queens should be treated along with their offspring, since they often develop patent infections at that period of time. In animals older than 6 months, faecal examinations should be performed two to four times yearly, followed by the appropriate parasiticidal medication. In dogs which regularly receive heartworm preventatives, deworming schedules have to be modified according to their anthelmintic spectrum.
... One notable feature is the low efficacy of the classical GABA receptor antagonist bicuculline on the Cysloop GABA receptors UNC-49 and EXP-1 from Caenorhabditis elegans (Bamber et al., 2003;Beg and Jorgensen, 2003). This phenomenon has also been observed in the analysis of Cysloop GABA receptors from somatic muscles of the parasitic nematode Ascaris suum (Wann, 1987;Martin, 1993). From these observations, it has been suggested that the agonist binding site of nematode GABA receptors exhibit structural differences compared with mammalian GABAA receptors (Bamber et al., 2003). ...
... There is now a sizable body of evidence suggesting that there is a diversity of invertebrate and bacterial GABA-gated ion channels that do not fall under the umbrella categorization of any vertebrate Cys-loop GABA receptor (Martin, 1993;Hosie and Sattelle, 1996;Bamber et al., 2003;Thompson et al., 2012). In this study, there were several noticeable differences between the profiles of the nematode in comparison with vertebrate GABA receptors. ...
Cys-loop GABA receptors represent important targets for human chemotherapeutics and insecticides and have potential as targets for novel anthelmintics (nematocides). However, compared to insect and mammalian receptors relatively little is known regarding the pharmacological characteristics of nematode cys-loop GABA receptors. Our aim was to investigate the agonist binding site of the cys-loop GABA receptor UNC-49 (Hco-UNC-49) from the parasitic nematode Haemonchus contortus.
We used two-electrode voltage clamp electrophysiology to measure channel activation from classical GABA receptor agonists on Hco-UNC-49 expressed in Xenopus laevis oocytes. This was complimented with site-directed mutagenesis and in silico homology modeling.
The sulphonated molecules P4S and taurine display no effect on Hco-UNC-49. Other classical cys-loop GABAA receptor agonists tested on the Hco-UNC-49B/C heteromeric channel had a rank order efficacy of GABA > TACA > Isoguvacine > IMA > R(-)-GABOB > S(+)-GABOB > GAA > Isonipecotic acid > DAVA (partial agonist) > β-alanine (partial agonist). In silico ligand docking also revealed some variation in binding between agonists. Mutagenesis of a key serine residue in binding loop C to threonine had minimal effects on GABA and IMA but significantly increased the maximal response of DAVA and caused a 2-fold decrease in the EC50 for R(-)-GABOB and S(+)-GABOB.
Our data has revealed differences in the pharmacological profile of Hco-UNC-49 when compared to insect RDL and vertebrate cys-loop GABA receptors suggesting differences in the agonist binding pocket. These findings could possibly be exploited to develop new drugs that specifically target GABA receptors of parasitic nematodes.
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... The imidazothiazole group acts as nicotinic cholinergic agonists in the neuromuscular coordination of parasites. They penetrate the parasite through the cuticle, binding to acetylcholinergic neurotransmitter receptors, producing their activation, leading to the accumulation of acetylcholine in the synaptic cleft and consequently, the parasite shows hyperexcitability and spastic paralysis (MARTIN, 1993). ...
Cattle farming is of paramount importance to the national economy, being represented by 214 million animals. Helminths are a major obstacle to animal production and represent one third of the parasitic diseases that affect cattle. The main form of control of this pathology is from the use of chemotherapy drugs, however, the easy access of the producer to anthelmintics, combined with the lack of adequate professional guidance, has led to the indiscriminate use of these drugs and, consequently, the increase of parasitic resistance. Due to this growth, the emergence of promising alternative control strategies for worms has been observed, such as biological control using nematophagous fungi, as well as the use of herbal medicines.
... Acetylcholine (ACh) and its receptor targets are essential for growth, development, and neuromuscular function in the clade V model nematode Caenorhabditis elegans (42,43). The contribution of nicotinic acetylcholine receptors (nAChRs) to cholinergic signaling is underscored by the successful development of nicotinic channel agonists as antiparasitics (44)(45)(46)(47)(48)(49)(50), but much less is known about the druggability of muscarinic acetylcholine receptors (mAChRs), which are associated with slower but more sustained synaptic and extrasynaptic transmission. The C. elegans genome encodes three known G protein-linked acetylcholine receptors (GARs) (51)(52)(53)(54) that are widely expressed in the nervous system and muscle tissues (53,55) and are involved in the regulation of feeding, mating, egg laying, and locomotion (32,52,(56)(57)(58). ...
The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor of B. malayi (Bma-GAR-3) that is highly expressed across intramammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Finally, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.
... Martin (1980) showed that ionotropic GABA receptors are also present extrasynaptically in the A. suum muscle cell bag region. Various researchers have used two-electrode current-clamp technique to show the unique pharmacology of Ascaris inhibitory GABA channels than vertebrate receptors (Martin, 1980(Martin, , 1993Holden-Dye et al., 1988Duittoz and Martin, 1991). The Ascaris GABA receptor was insensitive to the vertebrate GABA A receptor agonists sulphonic acid derivatives such as piperidine-4-sulphonic acid. ...
Ion channels are specialized multimeric proteins that underlie cell excitability. These channels integrate with a variety of neuromuscular and biological functions. In nematodes, the physiological behaviors including locomotion, navigation, feeding and reproduction, are regulated by these protein entities. Majority of the antinematodal chemotherapeutics target the ion channels to disrupt essential biological functions. Here, we have summarized current advances in our understanding of nematode ion channel pharmacology. We review cys-loop ligand gated ion channels (LGICs), including nicotinic acetylcholine receptors (nAChRs), acetylcholine-chloride gated ion channels (ACCs), glutamate-gated chloride channels (GluCls), and GABA (γ-aminobutyric acid) receptors, and other ionotropic receptors (transient receptor potential (TRP) channels and potassium ion channels). We have provided an update on the pharmacological properties of these channels from various nematodes. This article catalogs the differences in ion channel composition and resulting pharmacology in the phylum Nematoda. This diversity in ion channel subunit repertoire and pharmacology emphasizes the importance of pursuing species-specific drug target research. In this review, we have provided an overview of recent advances in techniques and functional assays available for screening ion channel properties and their application.
... Levamisole is able to paralyze nematode muscles, leaving the worms unable to attach themselves to the mucous membranes, and causing them to be expelled through the intestine. 8 Levamisole has several other effects on human organisms: it exerts immunomodulatory properties and acts on the dopaminergic, cholinergic and noradrenergic systems. 9 Levamisole was subsequently used for its immunomodulatory action in certain forms of rheumatoid arthritis and in association with 5-fluorouracil in patients with colon cancer or melanoma. ...
Levamisole was initially prescribed for the treatment of intestinal worms. Because of immunomodulatory properties, levamisole has been used in inflammatory pathologies and in cancers in association with 5‐fluorouracil. Levamisole is misused as a cocaine adulterant. Post‐marketing reports have implicated levamisole in the occurrence of adverse drug reactions (ADRs) and its use is now limited in Europe and North America. In contrast, all other parts of the World continue to use single‐dose levamisole as an anthelmintic. The aim of this study was to identify ADRs reported after levamisole exposure in VigiBase, the World Health Organisation's pharmacovigilance database, and analyse their frequency compared to other drugs and according to levamisole type of use.
Methods
All levamisole‐related ADRs were extracted from VigiBase. Disproportionality analyses were conducted to investigate psychiatric, hepatobiliary, renal, vascular, nervous, blood, skin, cardiac, musculoskeletal and general ADRs associated with levamisole and other drugs exposure. In secondary analyses, we compared the frequency of ADRs between levamisole and mebendazole and between levamisole type of use.
Results
Among the 1763 levamisole‐related ADRs identified, psychiatric disorders (reporting odds ratio with 95% confidence intervals: 1.4 [1.2–2.6]), hepatobiliary disorders (2.4 [1.9–4.3]), vasculitis (6.5 [4.1–10.6]), encephalopathy (22.5 [17.4–39.9]), neuropathy (4.3 [2.9–7.1]), haematological disorders, mild rashes and musculoskeletal disorders were more frequently reported with levamisole than with other drug. The majority of levamisole‐related ADRs occurred when the drug was administrated for a non–anti‐infectious indication.
Conclusion
The great majority of the levamisole‐related ADRs concerned its immunomodulatory indication and multiple‐dose regimen. Our results suggest that single‐dose treatments for anthelmintic action have a good safety profile.
... This action results in the paralysis and expulsion of the nematodes from the mammalian hosts (Arena et al. 1992;Cully et al. 1994;Bloomquist 2003). Piperazine citrate is also a GABA agonist and expel nematodes from host intestine (Martin 1993). Although the effective deworming dosage of ivermectin and piperazine citrate generally have no toxicity for most mammalian hosts, it was observed that even at a low concentration, these drugs can kill the cockroach hosts. ...
The efficacy of pyrantel pamoate, pyrvinium pamoate, ivermectin, and piperazine citrate against pinworm in cockroach was evaluated. Laboratory-reared German cockroaches naturally infected with Blatticola blattae were treated with the anthelmintics and necropsied at 3 to 35 days after treatment. Ivermectin at over 5 ppm and piperazine citrate at over 2000 ppm killed all the treated cockroaches. Pinworms were still detected in cockroaches given lower concentration of the aforementioned drugs. Administration of pyrantel pamoate (100–1000 ppm) and pyrvinium pamoate (2000 ppm) did not kill the cockroaches, and no pinworms were detected at 3 and 17 days after treatment. Thus, pyrantel pamoate and pyrvinium pamoate were found to be effective for deworming B. blattae in the German cockroaches, without causing mortality for the host. Our results showed that anthelmintics selection is essential for eradication of pinworms in cockroaches because of the toxicity for the host such as ivermectin or piperazine citrate. This is the first report of piperazine citrate toxicity in cockroaches.
... Parasitic infections by soil-transmitted helminths (STHs: Ascaris, Trichuris and 58 hookworm) are a major medical and public health concern in many developing (Aubry, 1970;Aceves, 1970;Martin and Robertson, 2007). Frequent use of these 78 anthelmintics has led to the emergence of widespread drug resistance in animal 79 parasites (Prichard, 1994) and there is concern about the development of resistance in We used Ascaris suum for our studies (Martin, 1993). This parasite is closely related to 118 Ascaris lumbricoides seen in humans (Boes and Helwigh, 2000;Nejsum et al., 2005). ...
Cholinergic agonists, like levamisole, are a major class of anthelmintic drug 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).
... Therefore, different compounds/active principles of plant extracts/fractions may have different targets to exert anthelmintic effect on eggs, larvae and adults. The known target sites on parasites are solely proteins and include ion channels, enzymes, structural proteins, transport molecules, etc. (Lacey, 1988;Geary et al., 1992;Martin, 1993;Kohler, 2001). ...
This study was carried out to assess the anthelmintic activity of Acacia nilotica bark and leave extracts in different solvents. Adult motility assay, egg hatch test and fecal egg count reduction test were carried out to evaluate the anthelmintic activity. Effect of plant extracts both of leaves and bark of A. nilotica was dose-dependent. Highest mortality of worms was observed 12 hours post-exposure @ 25 mg/ml. Extracts of leaves were more potent than the bark extracts. Ethyle acetate fractions both of bark and leaves exhibited higher anthelmintic effects compared with chloroform, petroleum spirit and aqueous fractions. Crude aqueous methanol extract (CAME) of bark (LC 50= 201.0032 μg/ml) had higher inhibitory effects compared with that of leaves (LC 50= 769.2485 μg/ml) on egg hatching. Likewise, chloroform and ethyle acetate fractions of A. nilotica bark exhibited higher ovicidal activity. In vivo, maximum reduction (72.01%) in fecal egg counts was recorded for CAME of bark followed by CAME of leaves (63.44%) @ 8 g/kg at day 12 post-treatment. Results suggest lipophilic nature of the active principles having anthelmintic efficacy in A. nilotica bark and leaves.
... Ascaris muscle was shown to contract in response to both acetylcholine and nicotine, and inhibition of ACh-induced contractions could be accomplished by application of tubocurarine and mecamylamine; this provided direct evidence for a nAChR involved in Ascaris muscle contractions (Baldwin & Moyle, 1949;Natoff, 1969;Rozhkova et al., 1980). Electrophysiological techniques have also been widely used to study the pharmacology of native Ascaris nAChRs; these recordings utilize extrasynaptic receptors present on the muscle bag cell (Colquhoun et al., 1991;Martin, 1993). ...
... This is consistent with our EC 50 data for the Hco-UNC-49 channel which clearly shows piperazine to be about 100-130 fold less potent compared to GABA. Furthermore, it has been noted that the GABA receptor characterized in the piperazine-sensitive A. suum muscle tissue shares a similar pharmacological profile with UNC-49 (Bamber et al., 2003;Martin, 1993;Siddiqui et al., 2010). Additionally, there is evidence for the presence of an unc-49-like coding sequence in the A. suum genome (GenBank Accession Number: BM319703). ...
... Different compounds/ active principles of essential oils may have different targets to exert toxicity and repellency on insects. The known target sites on parasites are solely proteins and include ion channels, enzymes, structural proteins, transport molecules, etc. [18][19][20][21][22]. Sonia et al determined effect of Anthumgraveolens L. extract on biochemical and histopathological alteration of deltamethrin in rats [23]. ...
A study was conducted to evaluate the insecticidal activity of essential oils obtained from root, stem and leaves
of Boenninghausenia albiflora (Sapindales: Rutaceae) against Black garden ant Lasius Niger L. (Hymenoptera:
Formicidae). The major compounds in these essential oils were identified using gas chromatography-mass
spectrometry and their insecticidal activity was tested at three concentrations i.e. 1, 5 and 10% in ethanol. All
essential oils showed similar insecticidal and repellent activity at each concentration but significantly different at
p≤0.05 from controls with LC50=12.35 μl, while dose dependent effect was significant with R2=0.803. It can be
concluded that the three essential oils in this study have both insecticidal as well as repellent effect.
... This group is used in cattle by subcutaneous rout, at the dose of 9.4 mg/kg and it acts mainly in the neuromuscular coordination of parasites, as a nicotinic cholinergic agonist. They penetrate the parasite through the cuticle and bind to acetylcholinergic neurotransmitters, causing excessive hyperpolarization of the post-synaptic membrane and spastic paralysis of the parasites (Martin, 1993). As to disophenol, the levamisole is a drug with reduced safety index, and even at therapeutic doses, the animals may exhibit excitement and drooling. ...
The current study evaluated, in vivo, the clinical safety and the anthelmintic efficacy of 24% aurixazole (24 mg/kg), administered orally, in bovines. Two experiments were conducted: the first one evaluating the clinical safety of 24% aurixazole (24 mg/kg) in cattle, and a second one evaluating the anthelmintic efficacy of aurixazole (24 mg/kg) against gastrointestinal nematodes on naturally infected cattle. Based on the results of clinical safety, no alterations on clinical and haematological signs and on the biochemical values obtained in animals treated orally with aurixazole 24 mg/kg were observed. Regarding the results of reduction or efficacy, obtained by eggs per gram of faeces (EPG) counts, the formulation of aurixazole reached values superior to 99% (arithmetic means) in all post-treatment dates. In two occasions, this formulation reached maximum efficacy (100%). Comparing these results with the reduction percentages obtained by EPG counts, it is possible to verify that the values obtained by all three formulations were compatible with the efficacy results. Aurixazole reached maximum efficacy (100%) against Haemonchus placei, Cooperia spatulata and Oesophagostomum radiatum. Against Cooperia punctata, this formulation reached an efficacy index of 99.99%. Regarding aurixazole, no specific trials were conducted on the field in order to evaluate the behaviour of this molecule against helminths that are resistant to other molecules, specially isolated levamisole and disophenolat. Due to this fact, future studies will be necessary to assess the effectiveness of aurixazole against strains of nematodes that are resistant to levamisole and disophenolat, but the results of clinical safety and efficacy described in this study allow us to conclude that the aurixazole molecule, concomitantly with other measures and orally administered formulations, can be another important tool in the control of nematodes parasitizing bovines.
... This group acts mainly in the neuromuscular coordination of parasites, as a nicotinic cholinergic agonist. They penetrate in the parasite through the cuticle and bind to acetylcholinergic neurotransmitters, causing excessive hyperpolarization of the post-synaptic membrane and spastic paralysis of the parasites (Martin, 1993). ...
... In vitro treatment of fish leukocytes with levamisole enhanced phagocytic cell activities (chemotactic activity, phagocytosis, respiratory burst and myeloperoxidase activity) or natural cytotoxic activity in carp (Siwicki, 1987(Siwicki, , 1989Baba et al., 1993), rainbow trout (Kajita et al., 1990), coho salmon (Olivier et al., 1985) and gilthead seabream (Meseguer et al., 1997;Mulero et al., 1998aMulero et al., , 1998bCuesta et al., 2002). Levamisole acts on nicotinic receptors of Ascaris suum and other large nematodes (Martin, 1993). Levamisole is found to be effective in a freshwater bath against the nematode Anguillicola crassus, pathogenic in eels under in vivo conditions . ...
The purpose of this paper is to provide a general overview of important manifested parasitic diseases of Mediterranean fish and their control methods. Currently available treatments practised in Mediterranean mariculture are explained briefly under two main groups: parasites located on the skin and gills, and those located in internal organs. Available pharmaceutical and biological products for the treatment of Monogenea, Isopoda, Copepoda, Cestoda and Protozoa are described, and problems related to their therapy are discussed. The mode of action of these drugs is also explained and new research developments in this area are presented.
... Mode of action. Binds to acetylcholine receptors and inhibits the production of succinate dehydrogenase, causing spastic paralysis and passive elimination of the worms (Martin, 1993). ...
... Furthermore, unlike mammalian GABA A receptors, the UNC-49 receptor is relatively insensitive to bicuculline inhibition or benzodiazepine enhancement and, unlike the Drosophilia RDL receptors, is insensitive to dieldrin (Bamber et al. 2003;Brown et al. 2012). Overall, the unique pharmacology of the UNC-49 receptor mostly resembles that of the GABA receptor characterized in Ascaris muscle (Holden-Dye et al. 1989;Walker et al. 1992;Martin 1993). Interestingly, the GABA A -q receptor has also been shown to be insensitive to bicuculline and many other traditional GABA A allosteric modulators such as benzodiazepines (i.e. ...
Parasitic nematode infection of humans and livestock is a major problem globally. Attempts to control nematode populations have led to the development of several classes of anthelmintic, which target cys-loop ligand-gated ion channels. Unlike the vertebrate nervous system, the nematode nervous system possesses a large and diversified array of ligand-gated chloride channels that comprise key components of the inhibitory neurotransmission system. In particular, cys-loop GABA receptors have evolved to play many fundamental roles in nematode behaviour such as locomotion. Analysis of the genomes of several free-living and parasitic nematodes suggests that there are several groups of cys-loop GABA receptor subunits that, for the most part, are conserved among nematodes. Despite many similarities with vertebrate cys-loop GABA receptors, those in nematodes are quite distinct in sequence similarity, subunit composition and biological function. With rising anthelmintic resistance in many nematode populations worldwide, GABA receptors should become an area of increased scientific investigation in the development of the next generation of anthelmintics.
... Il existe à ce jour trois grandes familles de molécules anthelminthiques efficaces contre les strongles gastro-intestinaux des ovins (Lanusse et Prichard, 1993) Les benzimidazoles agissent en bloquant certains systèmes enzymatiques du métabolisme anaérobie des parasites, les privant ainsi de leurs ressources énergétiques (Prichard, 1973), mais également en inhibant la formation des microtubules du cytosquelette des parasites, sans altérer ceux de l'hôte (Lacey, 1988 ;Martin et al., 1997) Le lévamisole (famille des imidazothiazoles) est un agoniste de l'acétylcholine ; en se fixant sur les récepteurs nicotiniques du parasite, il entraîne une paralysie spastique de celui-ci et sa mort (Martin, 1993(Martin, et 1997Kohler, 2001). Cette molécule n'a en revanche aucun effet sur les larves inhibées. ...
Les Nématodes gastro-intestinaux sont des parasites majeurs en élevage ovin. Actuellement, leur contrôle repose sur l'utilisation de molécules anthelminthiques. L'extension de résistances à ces molécules dans les populations de strongles rend urgente la recherche de solutions alternatives comme la sélection d'animaux résistants. Toutefois, la méconnaissance des mécanismes de la réponse immunitaire des ovins contre ces strongles reste un obstacle à son développement. La première partie de nos travaux a démontré une orientation Th2 claire lors d'infestation par Haemonchus contortus et plus équivoque lors d'infestation par Trichostrongylus colubriformis. Dans un second temps, les réponses immunes adaptatives de moutons de race sensible (INRA 401) et résistante (Barbados Black Belly) lors d'infestation par H. contortus ont été comparées. Des différences d'expression des gènes de l'IL-4, IL-5 et de l'IL-13, et d'éosinophilie sanguine ont été enregistrées entre les deux races. ABSTRACT : Gastrointestinal nematodes are major parasites of sheep industry. Current control is mainly based on chemical molecules. Due to the extension of anthelmintic resistance in nematode populations all around the world, alternative strategies, such as selective breeding of resistant sheep, are now necessary. Nevertheless, the imprecise knowledge of sheep immune mechanisms towards these parasites limits their development. The first part of this study demonstrated a clear Th2 polarization of the sheep immune response to Haemonchus contortus infection. In another hand, the response to Trichostrongylus colubriformis was more equivocal. In a second time, adaptative immune responses to H. contortus infection were compared between a susceptible breed (INRA 401) and a resistant one (Barbados Black Belly). Differences in IL-4, IL-5 and IL-13 gene expressions and blood eosinophilia were noticed between the two breeds.
... Determining the role of GluClα is complicated further by indications that avermectins potentiate gating of GABA-gated channels by GABA (Pong and Wang, 1982;Sigel and Baur, 1987) or reduce the mean channel current (Martin and Pennington, 1989). Since GABA-gated channels are expressed in nematodes (see Martin, 1993;McIntire et al., 1993a,b) they are also potentially relevant targets of avermectins. ...
Ivermectin is a widely used anthelmintic drug whose nematocidal mechanism is incompletely understood. We have used Caenorhabditis elegans as a model system to understand ivermectin's effects. We found that the M3 neurons of the C.elegans pharynx form fast inhibitory glutamatergic neuromuscular synapses. avr-15, a gene that confers ivermectin sensitivity on worms, is necessary postsynaptically for a functional M3 synapse and for the hyperpolarizing effect of glutamate on pharyngeal muscle. avr-15 encodes two alternatively spliced channel subunits that share ligand binding and transmembrane domains and are members of the family of glutamate-gated chloride channel subunits. An avr-15-encoded subunit forms a homomeric channel that is ivermectin-sensitive and glutamate-gated. These results indicate that: (i) an ivermectin-sensitive chloride channel mediates fast inhibitory glutamatergic neuromuscular transmission; and (ii) a nematocidal property of ivermectin derives from its activity as an agonist of glutamate-gated chloride channels in essential excitable cells such as those of the pharynx.
... The nematode neuromuscular system is relatively simple in construction (Goldschmidt, 1908;Stretton, 1976; for review see Martin, 1993). In the parasitic nematode, kscaris suum, the nervous system comprises 298 nerve cells, 162 of which lie in the head region (including the retrovesicular ganglion) (Stretton et al., 1992). ...
A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated from Ascaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also depended on external Ca++ and were reduced by the Ca(++)-channel blocker verapamil, observations consistent with the finding that nematode NOS is Ca(++)-dependent. KSAYMRFamide (PF3), KNIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A. suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.
The diversification of anthelmintic targets and mechanisms of action will help ensure the sustainable control of nematode infections in response to the growing threat of drug resistance. G protein-coupled receptors (GPCRs) are established drug targets in human medicine but remain unexploited as anthelmintic substrates despite their important roles in nematode neuromuscular and physiological processes. Bottlenecks in exploring the druggability of parasitic nematode GPCRs include a limited helminth genetic toolkit and difficulties establishing functional heterologous expression. In an effort to address some of these challenges, we profile the function and pharmacology of muscarinic acetylcholine receptors in the human parasite Brugia malayi, an etiological agent of human lymphatic filariasis. While acetylcholine-gated ion channels are intensely studied as targets of existing anthelmintics, comparatively little is known about metabotropic receptor contributions to parasite cholinergic signaling. Using multivariate phenotypic assays in microfilariae and adults, we show that nicotinic and muscarinic compounds disparately affect parasite fitness traits. We identify a putative G protein-linked acetylcholine receptor (Bma-GAR-3) that is highly expressed across intra-mammalian life stages and adapt spatial RNA in situ hybridization to map receptor transcripts to critical parasite tissues. Tissue-specific expression of Bma-gar-3 in Caenorhabditis elegans (body wall muscle, sensory neurons, and pharynx) enabled receptor deorphanization and pharmacological profiling in a nematode physiological context. Lastly, we developed an image-based feeding assay as a reporter of pharyngeal activity to facilitate GPCR screening in parasitized strains. We expect that these receptor characterization approaches and improved knowledge of GARs as putative drug targets will further advance the study of GPCR biology across medically important nematodes.
Infection with Ascaris lumbricoides constitutes one of the most common helmintic diseases in the world, especially in tropical and subtropical regions. Transmission of this disease involves environmental contamination with eggs, and therefore, is classified as a soil-transmitted disease. The public health importance of ascariasis is made evident by the high number of infected individuals suffering its consequences, which can become severe, depending on the worm burden. Since the introduction of benzimidazoles, chemotherapy has improved significantly, offering fast and direct effects at relatively low cost, as a result of the simple and safe application of these drugs. Unfortunately, the effects are not long-lasting or permanent. The intervention with chemotherapy alone constitutes a fast and efficient way to reduce the worm burden in a population of high prevalence, but does not avoid reinfection. Therefore, the option of integrated control programmes based on chemotherapy in combination with sanitation and health education, together with strong community involvement, must be considered in order to ensure the positive long-term effects of such programmes.
The structural simplicity of nematode nervous systems has provided a useful resource for studies on basic neurobiology. Two species have underpinned basic neuroscience research in phylum Nematoda, the free-living nematode Caenorhabditis elegans and the pig parasitic nematode Ascaris suum due to their genetic tractability and large size, respectively. The fact that most of the chemotherapeutic targets for the treatment of roundworm parasite infections of animals and man associate with neuromuscular function means that nematode neurobiology research resonates beyond discovery biology and informs parasite control. These diverse drivers for the interrogation of nematode neurobiology have exposed a unique approach to the regulation of complex behavioral traits, encompassing structural simplicity with profound chemical complexity. The latter embraces extreme diversity in both the ligand-gated ion channel subunit complements for classical neurotransmitters and in the neuropeptide ligands that signal mostly via G-protein coupled receptors, offering compelling opportunities for parasite control-target discovery and validation.
The goal of widespread acceptance of sustainable, evidence-based parasite control programs can be accomplished through continuing education of veterinary practitioners and distribution of high-quality information to horse owners. The objective of this chapter is to provide the veterinary practitioner with the tools and information needed to achieve that goal. An important principle for evidence-based management of parasite populations is maintenance of healthy herd refugia. Practitioners should always refer to the manufacturer's recommendations concerning dosing, spectrum of activity, efficacy, safety, and contraindications, because availability and labeling claims vary between countries and change over time. The chapter talks about macrocyclic lactones, moxidectin, benzimidazoles, and tetrahydropyrimidine anthelmintics. It concludes with a discussion on broad-spectrum combination anthelmintic products.
Molecular cloning has introduced an unexpected, large diversity of neurotransmitter hetero- oligomeric receptors. Extensive research on the molecular structure of the γ-aminobutyric acid receptor (GABAR) has been of great significance for understanding how the nervous system works in both vertebrates and invertebrates. However, only two examples of functional homo-oligomeric GABA-activated Cl(-) channels have been reported. In the vertebrate retina, the GABAρ1 subunit of various species forms homo-oligomeric receptors; in invertebrates, a cDNA encoding a functional GABA-activated Cl(-) channel has been isolated from a Drosophila melanogaster head cDNA library. When expressed in Xenopus laevis oocytes, these subunits function efficiently as a homo-oligomeric complex. To investigate the structure-function of GABA channels from the crayfish Procambarus clarkii, we cloned a subunit and expressed it in human embryonic kidney cells. Electrophysiological recordings show that this subunit forms a homo-oligomeric ionotropic GABAR that gates a bicuculline-insensitive Cl(-) current. The order of potency of the agonists was GABA > trans-4-amino-crotonic acid = cis-4-aminocrotonic acid > muscimol. These data support the notion that X-organ sinus gland neurons express at least two GABA subunits responsible for the formation of hetero-oligomeric and homo-oligomeric receptors. In addition, by in situ hybridization studies we demonstrate that most X-organ neurons from crayfish eyestalk express the isolated pcGABAA β subunit. This study increases the knowledge of the genetics of the crayfish, furthers the understanding of this important neurotransmitter receptor family, and provides insight into the evolution of these genes among vertebrates and invertebrates. © 2015 Wiley Periodicals, Inc.
This chapter discusses the interactions between drugs and gene products in Caenorhabditis elegans in genetic pharmacology. Caenorhabditis elegans has been a popular organism for the study of drug action. This chapter discusses the methods that used in compound-based studies of C. elegans and evaluates the effects of compounds on C. elegans growth, development, metabolism, and behavior. The strategies for the isolation, and analysis of drug-resistant and hypersensitive mutants are discussed. Studies combining bioactive compounds and C. elegans can be separated based on experimental strategy. The first strategy employs compounds with known modes of action to characterize particular aspects of C. elegans biology in wild type and mutant animals. The second strategy uses active compounds as screening or selective agents to isolate new drug-resistant or hypersensitive mutants and, thus, to identify genes with altered drug responses. Study of such compound-specific mutants can identify specific drug targets, and/or provide insight into the mechanism of drug action and the sites of drug action. The third strategy involves the use of C. elegans, both wild type and selected mutants, to analyze the mechanism of action of uncharacterized or poorly characterized compounds. This has led to the use of C. elegans as a primary screen for compounds active against parasitic nematodes.
A study was conducted to evaluate the toxicity and repellency of essential oils from leaves of Skimmia laureola (DC.), Murraya paniculata (Linn) cv. Desi, Murraya paniculata (Linn) cv. China and Murraya koenigii (Linn.) of family Rutaceae in Pakistan. The oils were tested at three concentrations i.e. 1, 5 and 10 %. Common ant species Lasius niger (black ant) of Pakistan were selected and subjected to force-feeding at room temperature. Results showed that insecticidal activity of each tested essential oil was significantly different from each other. Murraya paniculata (both varieties) was the most effective tested oil (LC50 = 6.58 μL) while Skimmia laureolas showed least repellent activity (LC50 = 10.15 μL).
Ivermectin is a potent antiparasitic drug against nematode and arthropod parasites. In this study, we examined the lethal and sublethal effects of ivermectin in a freshwater oligochaete, Lumbriculus variegatus. The median lethal concentration (LC50) at 72 h after ivermectin exposure was 560 nM. Sublethal endpoints focused on several stimulus-evoked locomotor behaviors: escape reflexes controlled by giant interneuron pathways, swimming and reversal, and crawling. Swimming, reversal, and crawling are controlled by nongiant interneuron pathways. Ivermectin inhibited swimming, reversal, crawling frequency, and crawling speed in a time- and concentration-dependent manner with a mean inhibitory concentration (IC50) at 3 h of 1.1, 16, 91, and 51nM, respectively. Ivermectin at 0.3 nM also significantly decreased the frequency of helical swimming waves. Picrotoxin, a Cl− channel blocker, antagonized the ivermectin-induced decrease in swimming frequency, crawling frequency, and crawling speed. There were no adverse effects on escape reflex 3 h after exposure to 300 nM ivermectin. Electrophysiological recordings showed that ivermectin had no effects on the conduction velocity of giant fiber systems. The results indicated that locomotor behaviors controlled by nongiant locomotor pathways were more sensitive to ivermectin than pathways controlled by giant interneurons and that Cl− channels may be involved in mediating ivermectin's inhibitory effects.
Nematodes represent a serious threat to both plants and animals. New methods of control of theseparasites are being sought since a number of soil-applied commercial nematocides are being phased out and resistance to anthelmintics is an increasing problem. Integrated pest management, transgenic plant resistance and biological control strategies are being investigated as methods of control. At the same time, the search for naturally occurring antinematodal compounds continues unabated. The discovery of the potent anthelmintic activity of the avermectins and milbemycins has stimulated interest in the identification of other natural products with high activity and selective mode of action towards nematodes. In this report, the wide range of metabolites shown to have antinematodal activity are reviewed. Aspects of their chemistry and activity are outlined and, wherever possible, the relationships between structure and bioactivity are emphasised.
Invertebrate ligand-gated chloride channels are well recognized as important targets for several insecticides and anthelmintics. Hco-UNC-49 is a GABA-gated chloride channel from the parasitic nematode Haemonchus contortus and is an orthologue to the neuromuscular receptor (Cel-UNC-49) from the free-living nematode Caenorhabditis elegans. While the receptors from the two nematodes are similar in sequence, they exhibit different sensitivities to GABA which may reflect differences in in vivo function. The aim of the current study was to further characterize the pharmacology of the Hco-UNC-49 receptor by examining its sensitivity to various insecticides and anthelmintics using two-electrode voltage clamp. Specifically, the insecticides fipronil and picrotoxin appear to inhibit the channel in a similar manner. The IC(50) of picrotoxin on the homomeric channel was 3.65 ± 0.64 μM and for the heteromeric channel was 134.56 ± 44.12 μM. On the other hand, dieldrin, a well-known insect GABA receptor blocker, had little effect on the UNC-49 channel. The anthelmintics ivermectin and moxidectin both moderately potentiated the activation of Hco-UNC-49 by GABA, while piperazine was able to directly activate both the Hco-UNC-49 homomeric and heteromeric channels with EC(50) values of 6.23 ± 0.45 mM and 5.09 ± 0.32 mM, respectively. This piperazine current was reversibly blocked by picrotoxin which demonstrates that the anthelmintic specifically targets Hco-UNC-49. These results demonstrate that Hco-UNC-49 exhibits binding sites for several molecules including piperazine and macrocyclic lactone anthelmintics. In addition, this is the first report of the heterologous expression and subsequent characterization of a receptor for piperazine.
Many drugs with very different affinity to a large number of receptors are described. Thus, in this work, we selected drug-target pairs (DTPs/nDTPs) of drugs with high affinity/nonaffinity for different targets. Quantitative structure-activity relationship (QSAR) models become a very useful tool in this context because they substantially reduce time and resource-consuming experiments. Unfortunately, most QSAR models predict activity against only one protein target and/or they have not been implemented on a public Web server yet, freely available online to the scientific community. To solve this problem, we developed a multitarget QSAR (mt-QSAR) classifier combining the MARCH-INSIDE software for the calculation of the structural parameters of drug and target with the linear discriminant analysis (LDA) method in order to seek the best model. The accuracy of the best LDA model was 94.4% (3,859/4,086 cases) for training and 94.9% (1,909/2,012 cases) for the external validation series. In addition, we implemented the model into the Web portal Bio-AIMS as an online server entitled MARCH-INSIDE Nested Drug-Bank Exploration & Screening Tool (MIND-BEST), located at http://miaja.tic.udc.es/Bio-AIMS/MIND-BEST.php . This online tool is based on PHP/HTML/Python and MARCH-INSIDE routines. Finally, we illustrated two practical uses of this server with two different experiments. In experiment 1, we report for the first time a MIND-BEST prediction, synthesis, characterization, and MAO-A and MAO-B pharmacological assay of eight rasagiline derivatives, promising for anti-Parkinson drug design. In experiment 2, we report sampling, parasite culture, sample preparation, 2-DE, MALDI-TOF and -TOF/TOF MS, MASCOT search, 3D structure modeling with LOMETS, and MIND-BEST prediction for different peptides as new protein of the found in the proteome of the bird parasite Trichomonas gallinae, which is promising for antiparasite drug targets discovery.
Abstract: Baylisascaris transfuga, the bear roundworm is an ascaroid parasite that has been reported in all species of bears (giant panda, Ailuropoda melanoleuca; Malayan sun bears, Helarctos malayanus; sloth bears, Melursus ursinus; American black bears, Ursus americanus; brown bear, Ursus arctos; polar bears, Ursus maritimus; Asiatic black bear, Selenarctos thibetanus; and Andean bears, Tremarctos ornatus). This ubiquitous nematode of bears is particularly problematic for captive populations. Baylisascaris species have been implicated in clinical and subclinical disease in natural hosts including bears, as well as lethal larval migrans syndromes in a number of domestic species, alternative livestock, and captive and free ranging incidental hosts, including humans. Eradication or improved control measures for addressing contaminated bear enclosures will heighten biosecurity for this infectious pathogen and reduce the risk of potential public health threats associated with Baylisascaris species. Impediments to control efforts include ineffective sanitation and disinfection of contaminated and re-contaminated exhibits, and concerns regarding the development of parasite resistance in helminth parasites of bears treated routinely with anti-helminthic pharmacological agents. The development of naturalistic exhibits and environmental enrichment programs in zoos and other captive wildlife facilities, enhances the educational mission of zoos and captive wildlife display facilities, but impedes husbandry practices and preventive medicine programs aimed at prevention, control and eradication of infectious pathogens in captive enclosures. The first objective of this study was to obtain information on husbandry and preventive health programs from captive bear-holding facilities for evaluating factors which influence parasite contamination, persistence, and transmission among individual bears, bears species, other collection animals and people. The second objective was to obtain data on coprodiagnostic tests used by institutions to detect parasite ova prevalence in captive collections and conduct baseline studies of current roundworm prevalence and intensity levels in bear holding collections. For this second objective, two coprodiagnostic techniques were employed to compare sensitivities of tests typically employed by captive facilities and more sophisticated tests known to yield higher recoveries of parasite ova. A final objective was to compare the prevalence and intensity levels of roundworms among wild populations of bears and between captive and free-ranging populations. Thesis (Ph. D.)--Ohio State University, 2006. System requirements: World Wide Web browser.
In an immunocytochemical study, using an antiserum and a monoclonal antibody specific for the amino acid, gamma-aminobutyric acid (GABA), GABA-like immunoreactivity (GLIR) has been demonstrated for the first time in parasitic flatworms. In Moniezia expansa (Cestoda), GLIR was seen in nerve nets which were closely associated with the body wall musculature and in the longitudinal nerve cords. In the liver fluke Fasciola hepatica (Trematoda), the GLIR occurred in the longitudinal nerve cords and lateral nerves in the posterior half of the worm. GLIR was also detected in subtegumental fibres in F. hepatica. The presence of GABA was verified, using high-pressure liquid chromatography coupled with fluorescence detection. The concentration of GABA (mean +/- S.D.) in M. expansa anterior region was 124.8 +/- 15.3 picomole/mg wet weight, while in F. hepatica it was 16.8 +/- 4.9 picomole/mg. Since several insecticides and antinematodal drugs are thought to interfere with GABA-receptors, the findings indicate that GABAergic neurotransmission may be a potential target for chemotherapy in flatworms too.
Two cDNAs (Ce21 and Ce13) were isolated from a Caenorhabditis elegans library screened with a probe encoding conserved domains of the avian alpha5 neuronal nicotinic acetylcholine receptor (nAChR). Alignments to all nAChR subunits in the EMBL/Swissprot data base demonstrate that the Ce21 protein most resembles the vertebrate alpha7 subunit, whereas Ce13 is closest to the ARD subunit of Drosophila. The corresponding genes were isolated and hybridized to YAC grids: Ce21 maps on chromosome V near the his-23 gene, and Ce13 on chromosome I very near or at unc-29. The structure of the Ce21 gene was compared with that of other vertebrate and invertebrate nAChR genes and found to share by far the largest number of conserved splice sites with the vertebrate alpha7 gene. Upon expression in the Xenopus oocyte system, the Ce21 subunit assembled into a functional homomeric nAChR, whose properties were compared with those of the chicken alpha7 receptor. The anthelmintic nicotinic agonist levamisole is unable to activate the Ce21 and alpha7 receptors, but efficiently antagonises their responses to ACh. Both receptors desensitise quickly upon agonist application, are more sensitive to nicotine than to acetylcholine, and are efficiently blocked by dihydro-beta-erythroidine. Unlike the alpha7 receptor, however, the Ce21 receptor is relatively insensitive to methyllycaconitine and to alpha-bungarotoxin. The similarities in protein sequence, gene structure and physiological properties between alpha7 and Ce21 suggest a very ancient lineage for the alpha7 class of nAChR subunits.
The chemotherapeutic control of helminth parasites is compromised by the limited number of classes of anthelmintic drugs. Discovery of novel anthelmintics is impeded by the lack of novel screening technologies that overcome the difficulties inherent in screens based on whole organism toxicity. The development and implementation of mechanism-based screens for new anthelmintics offers great promise for the revitalization of antiparasitic drug discovery. However, mechanism-based screens must be based on a thorough understanding of the proteins or processes that offer the best chance for selective chemotherapeutic intervention. Basic research on the characterization of nematode FMRFamide-related peptides (FaRPs) has revealed that these peptides are ubiquitously distributed in helminths. Chemical identification of a number of nematode FaRPs has been achieved, and these peptides have potent and profound effects on the nematode neuromuscular system. Physiological processes mediated by nematode FaRPs (and other helminth neuropeptides) offer potential targets for the discovery of novel anthelmintics.
The induction of vancomycin resistance in enterococci containing the vanA gene cluster is thought to be controlled by a two-component sensor-response regulator system encoded by vanR and vanS. Eight inducing compounds were identified by screening a panel of more than 6,800 antibiotics and synthetic compounds including the three tested glycopeptides (vancomycin, avoparcin, and ristocetin), two other cell wall biosynthesis inhibitors (moenomycin and bacitracin), two cyclic peptide antibiotics (antibiotic AO341 beta and polymyxin B), and a macrocyclic lactone antibiotic (moxidectin). Induction activity by structurally unrelated antibiotics suggests that the induction signal is not a structural feature of vancomycin.
Isolated muscle bags from the parasitic nematode Ascaris suum were prepared by collagenase treatment and dissection. Single bags were mounted in a V-shaped plastic pipette for voltage clamp application and intra- and extracellular perfusion. With 'physiological' intra- and extracellular solutions, depolarizing voltage steps from near the normal resting membrane potential, -40 mV, produced in leak-corrected currents, a slowly activating outward current at potentials more positive than -20mV. At the end of the depolarizing pulse there was a slow inward tail current with a reversal potential near -20mV. Hyperpolarizing voltage steps produced an outward current relaxation and an outward tail current with the same reversal potential. The observations can be explained by the presence in the bag of a non-selective cation channel current, Ibcat, that activates spontaneously at the holding potential; depolarization increases opening of the channel and hyperpolarization decreases opening. Bath-applied acetylcholine in concentrations greater than 10(-7) M produced an increase in the amplitude of Ibcat. The effect of acetylcholine was not antagonized or prevented by 100 microM tubocurarine, suggesting the presence of a non-nicotinic acetylcholine receptor. Atropine (100 microM) had no detectable influence on the effect of acetylcholine but the FMRFamide peptide, PF1, in concentrations > 1 microM, reduced the amplitude of Ibcat. Ibcat was maintained when Cs+ was used to replace intra- and extracellular cations, showing that the channels were permeable to Cs+. It is concluded that the bag membrane possesses a slow voltage-activated non-selective cation channel current, Ibcat. The effect of acetylcholine in the presence of nicotinic antagonist indicates the presence of non-nicotinic acetylcholine receptors on the bag membrane. The effect of PF1 indicated the presence of PF1 receptors on the bag membrane.
Commonly used anthelmintic agents act on the muscle cells of parasitic nematodes to cause paralysis of the parasite and its expulsion from the host.
The motonervous system of nematodes contains neuropeptides, many of which are myoactive and elicit prolonged worm paralysis. Here we describe the actions of a novel peptide, KPNFIRFamide (Lys‐Pro‐Asn‐Phe‐Ileu‐Arg‐Phe‐amide; PF4), which mediates relaxation of the somatic muscle of the parasitic nematode Ascaris suum. Its mechanism of action is compared to that of the inhibitory neuromuscular junction transmitter, γ‐aminobutyric acid (GABA), which gates a chloride channel on Ascaris muscle.
Both PF4 and GABA hyperpolarized the muscle cells (EC 50 values 98 nM and 59 μM, respectively; n = 6) and this was accompanied by an increase in input conductance.
The increase in input conductance elicited by PF4 and a supramaximal concentration of GABA were additive (10 μm PF4, 7.78 ± 1.88 μS; 10 mM GABA, 4.68 ± 1.39μS; 10 mM GABA and 10μm PF4 12.05±2.6 μS, n = 6, P <0.02 with respect to PF4 alone; P<0.01 with respect to GABA alone).
The membrane potential response to 10 μm PF4 initially consisted of a fast hyperpolarization that occurred within 1 min of PF4 application. The reversal potential for this early response to PF4 (PF4‐early) was determined at different extracellular chloride concentrations. Linear regression analysis of the natural logarithm of the extracellular chloride concentration against the reversal potential for PF4‐early yielded a straight line with a slope of −29.6±2.4 (‐34.4 to −24.9, 95% confidence limits; r ² = 0.82). This is close to the slope of −26.5 for a chloride‐dependent event, as predicted by the Nernst equation. There was a significant correlation between the reversal potential for this event and the reversal potential for GABA ( r = 0.94; P <0.001; n = 12).
The late response to PF4 (PF4‐late) appeared after 1 min and consisted of a slow reduction in the hyperpolarization to a plateau level, before the return of the membrane potential to the resting value. PF4‐late is not likely to be a chloride‐dependent event as during the hyperpolarization caused by a supramaximal concentration of GABA the muscle cells depolarized when a supramaximal concentration of PF4 was added to the perfusate. The membrane potential in the presence of 1 mM GABA was −61.8±4.8 mV and in the presence of 1 mM GABA with 10 μm PF4 was −47.5± 1.5 mV ( P <0.02; n = 6).
The conductance increase elicited by 30 μm GABA was blocked by 10 μm ivermectin (before ivermectin 0.97 ±0.2 μS, after ivermectin 0.33 ±0.12 μS; n = 5; P<0.05; Student's paired t test) but the conductance increase elicited by 1 μm PF4 was not (before ivermectin 0.96±0.14 μS, after ivermectin 1.07±0.19 μS; n = 0.34; Student's paired t test).
These data indicate that PF4 elicits a potent, inhibition of Ascaris muscle cells which is partially mediated by chloride and which is independent of the inhibitory GABA receptor.
Rational approaches to anthelmintic discovery include the design of screens for compounds directed at specific proteins in helminths that are pharmacologically distinguishable from their vertebrate homologues. The existence of several anthelmintics that selectively target the neuromusculature of helminths (e.g. levamisole, ivermectin, praziquantel, metrifonate), together with recent basic research in helminth physiology, have contributed to the recognition that neurobiology distinguishes these organisms from their vertebrate hosts. In this survey, we focus on mechanism-based screening and its application to anthelmintic discovery, with particular emphasis on targets in the neuromusculature of helminths. Few of these proteins have been exploited in chemotherapy. However, recent studies in comparative pharmacology and molecular biology, including the C. elegans genome project, have provided insights on potential new targets and, in some cases, molecular probes useful for their incorporation in mechanism-based screens.
The nematode nervous system employs many of the same neurotransmitters as are found in higher animals. The inactivation of neurotransmitters is absolutely essential for the correct functioning of the nervous system. In this article we discuss the various mechanisms used generally in animal nervous systems for synaptic inactivation of neurotransmitters and review the evidence for similar mechanisms operating in parasitic and free-living nematodes. The sequencing of the entire Caenorhabditis elegans genome means that the sequence of nematode genes can be accessed from the C. elegans database (ACeDB) and this wealth of information together with the increasing knowledge of the genetics of this free-living nematode will have great impact on all aspects of nematode neurobiology. The review will provide an insight into how this information may be exploited to identify and characterize target proteins for the development of novel anti-nematode drugs.
Three groups of anthelmintic drugs act directly and selectively on muscle membrane receptors of parasitic nematodes. These groups of anthelmintics are: (1) The Nicotinic Agonists (levamisole, pyrantel, morantel and oxantel) that act on acetylcholine receptors of nematode somatic muscle; (2) The GABA Agonist, piperazine, that acts on nematode muscle GABA receptors; and (3) The Avermectins that open glutamate gated Cl- channels on nematode pharyngeal muscle. The electrophysiology and pharmacology of muscle and neuromuscular transmission the nematode parasite, Ascaris suum, is outlined and effects of anthelmintics that interfere with transmission described. Resistance to anthelmintics has appeared in some parasitic nematodes but the mechanisms of this resistance remain to be determined.
This paper reviews sites of action of anthelmintic drugs including: (1) levamisole and pyrantel, which act as agonists at nicotinic acetylcholine receptors of nematodes; (2) the avermectins, which potentiate or gate the opening of glutamategated chloride channels found only in invertebrates; (3) piperazine, which acts as an agonist at GABA gated chloride channels on nematode muscle; (4) praziquantel, which increases the permeability of trematode tegument to calcium and results in contraction of the parasite muscle; (5) the benzimidazoles, like thiabendazole, which bind selectively to parasite beta-tubulin and prevents microtubule formation; (6) the proton ionophores, like closantel, which uncouple oxidative phosphorylation; (7) diamphenethide and clorsulon, which selectively inhibit glucose metabolism of Fasciola and; (8) diethylcarbamazine, which appears to interfere with arachidonic acid metabolism of filarial parasites and host. The review concludes with brief comments on the development of anthelmintics in the future.
We have used GABA-specific antisera to detect GABA-immunoreactivity in the motor neurons of the ventral nerve cord of Ascaris. We find that a subset of the individually identifiable commissures of motor neurons is specifically stained. On the basis of the location and morphology of stained commissures and of the location of stained cell bodies in the ventral nerve cord, we conclude that the labeled neurons comprise all members of the VI (inhibiting ventral muscle; 13 cells) and DI (inhibiting dorsal muscle; 6 cells) classes of inhibitory motor neurons (Stretton et al., 1978; Walrond et al., 1985). This result supports previous suggestions (e.g., del Castillo et al., 1964b) that GABA is the neurotransmitter released by the inhibitory motor neurons of nematodes. In the anterior part of the animal, the inhibitory motor neuron commissures have small branches in the sublateral nerve cords that have not been previously described: VI commissures have dorsal sublateral branches, while DI cells have ventral branches. Posterior VI neurons have branches in the lateral nerve cords.
Parasitic diseases are the most widespread of all the major diseases, currently 9 affecting about 3 x 10 people and innumerable domestic animals. There is no doubt that among these parasitic diseases, the helminthic infections of the gastrointestinal tract are about the most important because of their global distribution, their high prevalence, their effects on the nutritional status of men and animals, their effects on the physical and mental development of children, and their economic effects on the production of animals. Anthelmintics are important elements in the control of these gastrointestinal helminthic infections. In this volume the editors and authors have tried to find a way through the immense amount of information on anthelmintic drugs that is scattered throughout the literature. Different authors have critically examined this information from different angles. However, the aim of all has been to provide the information needed by veterinarians, physicians, and public health workers to select the most suitable drug for a given situation.
Quarterly Journal of Experimental Physiology 72 (1987) Page 44, eqn. (10), should read: Pr = n! / r!(n‐r)! Pr(1‐P)n‐r
Recombinant DNA technology has made it possible to clone receptors from many organisms by cross-hybridization or by the polymerase chain reaction. It may be difficult, though, to establish the functional importance of any clone obtained. We describe the cloning of nematode acetylcholine receptor genes by selection for resistance to levamisole, a scheme providing assurance that the clones obtained are functionally related.
The existence of genetic drug resistance to existing anthelmintics presents a serious challenge, and emphasizes the urgent need for new generations of drugs. Fortunately, the potential for developing new drugs, acting on so far untargeted systems in parasites, seems high.
The biological effect of chemical agents can only be the result of an interaction of the molecules of the active agent and particular molecules or molecular complexes present in the biological object. The same holds true for endogenous bioactive agents such as hormones, neurotransmitters, etc. Often these messengers have their molecular sites of action, referred to as their specific receptors, located on the outer cell membrane (Lefkowitz et al., 1976). They then transmit information from remote or nearby cells to the surface of the target cells where they trigger these cells, thus inducing certain changes in cell function. The interaction between the bioactive agent and its specific receptors initiates the transduction of information received from outside to the cell interior. An example is the generation of intracellular mediators such as cyclic AMP. Similarly, the hormone-receptor complex, in the case of the interaction of steroid hormones and their specific proteins in the cytoplasm, is involved in the transduction of information to the cell nucleus. The presence of specific receptor for the bioactive agents either on the outer cell membrane or in the cytoplasm of only particular tissue cells accounts for a selectivity in action on these tissues.
No one who has made even a casual study of the Nematoda can fail to have been struck by the extraordinary similarity in form and organization between the very large number of species, genera, orders and families which constitute this class of the very diversiform phylum Aschelminthes. Though there are species which show some variation from the normal ‘nematode facies’, they are few in number and merely seem to emphasize still further the great uniformity of the group as a whole. The characteristic features of the nematodes are present in fresh-water, marine and parasitic forms; they are largely independent of size, of diet and of stage of development.
Vialli (1923, see Schopfer, 1927), Duval & Courtois (1928) and Schopfer (1924, 1925, 1926, 1927, 1932) have shown by means of freezing-point determinations that the osmotic pressure of the body fluid of Parascaris megalocephala and Ascaris vitulorum is lower than that of their hosts. Schopfer (1932) found that the depression of the freezing point of extracts of whole specimens of A. ovis and A. lumbricoides was in each case less than that of the intestinal fluid of the host. He (1932) also found a similar relationship in the case of Proleptus obtusus, an intestinal parasite of Scyliorhinus caniculus, and Panikkar & Sproston (1941) came to the same conclusion as the result of their work on an unidentified species of Angusticaecum from the tortoise. In free-living land or fresh-water nematodes the body fluid is presumably hypertonic to the environment, and Stephenson’s work (1942) indicates that in Rhabditis terrestris this is, in fact, the case, and that there is an active process of osmotic regulation by means of which this difference in osmotic pressure is maintained.
Segments of the obliquely striated body muscle of Ascaris were fixed at minimum body length after treatment with acetylcholine and at maximum body length after treatment with piperazine citrate and then studied by light and electron microscopy. Evidence was found for two mechanisms of length change: sliding of thin filaments with respect to thick filaments such as occurs in cross-striated muscle, and shearing of thick filaments with respect to each other such that the degree of their stagger increases with extension and decreases with shortening. The shearing mechanism could account for great extensibility in this muscle and in nonstriated muscles in general and could underlie other manifestations of "plasticity" as well. In addition, it is suggested that the contractile apparatus is attached to the endomysium in such a way that the sarcomeres can act either in series, as in cross-striated muscle, or individually. Since the sarcomeres are virtually longitudinal in orientation and are almost coextensive with the muscle fiber, it would, therefore, be possible for a single sarcomere contracting independently to develop tension effectively between widely separated points on the fiber surface, thus permitting very efficient maintenance of isometric tension.
Nematodes are members of a phylum which has made an important impact on medicine and agrochemistry. Out of a total of some 30 000 species described, about half are parasitic. Their occurrence world-wide is phenomenal: a quarter or more of the world's population are afflicted with nematode infections, they cause considerable losses in livestock, and the small plant parasitic nematodes are responsible for widespread crop damage.
The venoms of several species of Araneidae spiders contain components which specifically inhibit the postsynaptic response of cells to glutamate stimulation. Argiopine is the active component identified in the venom of the spider Argiope lobata. Synthetically prepared argiopine is a competitive inhibitor of specific [3H]glutamate binding to membranes prepared from the nematode Caenorhabditis elegans with an inhibition constant of 8 μM. The in vivo nematocidal activity of argiopine was assessed by incubating C. elegans in various concentrations of the toxin and quantitating its effect on motility. Under these conditions, the ED50 value for argiopine is 25 μM. However, argiopine has no inhibitory effect on [3H]glutamate binding to membranes prepared from rat brain. Our results demonstrate that argiopine interacts with a glutamate binding site in C. elegans pharmacologically distinct from that present in mammalian brain tissue.
The Ascaris motor nervous system contains about 90 motorneurons and interneurons. In order to understand its function, our strategy has been to start with an extensive morphological analysis of the wiring diagram, and then to use this information as a framework for the electrophysiological and biochemical analysis of cellular properties and interactions. In this article we discuss the signalling mechanisms, predominantly passive, used by this nervous system. We also discuss a model for nematode locomotion that emerges from our work.
Gamma-aminobutyric acid (GABA), glutamate decarboxylase and GABA-transaminase were identified in the nematode . The concentration of GABA in (0.14 μg/mg protein) is approximately 10-fold lower than the concentration of GABA in rat brain. Glutamate decarboxylase and GABA-transaminase, the GABA anabolic and catabolic enzymes, are also present in . Crude membrane fractions were prepared from and used to study specific [3H] GABA binding sites. GABA binds to membranes with high affinity (37 nM) and low capacity (Bmax = 2.25 pmol/mg protein). Muscimol is a competitive inhibitor of specific GABA binding with a KI value of 120 nM. None of the other GABA agonists or antagonists inhibited greater than 40% of the specific GABA binding at concentrations up to 10−4 M. Thirteen spider venoms were examined as possible GABA agonists or antagonists, the venom from inhibits specific GABA binding with a KI value of 6 nl/ml. These results suggest that GABA has a physiological role as a neurotransmitter in .
1.1. The small round worm Nippostrongylus brasiliensis is paralysed by the anthelmintic levamisole, but if the worms are left in the solution the paralysis passes off. This phenomenon was not observed with a series of other anthelmintics.2.2. A small percentage of Ascaris lumbricoides var suum also resume movement in levamisole; such worms do not exhibit the expected contraction when injected with the anthelmintics bephenium, methyridine and pyrantel.3.3. Since the autonomic blocking agents mecamylamine and pempidine block levamisole contractions in normal Ascaris, it is suggested that all four anthelmintics may act in a similar way.
1. An outside-out patch-clamp technique was used to examine the actions of SR95103 (30 μM to 2 mM) on GABA-activated single-channel currents recorded from the bag region of somatic muscle of Ascaris suum.2. Effects of SR95103 on open-probability and the distributions of open-, closed- and burst-durations were examined quantitatively.3. The mode of action of SR95103 is discussed; it is suggested that most of the antagonism is competitive and produced by a single molecule of SR95103 combining with the GABA receptor-channel complex; there is an additional, small non-competitive component, possibly a channel-blocking action.
Nematodes are known to be unique among animals both with respect to the structure of their somatic muscle cells and to the spatial relations these cells have with the nervous system. It has been suggested that the nematode neuromuscular junction might also be unique. Although a century of investigation of the anatomy and physiology of round worms had shed little light on the question, recent work in electronmicroscopy and electrophysiology indicates that nematode neuromuscular junctions are not fundamentally different, either structurally or functionally, from those of other animals.
The results obtained in this investigation are admittedly not as extensive as is desirable but they allow certain conclusions to be drawn.
1. The sodium and potassium contents of the body fluid of Ascaris lumbricoides are somewhat variable, but these variations do not seem to be dependent upon those of the external medium.
2. The calcium and magnesium contents of the body fluid are relatively constant and are not affected by those of the external medium.
3. The chloride concentration of the body fluid is closely related to and always remains lower than that of the external medium.
4. As shown in Table 2, there is a large gap between the total concentrations of inorganic cations and anions in the intestinal fluid of the pig. Presumably a considerable proportion of the inorganic cations are combined with organic anions, at present undetermined. Exposing the worms to saline media composed of chloride caused a large rise in the internal chloride concentration. This may well be a limiting factor in the life of the animals in such media, and the next step forward would seem to be the fuller analysis of the environment to which they are normally exposed.
DURING studies on anthelmintic activity which have been in progress in these laboratories during the past few years, we have encountered an interesting degree of activity among a limited number of substituted pyridines. Nothing of practical value emerged from the early observations, but they caused us to retain an interest in related substances. Recently, through the courtesy of The Midland Tar Distillers, Ltd., we had the opportunity of examining some pyridine compounds made in their Research and Development Department, and one of them, prepared by Mr. P. Araall, has proved of exceptional interest. This compound is 2-(β-methoxyethyl)-pyridine (U.K. Patent Application 40845/59 ; 40931/59 ; 20099/60) : It is a liquid and extremely soluble in water.
1. The distribution of non-specific esterase and of cholinesterase in Ascaris lumbricoides has been determined using histochemical methods.
2. Non-specific esterase has been shown to be present in the cuticle, in the walls of the excretory canals, in the innervation processes of the muscles, in the coelomocytes, in the oesophageal glands, in the intestine, in the rectal glands, in the male and female reproductive systems, in parts of the nervous system and as 'caps' of enzyme on fat globules in the hypodermis and muscles.
3. Cholinesterase has been detected in the innervation processes and sheath of the muscles, in the sphincter muscles, in the intestine, in the sensory papillae and amphids, and in parts of the nervous system, including a nerve in the spicules.
4. Parts of the nervous system and the innervation processes of the muscles contain an esterase which is not inhibited by cholinesterase inhibitors and it is suggested that there may be esterase, distinct from cholinesterase, involved in nerve transmission.
5. Merocrine secretion of esterase has been observed in the intestine.
A FMRFamide-like peptide has been detected in the nematode Ascaris suum, using the peroxidase-anti-peroxidase (PAP) immunocytochemical technique. Positive reactions were obtained in both the central nervous system and the peripheral nervous system of the worm, the strongest reactions being in the anterior nerve ring, the cephalic papillary ganglia, the lateral ganglia and the dorso-rectal ganglion. Immunoreactivity was observed along the length of the main nerve cords of the worm and, to a lesser extent, in the pharyngeal nerve cords. The possible role of this neuropeptide in the physiology of the nematode is discussed.
Adult female Ascaris lumbricoides are poisoned on injection of certain organophosphates into the worms at a dose of 0.2 to 2.0 mg. per kg. The toxic compounds are potent inhibitors, both in vivo and in vitro, of an acetylcholinesterase (AChE) in the anterior end of the worms. Fifty-six compounds are included in this study on the relation among toxicity, potency as in vivo and in vitro AChE inhibitors, and sensitivity of other Ascaris esterases to inhibition. AChE activity in poisoned worms recovers only slowly from in vivo inhibition. Detoxication of organophosphates occurs at a very low rate in the worms as compared to insects or mammals; phosphorothionates and phosphorodithioates are not converted in vivo to the more toxic phosphate and phosphorothiolate analogs; other reactions of the organophosphates, including hydrolysis, proceed at a low rate in the worms or homogenates of the worms.
Ibotenic acid [2-(3-hydroxyisoxazol-5-yl)glycine] induced a dose-dependent increase in chloride ion conductance in locust muscle fibres which was not sensitive to 4-aminobutyric acid (GABA). This ibotenate response became rapidly desensitised and appeared to be due to activation of extrasynaptic glutamate H receptors. 22-23-Dihydroavermectin B1a (DHAVM) (500 pg to 1 μg ml−1) induced irreversible increases in permeability to the chloride ion and abolished ibotenate responses. DHAVM responses were not altered when glutamate H receptors were desensitised by glutamate (1 mM) or ibotenate (100 μM). Irreversible changes in input conductance caused by DHAVM were not affected by penicillin G (1 mM) or bicuculline (1 mM), but picrotoxin (1 mM) and zinc chloride reduced DHAVM responses by 23.7 and 52.6%, respectively. It is concluded that DHAVM has a number of sites of action on locust muscle that include effects on the glutamate H receptor–chloride ion channel complex, in addition to effects on the GABA receptor–chloride ion channel previously described.
This study describes the pharmacology of the Ascaris suum muscle GABA receptor using a range of GABA agonists and antagonists. The GABA receptor at this site does not readily fit into GABA-A or GABA-B classification. The effect of the potent anthehmintic, avermectin, on these cells was investigated. It was found to inhibit GABA-evoked increase in membrane conductance and membrane hyper polarisation on quiescent muscle cells with an apparent affinity constant in the mid-micromolar range. When applied to spontaneously active muscle cells, avermectin (I üM) inhibited the spiking activity after a delay of about 25 min.
Intracellular current and voltage clamp techniques were used to investigate the mode of action of the anthelmintics, morantel, pyrantel and levamisole applied to the bag region of Ascaris suum muscle cells. Microperfusion of the anthelmintics and of O-acetylcholine (ACh) increased the input conductance and depolarised the membrane potential of the muscle bags. The relative potencies of these drugs were determined from dose–conductance relationships and found to be: morantel = pyrantel > levamisole > ACh. High doses (>10μM) of morantel caused antagonism of ACh responses. ACh-induced currents were measured under voltage clamp (over the range −80 to +10mV). At membrane potentials between −80 and 0 mV, microperfusion of ACh induced a voltage-dependent inward current. The current–voltage relationship was linear for membrane potentials in the range −30 to +10mV. The reversal potential was measured directly and found to be about +10mV. The relationship became non-linear at membrane potentials more negative than −30 mV, and the degree of non-linearity was dependent upon the concentration of ACh. The current–voltage relationships for morantel, pyrantel and levamisole also possessed both linear (−30 to 0mV) and non-linear components. The reversal potential for each agonist, determined by extrapolation of the linear component of the current–voltage relationship, was approximately +10mV, indicating the same cation channels were activated both by ACh and the anthelmintics. Evidence for competition between ACh and pyrantel for the same membrane receptor was obtained using iontophoretic delivery of each agonist from a double-barrelled micropipette. It is concluded that the anthelmintics, morantel, pyrantel and levamisole act as potent agonists at ACh receptors on muscle bag membranes of A. suum.
The ionic dependence of the myogenic spike potentials and slow waves recorded fromAscaris lumbricoides somatic muscles has been investigated. Spikes appear to be mediated exclusively by calcium ions; the spike active potential varies with calcium concentration as expected for a calcium electrode and spikes persist in sodium-free media (Fig. 2). Slow waves can be mediated either by sodium or calcium; they persist when calcium or sodium are removed separately, but not when both are removed together (Figs. 3, 4, 6).In rhythmically active preparations, a burst of slow waves and spikes accompanies each contraction. Two phenomena may be related to the mechanism of this modulation:1) TEA, although it does not prolong slow waves or spikes, induces rhythmic bursts of activity similar to spontaneous modulation (Fig. 5). This TEA-induced modulation appears to be myogenic. 2) Under conditions where calcium influx is reduced (either by addition of EGTA to the bath or by replacement of calcium with barium or strontium), very long-duration square waves are observed (Figs. 4. 7. 8). The square waves resemble slow waves in their ionic dependence, but differ in their sensitivity to TEA and to variation in the external potassium concentration. It is suggested that modulation and square waves involve the same channels. The significance of these results in understanding the role of myogenic activity in nematode locomotion is discussed.
The functional muscle syncytium overlying the nerve cord inAscaris lumbricoides is preferentially excited by anodal stimulation with an extracellular electrode. Cathodal stimulation preferentially excites the nerve cord, allowing determination of separate conduction velocities for the nerve cord and syncytium. The propagation velocity of the nerve cord is 16.2±1.2 cm/s; that of the syncytium varies with Ca2+ concentration, being 21.6±1.3 cm/s under normal conditions (Figs. 5, 6). Both values are too high to account for the propagation of contractile waves in the intact animal.
Modulation of spontaneous electrical activity on a behaviorally significant time scale is observed. The modulation shows dorsal-ventral coordination if the right lateral line is intact, in accord with known nervous system asymmetry (Fig. 10).
The structure and connectivity of the nervous system of the nematode Caenorhabditis elegans has been deduced from reconstructions of electron micrographs of serial sections. The hermaphrodite nervous system has a total complement of 302 neurons, which are arranged in an essentially invariant structure. Neurons with similar morphologies and connectivities have been grouped together into classes; there are 118 such classes. Neurons have simple morphologies with few, if any, branches. Processes from neurons run in defined positions within bundles of parallel processes, synaptic connections being made en passant. Process bundles are arranged longitudinally and circumferentially and are often adjacent to ridges of hypodermis. Neurons are generally highly locally connected, making synaptic connections with many of their neighbours. Muscle cells have arms that run out to process bundles containing motoneuron axons. Here they receive their synaptic input in defined regions along the surface of the bundles, where motoneuron axons reside. Most of the morphologically identifiable synaptic connections in a typical animal are described. These consist of about 5000 chemical synapses, 2000 neuromuscular junctions and 600 gap junctions.
The transport properties of isolated cuticle from Ascaris suum were studied using standard two-chamber diffusion cells and a number of radiolabeled permeants which varied in molecular size, lipophilicity and electrical charge. The permeability coefficient of the collagen matrix (lipid-extracted cuticle) vs. molecular radius relationship showed the interdependence of molecular size and electrical charge of the permeants with respect to the aqueous pores of the negatively charged matrix. The permeability of neutral solutes decreased monotonically with size. Protonated amines permeated the aqueous pores faster than neutral solutes of comparable size, while the permeation of anions was slower. The average pore size was estimated to be 1.5 nm in radius. A biophysical model which accounted for diffusion of molecules within a fixed electrostatic field of force and for molecular sieving by the pore channels was used in the mechanistic interpretation of the data. The effective permeability coefficient of the non-lipid-extracted cuticle was delineated into the permeability coefficients of the water-filled collagen matrix and the lipoidal component of the cuticle to determine which layer was the rate-controlling barrier. While each solute was capable of penetrating the water-filled collagen matrix, the rate-determining step for the majority of compounds was passive diffusion across the lipid component, which controlled 75–99% of transport. The exception was water, for which transport kinetics was 75% matrix-controlled. In general, permeation across the lipid-filled tissue was more favorable for small lipophilic compounds because of molecular restriction not only in the aqueous pores, but also in the lipid-filled pores.
Avermectin B1a, a novel antiparasitic agent, paralyzes Ascaris suum without causing either flaccid paralysis or a hypercontraction. It reduces the lengthening of the acetylcholine-preconditioned A. suum muscle strip caused by γ-aminobutyric acid. It does not affect the contraction of the isolated muscle strip preparation caused by applying acetylcholine. However, preinjection with Avermectin B1a does significantly reduce the shortening caused by acetylcholine injection without affecting the paralysis of an intact ascarid worm. These results suggest that Avermectin B1a may act on the central nervous system of Ascaris sp. nematodes.
The basis for the comparative toxicity to parasitic nematodes and their mammalian hosts of the anthelmintics levamisole, pyrantel, and several related analogs on somatic nicotinic cholinergic transmission was examined. Measurements of muscle contractility and membrane potential were made using the isolated hemidiaphragm preparation of the rat and isolated axial muscle segments from the gastrointestinal nematode Haemonchus contortus. Pyrantel caused a dose- and time-dependent reduction of nerve-evoked twitches in the rat diaphragm. These effects were exacerbated by increasing the frequency of phrenic nerve stimulation from 0.5 to 50 Hz. Levamisole was less potent and the onset of its effects slower than pyrantel. Neither drug significantly affected twitches evoked from d-tubocurarine-blocked preparations following direct stimulation of the diaphragm. Twitch depression was reversed by washing, but not by application of physostigmine. In H. contortus, both drugs stimulated a spastic contraction and sustained paralysis in the concentration range of 1–10 μm, mimicking the action of nicotine. Neither nicotinic nor muscarinic antagonists blocked these responses. Moreover, neither nicotinic antagonists nor muscarinic agonists or antagonists had any independent effect on contractility of the parasite muscle segments. The blocking actions of levamisole and pyrantel on H. contortus axial muscle were associated with membrane depolarization at the muscle. In the rat-isolated hemidiaphragm, pyrantel, but not levamisole, depolarized end-plate regions of muscle fibers. d-Tubocurarine blocked the depolarizing action of pyrantel but not levamisole on rat-isolated hemidiaphragm. In axial muscle fibers of H. contortus, d-tubocurarine did not block the depolarizing actions of pyrantel, levamisole, or nicotine. 3-Bromo and 3-amino derivetives of levamisole were equipotent with and mimicked the actions of the parent compound on H. contortus axial muscle contractility. In the rat preparation, the 3-bromo derivative was more potent than levamisole or 3-amino-levamisole. 3-Amino-levamisole, but not 3-bromo-levamisole, depolarized muscle endplate membrane in the rat diaphragm. Results of the present study are consistent with the following conclusions: (a) both levamisole and pyrantel block contractility of nematode axial muscle by causing sustained depolarization of the muscle membrane; (b) both drugs block neuromuscular transmission at the mammalian neuromuscular junction but their mechanisms appear to differ; (c) levamisole and pyrantel are more potent blockers of neuromuscular transmission in H. contortus than in the rat. These results suggest that potentially important pharmacological differences exist between nematode and mammalian somatic nicotinic receptors.
1. GABA-induced changes in input conductance in the bag region of Ascaris suum muscle were monitored using a two-microelectrode current-clamp technique.2. Effects of the arylaminopyridazine-GABA derivatives, SR95103 and SR95531 on GABA conductance responses were observed.3. SR95103 was more potent than SR95531 as an antagonist; this potency order contrasts with vertebrate GABA, receptors.4. The antagonism of SR95103 was associated with a parallel shift to the right in the GABA dose-response relationships.5. A modified Schild plot was used to describe the action of SR95103: the data was consistent with 2 molecules of GABA but one molecule of antagonist interacting with the receptor.6. The KB for SR95103 was 64 ± 13 μM (mean ± SE, N = 14).
The ultrastructure of the excretory system, including the subventral glands, of the nematode Nippostrongylus brasiliensis has been described. The walls of the lateral excretory canals contain canaliculi which open into the lumen of the canal. It is suggested that these canals play a role in osmoregulation and excretion. The sub-ventral glands contain two types of secretory granule and contain non-specific esterase, cholinesterase and aminopeptidase. It is suggested that these glands are not excretory but play an important role in feeding.
The nervous system of Caenorhabditis elegans is arranged as a series of fibre bundles which run along internal hypodermal ridges. Most of the sensory integration takes place in a ring of nerve fibres which is wrapped round the pharynx in the head. The body muscles in the head are innervated by motor neurones in this nerve ring while those in the lower part of the body are innervated by a set of motor neurones in a longitudinal fibre bundle which joins the nerve ring, the ventral cord. These motor neurones can be put into five classes on the basis of their morphology and synaptic input. At any one point along the cord only one member from each class has neuromuscular junctions. Members of a given class are arranged in a regular linear sequence in the cord and have non-overlapping fields of motor synaptic activity, the transition between fields of adjacent neurones being sharp and well defined. Members of a given class form gap junctions with neighbouring members of the same class but never to motor neurones of another class. Three of the motor neurone classes receive their synaptic input from a set of interneurones coming from the nerve ring. These interneurones can in turn be grouped into four classes and each of three motor neurone classes receives its synaptic input from a unique combination of interneurone classes. The possible developmental and functional significance of these observations is discussed.
Dopamine is the putative transmitter of eight neurons in the hermaphrodite form of the nematode Caenorhabditis elegans. These include the cephalic and deirid neurons, which are believed to be mechanosensory. The male has an additional six dopaminergic neurons in the tail. Mutants have been selected which have defects in the formaldehyde induced fluorescence and lack dopamine to varying degrees, but they are not insensitive to touch. The dopaminergic neurons of C. elegans are compared with the homologous neurons in Ascaris lumbricoides.