Recording drug responses from adult Dirofilaria immitis pharyngeal and somatic muscle cells.

Despite being considered one of the most pathogenic helminth infections of companion animals, members of macrocyclic lactone class are the only drugs available for the prevention of heartworm disease caused by Dirofilaria immitis. Alarmingly, heartworm prevention is at risk; several studies confirm the existence of macrocyclic lactone resistance in D. immitis populations across the United States. To safeguard the long term prevention and control of this disease, the identification and development of novel anthelmintics is urgently needed. To identify novel, resistance-breaking drugs, it is highly desirable to: Unfortunately, none of the three above statements can be answered sufficiently for D. immitis and most of our hypotheses derive from surrogate species and/or in vitro studies. Therefore, the present study aims to improve our fundamental understanding of the neuromuscular system of the canine heartworm by establishing new methods allowing the investigation of body wall and pharyngeal muscle responses and their modulation by anthelmintics. We found that the pharynx of adult D. immitis responds to both ivermectin and moxidectin with EC50s in the low micromolar range. We also demonstrate that the somatic muscle cells have robust responses to 30 µM acetylcholine, levamisole, pyrantel and nicotine. This is important preliminary data, demonstrating the feasibility of electrophysiological studies in this important parasite.

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

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

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

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

Emodepside and SL0-1 potassium channels: a review.

Nematode parasites infect humans and domestic animals; treatment and prophylaxis require anthelmintic drugs because vaccination and sanitation is limited. Emodepside is a more recently introduced cyclooctadepsipeptide drug that has actions against GI nematodes, lungworm, and microfilaria. It has a novel mode of action which breaks resistance to the classical anthelmintics (benzimidazoles, macrocyclic lactones and cholinergic agonists). Here we review studies on its mode of action which suggest that it acts to inhibit neuronal and muscle activity of nematodes by increasing the opening of calcium-activated potassium (SLO-1) channels.

On the mode of action of emodepside: slow effects on membrane potential and voltage-activated currents in Ascaris suum.

BACKGROUND AND PURPOSE: Anthelmintics are required for treatment and prophylaxis of nematode parasites of humans and domestic animals. Emodepside, a cyclooctadepsipeptide, is a modern anthelmintic that has a novel mode of action involving a Ca-activated K channel (SLO-1) in Caenorhabditis elegans, sometimes mediated by a latrophilin (LAT) receptor. We examined mechanisms of action of emodepside in a parasitic nematode, Ascaris suum. EXPERIMENTAL APPROACH: RT-PCR was used to investigate expression of slo-1 and lat-1 in A. suum muscle flaps, and two-micropipette current-clamp and voltage-clamp techniques were used to record electrophysiological effects of emodepside. KEY RESULTS: Expression of slo-1 and lat-1 were detected. Emodepside produced a slow time-dependent (20 min), 4-aminopyridine sensitive, concentration-dependent hyperpolarization and increase in voltage-activated K currents. Sodium nitroprusside increased the hyperpolarizations and K currents. N-nitro-L-arginine inhibited the hyperpolarizations and K currents. Phorbol-12-myristate-13 acetate increased the K currents, while staurosporine inhibited the hyperpolarizations and K currents. Iberiotoxin reduced these emodepside K currents. The effect of emodepside was reduced in Ca-free solutions. Emodepside had no effect on voltage-activated Ca currents. CONCLUSIONS AND IMPLICATIONS: Asu-slo-1 and Asu-lat-1 are expressed in adult A. suum muscle flaps and emodepside produces slow activation of voltage-activated Ca-dependent SLO-1-like K channels. The effect of emodepside was enhanced by stimulation of protein kinase C and NO pathways. The data are consistent with a model in which NO, PKC and emodepside signalling pathways are separate and converge on the K channels, or in which emodepside activates NO and PKC signalling pathways to increase opening of the K channels.

Effects of SDPNFLRF-amide (PF1) on voltage-activated currents in Ascaris suum muscle.

Helminth infections are of significant concern in veterinary and human medicine. The drugs available for chemotherapy are limited in number and the extensive use of these drugs has led to the development of resistance in parasites of animals and humans (Geerts and Gryseels, 2000; Kaplan, 2004; Osei-Atweneboana et al., 2007). The cyclooctadepsipeptide, emodepside, belongs to a new class of anthelmintic that has been released for animal use in recent years. Emodepside has been proposed to mimic the effects of the neuropeptide PF1 on membrane hyperpolarization and membrane conductance (Willson et al., 2003). We investigated the effects of PF1 on voltage-activated currents in Ascaris suum muscle cells. The whole cell voltage-clamp technique was employed to study these currents. Here we report two types of voltage-activated inward calcium currents: transient peak (I(peak)) and a steady-state (I(ss)). We found that 1microM PF1 inhibited the two calcium currents. The I(peak) decreased from -146nA to -99nA (P=0.0007) and the I(ss) decreased from -45nA to -12nA (P=0.002). We also found that PF1 in the presence of calcium increased the voltage-activated outward potassium current (from 521nA to 628nA (P=0.004)). The effect on the potassium current was abolished when calcium was removed and replaced with cobalt; it was also reduced at a higher concentration of PF1 (10microM). These studies demonstrate a mechanism by which PF1 decreases the excitability of the neuromuscular system by modulating calcium currents in nematodes. PF1 inhibits voltage-activated calcium currents and potentiates the voltage-activated calcium-dependent potassium current. The effect on a calcium-activated-potassium channel appears to be common to both PF1 and emodepside (Guest et al., 2007). It will be of interest to investigate the actions of emodepside on calcium currents to further elucidate the mechanism of action.

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

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

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

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

Use of reverse osmosis membranes to remove perfluorooctane sulfonate (PFOS) from semiconductor wastewater.

Perfluorooctane sulfonate (PFOS) and related substances are persistent, bioaccumulative, and toxic, and thus of substantial environmental concern. PFOS is an essential photolithographic chemical in the semiconductor industry with no substitutes yet identified. The industry seeks effective treatment technologies. The feasibility of using reverse osmosis (RO) membranes for treating semiconductor wastewater containing PFOS has been investigated. Commercial RO membranes were characterized in terms of permeability, salt rejection, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and membrane surface zeta potential (streaming potential measurements). Filtration tests were performed to determine the membrane flux and PFOS rejection. Over a wide range of feed concentrations (0.5 - 1500 ppm), the RO membranes generally rejected 99% or more of the PFOS. Rejection was better for tighter membranes, but was not affected by membrane zeta potential. Flux decreased with increasing PFOS concentration. While the flux reduction was severe for a loose RO membrane probably due to its higher initial flux, very stable flux was maintained for tighter membranes. At a very high feed concentration (about 500 ppm), all the membranes exhibited an identical stable flux. Isopropyl alcohol, present in some semiconductor wastewaters, had a detrimental effect on membrane flux. Where present it needs to be removed from the wastewater prior to using RO membranes.

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

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

The time-course of the response to the FMRFamide-related peptide PF4 in Ascaris suum muscle cells indicates direct gating of a chloride ion-channel.

We investigated the effects of PF4 on Ascaris suum somatic muscle cells using a 2 electrode current-clamp technique. PF4 is a FaRP (FMRFamide-related peptide), originally isolated from the free-living nematode Panagrellus redivivus. PF4 caused hyperpolarization and an increase in chloride ion conductance when it was applied to the muscle cells of the Ascaris body wall. The delay between the application of the peptide and the appearance of the response was measured and compared with that of gamma-amino butyric acid (GABA), a compound that directly gates ion channels, and with PF1, a FaRP that acts via an intracellular signal transduction mechanism. The PF4 and GABA delay times were not significantly different; they were 1.51+/-0.11 sec and 1.22+/-0.10 sec respectively. The delay following application of PF1, 3.75+/-0.51 sec, was significantly longer. The rapid response to PF4 is consistent with direct gating of a chloride ion channel, which has not been described elsewhere in the literature.

Electrophysiological investigation of anthelmintic resistance.

It is pointed out that two of the three major groups of anthelmintic act by opening membrane ion-channels. It is appropriate, therefore, to use electrophysiological methods to study the properties of the sites of action of these drugs and the changes in the properties of these receptor sites associated with resistance. This paper describes the use of the patch-clamp technique to observe the currents that flow through the levamisole-activated channels as they open and close in levamisole-sensitive and levamisole-resistant isolates. It was found that, on average, the proportion of time the channels are open, is less in the resistant isolate. The patch-clamp technique also showed that the ion-channels are heterogeneous and that one of the subtypes is lost with the appearance of resistance. The use of the current clamp technique is illustrated to record a site of action of ivermectin in the pharyngeal muscle of Ascaris.

Pyrantel resistance alters nematode nicotinic acetylcholine receptor single-channel properties.

Resistance to the anthelmintics pyrantel ((E)-1,4,5, 6-tetrahydro-1-methyl-2-[2-(2thienyl)ethenyl]pyrimidine) and levamisole ((S)-2,3,5,6-tetrahydro-6-phenylimidazo[2,1-b]thiazole) is an increasingly widespread problem in gastro-intestinal nematode infestations. Both compounds act on the nicotinic acetylcholine receptors on the surface of nematode somatic muscle. The patch-clamp technique was used to measure nematode nicotinic acetylcholine receptor properties at 75, 50, -50 and -75 mV in a pyrantel-resistant isolate of Oesophagostomum dentatum. Patch pipettes contained 30 microM levamisole as agonist. We found that 28. 1% of membrane patches contained active receptors. At -50 mV, the single-channel conductance was 36.2+/-1.4 pS, the mean open-time (tau) was 1.45+/-0.14 ms and the mean probability of opening (P(o)) was 0.004+/-0.002. We compared these results with previous work on an anthelmintic sensitive isolate and a levamisole-resistant isolate [Robertson, A.P., Bjorn, H.E., Martin, R.J., 1999. Levamisole resistance resolved at the single-channel level. FASEB J. 13, 749-760.]. We found that pyrantel-resistant parasites had a reduced percentage of active patches and a reduced P(o) value when compared to anthelmintic sensitive worms. We concluded that pyrantel resistance is associated with a modification of the target nicotinic receptor properties.

Resistance to levamisole resolved at the single-channel level.

Levamisole is commonly used to treat nematode parasite infections but therapy is limited by resistance. The purpose of this study was to determine the mechanism of resistance to this selective nicotinic drug. Levamisole receptor channel currents in muscle patches from levamisole-sensitive and levamisole-resistant isolates of the parasitic nematode Oesophagostomum dentatum were compared. The number of channels present in patches of sensitive and resistant isolates was similar at 10 microM levamisole, but at 30 microM and 100 microM the resistant isolate contained fewer active patches, suggesting desensitization. Mean Po and open times were reduced in resistant isolates. The distribution of conductances of channels in the sensitive isolate revealed a heterogeneous receptor population and the presence of G25, G35, G40, and G45 subtypes. A G35 subtype was missing in the resistant isolate. Resistance to levamisole was produced by changes in the averaged properties of the levamisole receptor population, with some receptors from sensitive and resistant isolates having indistinguishable characteristics.

Anthelmintics and ion-channels: after a puncture, use a patch.

Two of three major types of anthelminitic, the avermectins and the nicotinic agonists, exert their therapeutic effect by an action on ligand-gated membrane ion-channels of nematodes. The avermectins, such as ivermectin, open glutamategated chloride channels which have so far been found only in invertebrate preparations; nicotinic anthelmintics, like levamisole, selectively gate nematode nicotinic acetylcholine receptors. We describe recent advances in the knowledge of the molecular structure of these ion-channel receptors in nematodes. Because opening of the ion-channels by these two groups of anthelmintic generates currents across cell membranes of nematodes, we can use electrophysiological methods to examine properties of the channels, the mode of action of the anthelmintics, and changes in the receptors associated with anthelmintic resistance. We illustrate some of our observations on these receptors using a two micro-electrode current-clamp technique to monitor membrane resistance (the puncture); and then some observations using The patch-clamp technique to monitor currents through individual ion-channels (the patch). The receptors for the two major groups of anthelmintics may not be homogeneous. Even in a single membrane patch from one muscle cell, nematode nicotinic acetylcholine receptors show evidence of heterogeneity and the avermectins may have multiple sites-of-action. If separate independent recessive genes are involved in production of different receptor subtypes, and if each subtype has to change to allow the development of resistance by the whole nematode, then the probability of resistance developing would be smaller than for anthelminitics with a single site-of-action. The MISER (multiple independent sites-of-action evading resistance) concept favours the development and use of anthelminitics with more than one site-of-action.

A vesicle preparation for resolving single-channel currents in tegument of male Schistosoma mansoni.

A tegumental vesicle preparation from adult male Schistosoma mansoni was developed that allows the resolution of single ion-channel currents. Adult male schistosomes were exposed to a low pH (3.75) medium for a period of approximately 30 min at 37 degrees C. During this period smooth vesicles formed from the tegument. Fluorescence microscopy following staining of the tegument with the dye, 5-N-[octadecanoyl]aminofluorescein (AF-18), transmission electron microscopy and scanning electron microscopy revealed that the vesicles were produced from the outer tegumental membrane. The fluorescence studies showed the presence of the double bilayer structure of the outer membrane in > 41% of the vesicles. These studies suggested that the preparation is suitable for single-channel recording with the patch-clamp technique. Cell-attached and isolated inside-out patch recordings of ion-channel activity were obtained with giga-ohm resistance seals. Different types of ion-channel were recorded from tegumental vesicles from male schistosomes, illustrating the potential of the technique. The channels observed included: a non-selective cation channel (360 pS); a K+ channel (with a conductance of 115 pS in high bath-K conditions); and a Cl- selective channel (20 pS). The currents of these ion-channels may cross the double bilayer of the outer tegumental membrane.

Heterogeneous levamisole receptors: a single-channel study of nicotinic acetylcholine receptors from Oesophagostomum dentatum.

A muscle vesicle preparation from Oesophagostomum dentatum, a 5 mm parasitic nematode, was developed for single-channel recording. Properties of nicotinic acetylcholine receptors activated by the anthelmintic levamisole (10 microM) were investigated using cell-attached and isolated inside-out patches. The current-voltage relationships of the single-channel currents were linear with conductances in the range 24.6-57.7 pS (mean 39.5 pS). The distributions of open times were fitted with a single exponential and mean open times were in the range 0.98-4.43 ms (mean 2.2 ms). The distributions of conductances and open times of the channels showed that the receptors could not be described as a single homogeneous population. There were two main channel subtypes: one subtype, designated G35, had a mean conductance of 35.2 pS and mean open time of 1.6 ms: another subtype designated G45, had a mean conductance of 44.6 pS and mean open time of 2.7 ms. A channel with a conductance near 25 pS. designated G25, and a channel with a conductance near 55 pS. designated G55, were also observed. The designations were based on the mean conductances. G. of the channel subtypes. A model for the heterogeneous population of nicotinic acetylcholine channels predicting four subtypes of receptor separated by their conductance is discussed and related to the development of levamisole resistance.

Target sites of anthelmintics.

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.

Effects of pH on a high conductance Ca-dependent chloride channel: a patch-clamp study in Ascaris suum.

Plasma membrane vesicles prepared from the bag region of the somatic muscle cells of the parasitic nematode Ascaris suum contain high conductance, voltage sensitive, Ca-dependent chloride channels, suggested to be involved in the excretion of carboxylic acids produced by the anaerobic respiration of glucose (Valkanov, Martin & Dixon, 1994). The effect of altered pH on this channel was investigated using the patch-clamp technique and isolated inside-out membrane patches. Changes in pH had little effect on channel conductances and only a small effect on reversal potentials. Under control conditions (symmetrical pH 7.2) the channel had the highest probability of opening at approximately -35 mV (the resting membrane potential of the cell). At positive membrane potentials the probability of opening decreased. The Boltzmann equation was used to describe the relationship between membrane potential and probability of channel opening, and to calculate the effective gating charge. Reduction of external pH produced an increase in the probability of channel opening at hyperpolarized membrane potentials. An increase in internal pH caused a voltage-independent increase in the probability of channel opening and made the effective gating charge less negative. The effect of reducing internal pH was marked: the channel then opened most frequently at positive membrane potentials and the probability of opening at -35 mV was greatly reduced. The decrease in internal pH changed the polarity of the effective gating charge. A simple model was constructed to describe the effects of pH on channel gating.

Chronic unilateral ureteral obstruction is associated with interstitial fibrosis and tubular expression of transforming growth factor-beta.

Chronic unilateral ureteral obstruction (UUO) results in interstitial fibrosis and nephron damage associated with irreversible loss of function. Collagen is increased in UUO, but detailed studies of rat renal extracellular matrix changes in UUO have not been carried out. Acute (3-day) obstruction results in increases in renal macrophages and the fibrogenic cytokine transforming growth factor-beta (TGF-beta), but their involvement in longer-term obstruction and fibrosis has not been studied. In the present experiments, kidneys of rats after UUO of 1, 2, 3, 7, 14, 21, and 28 days' duration were used. Trichrome staining, measurement of interstitial volume, and immunohistochemical studies localizing collagens I, III, and IV; laminin; fibronectin; TGF-beta; and macrophages were carried out. We found increases in the interstitial space in both cortex and medulla that (a) were significant by day 7 after UUO and (b) were accompanied by increased deposition of collagen I and collagen III. Collagen IV, laminin, and fibronectin, normally associated with the basement membrane, were found both in a thickened basement membrane and in the interstitial space. Macrophages, not found in sham-operated kidneys, were found in the interstitial space after UUO. TGF-beta was found in sham cortical tubules, but not in medullary tubules. UUO was associated with little change in cortical TGF-beta, whereas at 14 days, TGF-beta was found in dilated medullary tubules. Immunohistochemical findings were confirmed with measurements of tissue TGF-beta. In summary, UUO is associated with interstitial fibrosis. The increase in extracellular matrix is due both to increases in the interstitial collagens I and III and the basement membrane-associated collagen IV, laminin, and fibronectin. Macrophages are increased after UUO, but do not seem to be associated with the fibrogenic cytokine TGF-beta. Medullary tubular synthesis of TGF-beta may be a contributing factor in the fibrosis associated with UUO.