The Effect of the Anthelmintic Emodepside at the Neuromuscular

79 The eﬀect of the anthelmintic emodepside at the neuromuscular junction of the parasitic nematode Ascaris suum

J. WILLSON1,K.AMLIWALA1, A. HARDER2,L.HOLDEN-DYE1 and R. J. WALKER1* 1 Southampton Neuroscience Group, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, UK 2 Bayer AG, Business Group Animal Health, OP-B10-RD, D-51368 Leverkusen, Germany

(Received 21 June 2002; revised 22 August and 17 September 2002; accepted 17 September 2002)

SUMMARY

Here we report on the action of the novel cyclo-depsipeptide anthelmintic, emodepside, on the body wall muscle of the parasitic nematode, Ascaris suum. Emodepside caused (i) muscle relaxation, (ii) inhibition of muscle contraction elicited by either acetylcholine (ACh), or the neuropeptide, AF2 (KHEYLRFamide) and (iii) a rapid relaxation of muscle tonically contracted by ACh. The inhibitory action of emodepside on the response to ACh was not observed in a denervated muscle strip, indicating that it may exert this action through the nerve cord, and not directly on the muscle. Electrophysiological recordings showed emodepside elicited a Ca++ -dependent hyperpolarization of muscle cells. Furthermore, the response to emodepside was dependent on extracellular K+, similar to the action of the inhibitory neuropeptides PF1 and PF2 (SDPNFLRFamide and SADPNFLRFamide). Thus emodepside may act at the neuromuscular junction to stimulate release of an inhibitory neurotransmitter or neuromodulator, with a similar action to the PF1/PF2 neuropeptides.

Key words: anthelmintic, Ascaris suum, neuropeptide.

INTRODUCTION Although the identity of the neuropeptides that co- localize with ACh and GABA remains to be deter- Cyclo-octadepsipeptides are a new class of anthel- mined, there are a number of peptides that have mintic (Sasaki et al. 1992) which inhibit nematode potent activity on the muscle, strongly suggesting that motility (Samson-Himmelstjerna et al. 2000; Ter- they, or closely related sequences, have a physio- ada, 1992). The mechanism for this is poorly under- logical role in muscle regulation (Brownlee, Holden- stood (Chen, Terada & Cheng, 1996). A previous Dye & Walker, 2000). study has shown that PF1022A, the parent com- In the context of this study, the action of the pound for this class of anthelmintics, may have an inhibitory peptides, SDPNFLRF-amide (PF1) and action on the membrane properties of Ascaris suum SADPNFLRFamide (PF2), is of particular interest. body wall muscle (Martin et al. 1996). To provide Both these peptides have been biochemically isolated further insight into this we have used in vitro from 2 species of nematode (Geary et al. 1992; Rosoff preparations of body wall muscle of the parasitic et al. 1993), and have a potent inhibitory action on nematode A. suum. the somatic muscle of A. suum (Franks et al. 1994). The body wall muscle of Ascaris receives projec- At least part of this action is due to a direct effect on tions from excitatory and inhibitory motorneurones. the muscle (Holden-Dye et al. 1995), suggesting that The excitatory motorneurones use acetylcholine there is an inhibitory receptor for PF1-like peptides (ACh) as their transmitter. ACh acts on nicotinic on the somatic muscle. receptors on the muscle to elicit depolarization and Here we investigate the mechanism of action of the muscle contraction (Stretton et al. 1985; Pennington cyclodepsipeptide, emodepside, using in vitro muscle & Martin, 1990; Colquhoun, Holden-Dye & Walker, preparations of A. suum muscle to measure effects 1991). The inhibitory motorneurones release on muscle tension and membrane potential. In par- gamma-amino butyric acid (GABA) which activates ticular, we address the question of whether the effect ligand-gated chloride channels on the muscle to of emodepside is pre- or post-synaptic to the neuro- hyperpolarize and relax muscle (Martin, 1982; muscular junction, and how the action of emodepside Holden-Dye et al. 1989). In addition, both classes compares to the inhibitory neurotransmitter GABA of motorneurone have neuropeptide-like immuno- and the inhibitory neuropeptide, PF2. staining (Sithigorngul, Stretton & Cowden, 1990).

MATERIALS AND METHODS * Corresponding author: Neuroscience Research Group, University of Southampton, Bassett Crescent East, The methods used were similar to those described by Southampton SO16 7PX. Tel: +44 (0) 2380 594295. Fax: Trim et al. (1997). A. suum were obtained from a local +44 (0) 2380594319. E-mail: [email protected] abattoir and maintained for up to 5 days in artiﬁcial

Parasitology (2003), 126, 79–86. f 2003 Cambridge University Press DOI: 10.1017/S0031182002002639 Printed in the United Kingdom

Downloaded from https://www.cambridge.org/core. University of Athens, on 26 Sep 2021 at 00:34:34, subject to the Cambridge Core terms of use, available at https://www.cambridge.org/core/terms. https://doi.org/10.1017/S0031182002002639 J. Willson and others 80

perienteric ﬂuid (APF composition in mM, NaCl 67, . CH3COONa 67, CaCl2 3, MgCl2 15 7, KCl 3, Triz- 1 ma base 5, pH 7.6, with glacial acetic acid, 3 mM glucose at 37 xC).

Muscle tension measurements for the dorsal muscle strip Ascaris muscle strips were prepared by dissecting a 1 cm strip of the body wall muscle immediately an- terior to the genital pore. Dorsal muscle strips (DMS) were excised from this section by cutting along both Fig. 1. Emodepside causes a relaxation of the basal tone lateral lines. Dorsal muscle is devoid of motorneur- of Ascaris DMS. A representative response showing one somata, which are only present in the ventral relaxation of the basal tone of Ascaris DMS following nerve cord, however, it does contain the dorsal in- 10 mM application of emodepside. The bars indicate hibitory and dorsal excitatory motorneurone ter- duration of drug application. minals, which have reciprocal synapses with each other. For denervated muscle strips, the dorsal cord was removed from the muscle strip. This was ob- the experiment. Emodepside (supplied by Bayer AG, tained by cutting along a lateral line and opening up Leverkusen, Germany) was diluted in 100% ethanol the muscle exposing both nerve cords. The muscle at 10x1 M and then diluted down in APF so that the strip was then cut on the inner side of the nerve cords final concentration of ethanol in the organ bath or in so that only body wall muscle (consisting of half of electrophysiology experiments was no greater than the ventral muscle field and half of the dorsal muscle 0.1%. Neuropeptides, AF2 and PF2, were supplied field) between the nerve cords remained. by Alta Bioscience, Birmingham UK (>90% purity) Muscle strips were placed in a 15 ml organ bath and and were stored at x20 xCat10x3 M in 50 ml aliquots. connected by thread to a 2 g isometric transducer. All other drugs were obtained from Sigma Chemical The preparation was subject to a 1 g load and main- Co., Poole, UK. tained at 37 xC with a heated water jacket. Drugs were added in volumes of no greater than 1% of the bath Data analysis volume, and were rapidly mixed within the organ bath by gassing the bath with room air. Drugs were then For each individual muscle strip tension experiment a washed out by at least 3 times the bath volume of APF. consistent response to 30 mM ACh was obtained at the A hard copy of the data was obtained on a flat bed beginning of the experiment. The peak contractions chart recorder (BBC, Goerz, Metrawatt, Austria). of the responses were measured and deemed the ‘control’ response. Subsequent responses were nor- Electrophysiological studies from dorsal muscle malized with respect to the ‘control’ response. To test for statistical significance, the un- The DMS was pinned cuticle side down on a Syl- transformed data were used and analysed using the 1 gard elastomer 184 (Dow Corning Wiesbaden, Student’s t-test (two-tailed, and either paired or Germany) lined Perspex chamber and continuously unpaired, as appropriate). Significance was assumed perfused with APF at 32–34 xC, directed at the As- at P<0.05. caris muscle cell via a fine bore tube. Individual muscle bags were impaled with 2 microelectrodes RESULTS (10–30 MV;10mM KCl in 3 M CH3COOK) connected to an Axoclamp 2A amplifier. One electrode Muscle tension recordings was used to record membrane potential, and the se- cond to pass current pulses (20 nA, 0.2 Hz, 500 ms). Emodepside, at concentrations up to 10 mM, produced a small relaxation of basal tension of the DMS The chamber was grounded using a 3 M KCl agar . ¡ . bridge/AgCl electrode. Drugs were added in the (x0 19 0 04 g; n=22; Fig. 1). This effect was ir- perfusate for 2 min. Bath temperature was continu- reversible within the time-course of the experiment ously monitored with a temperature probe placed (i.e. no recovery after 30 min wash out). As the effect adjacent to the muscle strip. Hard copy of data was on basal tension was very small, and probably de- recorded on a Gould model 35, 2-channel chart re- pendent on the resting tension, we further investi- corder (Gould Instruments, Ohio, USA). gated the inhibitory action of emodepside against the contraction elicited by ACh. We measured the time- dependent and concentration-dependent effect of Drugs and materials emodepside on the contraction elicited by ACh. ACh Both ACh (Sigma Chemical Co., Poole, UK) and (30 mM) produced a rapid contraction of the DMS emodepside solutions were prepared on the day of which readily reversed on washing. Application of

for inhibition of the ACh contraction was around 1 mM, but the inhibition did not achieve signiﬁcance until 10 mM (P<0.05; n=5; Fig. 2C). To investigate relaxation of the muscle by emodepside more directly, the muscle was pre-contracted with ACh (30 mM). Emodepside was applied to the muscle at the peak of the contraction, and the time- course of the subsequent relaxation followed for 10 min, at which point the preparation was washed. The relaxation rate per minute was calculated as follows

PxR 1 % relaxation rate minx1= r , 10 100 where ‘P’ is the muscle tension during the peak contraction and ‘R’ is muscle tension immediately prior to the wash (Fig. 3A). At concentrations greater than 0.5 mM, emodepside significantly increased relaxation rate (n=5; P<0.05; Fig. 3B). In order to provide some insight into the mechanism for the emodepside induced relaxation, we compared the effect of emodepside with the action of the inhibitory neurotransmitter, GABA, and an inhibitory neuropeptide, PF2. GABA relaxes nematode muscle via a well-characterized GABA-gated chloride channel (Holden-Dye et al. 1989). There- fore we compared the response of muscle to GABA, and emodepside. In contrast to the slow, partial relaxation of the muscle observed with emodepside, the response to GABA was rapid and the relaxation was back to base-line tension (n=3; data not shown). To further compare the response to GABA and emodepside we investigated the effect of reduced Fig. 2. The time-dependent effect of emodepside (10 mM) on the response to ACh. (A) Example trace for the chloride APF. For these experiments chloride response to ACh after 10 min pre-incubation with salts were replaced with the impermeant anions, emodepside. The bars indicate duration of drug either isethionate or acetate. The response to GABA application. (B) Summary of the data from 6 experiments. changed from a relaxation to a contraction in low The ‘control’ contraction is the response to 30 mM ACh chloride. In contrast, emodepside caused a slow in the absence of emodepside. Emodepside was then relaxation (data not shown). Therefore the response applied to the DMS for 2, 5, 10 and 30 min before the to emodepside did not resemble the response to response to 30 mM ACh was measured. Data points are GABA. (This is further supported by the results of ¡ mean S.E. mean. (C) The effect of varying concentrations electrophysiological recordings, see later.) However, of emodepside on the 30 mM ACh induced contraction of the inhibitory neuropeptide PF2 relaxed muscle ton- Ascaris dorsal muscle. Contractions were obtained for ACh. Emodepside was then applied to the DMS at ically contracted with ACh, causing a slow and in- concentrations of 500 nM,1mM,5mM and 10 mM for complete relaxation at a similar rate to that observed 10 min before application of ACh. Data points are for emodepside (Fig. 3C). A similar reduction in the mean¡S.E. mean, no6. response to 30 mM ACh was observed in the presence of PF2 as for 10 mM emodepside (1 mM PF2 for 2 min emodepside (10 mM) to the muscle prior to addition of 36¡6%; n=10; Fig. 4). Previous studies have in- ACh (30 mM) caused a time-dependent reduction in dicated that the response to PF1-like peptides is the amplitude of the ACh contraction (Fig. 2A,B). long-lasting and requires more than a 30 min wash- This effect was not reversed following wash-out of out for recovery (Franks et al. 1994). In muscle that emodepside (Fig. 2A). The maximal effect was ob- was pre-treated with 1 mM PF2, there was no ad- served at 10 min (39¡6% inhibition; n=6), as there ditional reduction in the response to ACh on addition was no further reduction in the contraction when of emodepside (10 mM ; Fig. 4; n=4). emodepside was applied for 30 min (Fig. 2B). There- Emodepside also inhibited the response of the fore 10 min was selected as the time-point to inves- muscle to the neuropeptide, AF2. This peptide tigate the concentration-dependence of the inhibitory (2 min, 1 mM) elicited a biphasic effect on muscle action of emodepside. The threshold concentration tension, an initial relaxation followed by a prolonged

Fig. 4. PF2 (1 mM) relaxes Ascaris DMS in a similar manner to 10 mM emodepside. An example of the eﬀect of pre-treatment of the muscle with PF2 and subsequent application of emodepside on the response to ACh. The horizontal bars indicate the duration of application of the drugs.

Fig. 5. The effect of emodepside on the response to the excitatory neuropeptide AF2. AF2 was added to the preparation for 2 min, as indicated. After an initial Fig. 3. Effects of emodepside on muscle relaxation. relaxation, a characteristic increase in rhythmic activity (A) Example of the effect on muscle relaxation rate. was observed. In control experiments this persisted for The horizontal bar indicates the duration of application more than 1 h. Application of either vehicle or 10 mM of ACh. The arrows indicate the addition of either vehicle emodepside is indicated by the arrows. Emodepside caused (0.1% ethanol, indicated by arrow) or 10 mM emodepside a transient increase in the frequency of the rhythmic at the peak of the contraction. (B) Pooled data for different activity, followed by a complete cessation of activity. concentrations of emodepside (n=5). The relaxation was measured as the difference in tension at the peak of the response and prior to the wash, expressed as a percentage without nerve cord. For the ‘denervated’ prep- of the peak contraction and divided by 10, to yield the % arations the muscle was excised between the dorsal relaxation rate minx1. ‘Control’ is the relaxation rate with and ventral nerve cords so that neither of these were ACh alone. (C) Summary of the action of PF2 (1 mM) and present. However, it was not possible to remove the . emodepside (10 mM) on relaxation rate (n=6). * P<0 05; minor lateral cords and maintain a functional prep- ** P<0.01; *** P<0.001. aration, therefore this neural input was still intact. All 10 preparations, with or without nerve cord, re- sponded to ACh (30 mM) with a contraction. As with increase in rhythmic activity (Cowden & Stretton, previous experiments, the response to ACh was in- 1993) that persisted for more than 1 h. Emodepside hibited by emodepside in the intact preparations (0.5 mM) caused a relaxation (in 4 out of 6 prep- (Fig. 6A). The response to ACh in the denervated arations), but failed to completely inhibit the preparations was superimposed on a falling base-line rhythmic AF2 activity. At higher concentrations, tension, and was typically lower in amplitude than the emodepside caused a relaxation and abolished the response for intact preparations, reflecting damage AF2 induced rhythmic activity (1 mM, 6 out of 7 prep- to the muscle during dissection. Addition of emo- arations; 5 mM, 5 out of 5 preparations; and 10 mM,5 depside to the denervated preparations apparently out of 5 preparations). The effect of 10 mM emo- caused a slight reduction in baseline tension in 9 out depside on the response to AF2 is shown in Fig. 5. of 10 preparations (x0.25¡0.19 g), but this was To determine whether the inhibitory action of difficult to quantify because of the unstable base-line emodepside may involve neurotransmitter release tension. from the nerve cord onto the muscle, we compared Furthermore, the response to ACh in the dener- the action of emodepside on muscle strips with and vated preparations was not significantly inhibited by

Fig. 6. The eﬀect of removing the major nerve cords on the inhibitory action of emodepside and PF2. All 3 recordings are from muscle obtained from the same worm. The duration of application of drugs is indicated by the horizontal bars. (A) Recording from an intact muscle strip. (B) Recording from a ‘denervated’ muscle strip. (C) Recording from a denervated muscle strip showing the inhibitory action of PF2.

emodepside (10 mM for 10 min), if at all (the con- APF. In 7 out of 8 cells there was either very little or traction to ACh in the presence of emodepside was no change in membrane potential following addition 99.8¡4.8% of the contraction before addition of of emodepside in the absence of external Ca++ . emodepside; n=10; Fig. 6B). The inhibition of the Subsequent addition of Ca++ resulted in a rapid ACh response by PF2 was still observed in the de- hyperpolarization (Fig. 8). nervated preparation (Fig. 6C). The hyperpolarization caused by emodepside (10 mM for 2 min) was not observed in the presence of the K+ channel blockers 4-aminopyridine (4-AP, Electrophysiological recordings from muscle 250 mM) and tetraethylammonium (TEA, 5 mM). Co- Emodepside (10 mM for 2 min) caused a small, but application of both these blockers had a direct eﬀect signiﬁcant, slow and irreversible hyperpolarization on membrane potential causing a depolarization of of muscle cells with no detectable change in input 2.8¡0.5mV(n=5). Subsequent addition of emo- conductance (Fig. 7). After 30 min the average depside (10 mM for 2 min) in the presence of TEA and membrane potential change was x5.4¡1.0mV 4AP caused no change in muscle cell membrane (P<0.001, n=8; Fig. 7). The hyperpolarization elici- potential up to 10 min later (x0.1¡0.4mV;n=5). ted by 10 mM emodepside was reduced in Ca++ free Furthermore, in the presence of the channel blockers

Fig. 7. The effect of emodepside on the membrane potential of Ascaris muscle cells. The inset shows a representative response to 10 mM emodepside. The horizontal bar indicates the duration of application. Fig. 8. The effect of removing extracellular calcium on (Vehicle was without effect.) Scale bars are 10 mV and Ascaris muscle cell response to emodepside. The inset 2 min. The downward deflections are caused by injection of shows a representative experiment. The horizontal bar hyperpolarizing current pulses (0.1 Hz, 20 nA, 500 ms) indicates the duration of application. (Vehicle was without and provide a measure of cell input resistance. Note the effect.) Scale bars are 10 mV and 2 min. The downward decrease in ‘noise’ in the presence of emodepside. This is deflections are caused by injection of hyperpolarizing consistent with the abolition of spontaneous activity by current pulses (0.1 Hz, 20 nA, 500 ms) and provide a emodepside. The graph summarizes the data from 8 similar measure of cell input resistance. The arrows indicate the experiments. Resting membrane potential (#) 30 min removal and subsequent re-introduction of calcium ions. after a 2 min perfusion of emodepside ($). Each point The graph shows data from 8 similar experiments; indicates an individual muscle cell recording. The bars membrane potential in normal APF (#); membrane indicate the average value. potential 30 min after 2 min emodepside perfusion in the absence of calcium ($); membrane potential 30 min after re-introduction of calcium ions (%). there was an increase in the amplitude of spontaneous muscle potentials, and these persisted after the addition of emodepside. reduction in the amplitude of the ACh contraction. Further evidence for the involvement of K+ in This effect was significant at 10 mM emodepside and the emodepside-induced hyperpolarization was pro- maximal at 10 min incubation. The effect was irre- vided by experiments in K+-free APF. Perfusion of versible and it is likely that this reflects the highly K+-free APF resulted in muscle hyperpolarization lipophilic nature of emodepside. The ACh contrac- of x2.5¡1.0 mV after 10 min. Subsequent addition tion was not decreased by more than 40% of the of emodepside (10 mM for 2 min) resulted in a further control value, even at the highest concentration hyperpolarization of x12¡4 mV after 30 min (n=5; (10 mM) of emodepside tested, and higher con- compared to x5.4 mV in normal APF). centrations were not tested due to limitations of solubility of the compound. The inhibitory action of emodepside was also observed on muscle strips pre- DISCUSSION contracted with ACh. In the continued presence of Emodepside relaxes an isolated dorsal muscle strip ACh the initial, peak contraction is followed by a slow of Ascaris, reflecting the ability of this anthelmintic reduction in muscle tension, or ‘fade’ in the response, to paralyse parasitic nematodes. Here we have in- prior to the wash. This fade may be due to desensi- vestigated the cellular basis for this. As the effect of tization of the muscle. Addition of emodepside at the emodepside on basal muscle tension was very slight, peak of the contraction increased the relaxation rate the inhibitory action of the compound on the con- of the muscle with a threshold for the effect at less traction elicited by ACh was determined. ACh has than 1 mM. This was relatively slow in onset, and previously been shown to elicit contraction by acti- the tension did not relax right back to baseline before vation of a nicotinic receptor on the somatic muscle the wash. The inhibitory action of emodepside was (Colquhoun et al. 1991) and it would be expected that not specific for ACh-mediated contraction as it also an inhibitory compound would exert a physiological inhibited the action of the excitatory neuropeptide, antagonism of this response. Consistent with this, we AF2. found that pre-incubation of the muscle with emo- There are a number of possible cellular mechan- depside caused a concentration and time-dependent isms that may underly this inhibitory action of

emodepside and we undertook a systematic in- suggested, as one possiblity, that emodepside may act vestigation of these. For example, emodepside may by mimicking the action of the endogenous inhibitory exert its action either pre-synaptically or directly on neuromuscular junction transmitter, GABA (Martin the muscle. To test the latter possibility, we applied et al. 1996; Chen et al. 1996). For example, Martin emodepside to muscle from which the major nerve et al. (1996), observed a chloride-dependent in- cords had been removed. The muscle was probably crease in membrane input conductance that would compromised by the dissection procedure as the be consistent with activation of a GABA-gated chlor- base-line tension was not stable and the response to ide channel (Martin, 1982; Parri, Holden-Dye & ACh was reduced compared to an intact preparation. Walker, 1991; Holden-Dye et al. 1989). We there- In these preparations, emodepside caused a slight fore compared the inhibitory actions of GABA and decrease in base-line tension. This could be due to a emodepside on muscle tension. As would be expected mechanical artefact of drug addition, an action on of an inhibitory neurotransmitter, GABA elicited nerve endings of the minor nerves of the lateral cords, a fast relaxation of the muscle tonically contracted or a slight post-synaptic action. Most notably, how- with ACh back to base-line tension, quite different ever, emodepside did not inhibit the response to ACh from the slow incomplete relaxation caused by emo- when the nerve cord was removed. As a positive depside. The action of emodepside on the electro- control, intact dorsal muscle strips were taken from physiological properties of the muscle are also the same worms as the denervated muscle, and not like the actions of GABA. In contrast to the emodepside inhibited the response to ACh in these earlier study of Martin et al. (1996), we found that preparations. Furthermore, the nerve cord was not emodepside did not cause an increase in input required for the inhibitory response to PF2, con- conductance but rather it elicited a slow, K+-de- sistent with the post-synaptic action of this neuro- pendent hyperpolarization with no consistent peptide (Holden-Dye et al. 1995). change in input conductance. Taken together, our Further indirect evidence that the action of data do not support a role for GABA, or its receptor, emodepside may be mediated via an action at pre- in the action of emodepside. synaptic nerve terminals was provided from electro- A family of FMRF-amide like peptides are also physiological experiments. Intracellular recordings known to have potent effects on nematode muscle demonstrated that emodepside elicited a slow hyper- (Brownlee et al. 2000). Two of these peptides, PF1 polarization of muscle cells. The threshold con- and PF2 (Geary et al. 1992), are structurally very centration for the action of emodepside on muscle similar with the sequence SDPNFLRFamide and membrane potential was of the same order as that SADPNFLRFamide, and both of these have a potent observed for muscle relaxation, indicating that this inhibitory action on Ascaris somatic muscle (Holden- hyperpolarization is likely to be directly involved in Dye et al. 1995). To determine whether a PF1/2-like the inhibitory action of emodepside. As with the in- peptide may be involved in the response to emo- hibitory action of emodepside on muscle tension, this depside, we compared the action of emodepside with hyperpolarization did not reverse on washing. Re- that of PF2. As for emodepside, PF2 elicited a slow moval of calcium ions from the perfusate prevented but incomplete relaxation of dorsal muscle tonically the hyperpolarization in response to emodepside. contracted with ACh. Pre-incubation of the muscle This could be due to a requirement for calcium for with PF2 also reduced the amplitude of subsequent activation of a current on the muscle membrane e.g. contractions to ACh, in a similar manner to emo- a calcium-activated potassium channel. However, it depside. The receptors for PF2 are likely to be located is also consistent with a role for calcium-dependent post-synaptically on the muscle as the inhibitory ef- neurotransmitter release in the mechanism of action fect of PF2 on the ACh contraction was also observed of emodepside, and taken together with the exper- in a denervated muscle strip. iments of denervated muscle described above, we Electrophysiologically the response to PF2 is also favour this latter explanation. like the response to emodepside. PF2 causes a slow Intriguingly, the parent compound for emodep- hyperpolarization with no consistent change in input side, PF1022A, has been shown to interact with a conductance (Holden-Dye et al. 1995). The hyper- latrophilin receptor (Saeger et al. 2001). This may be polarization caused by PF2 is blocked by potassium relevant to the action of emodepside at the neuro- channel blockers (Walker et al. 2000). Similarly, the muscular junction as the latrophilin receptor is a G- hyperpolarization caused by emodepside was blocked protein coupled receptor and involved, in a not yet by potassium channel blockers, and enhanced in low fully characterized manner, in neurotransmitter re- external potassium. Furthermore, it has also been lease. In particular, latrophilin receptors regulate the shown that the response to PF1/PF2 is dependent on release of transmitters from dense-core vesicles e.g. calcium and that this reflects the involvement of a amines and neuropeptides (Davletov et al. 1998). nitric oxide signalling cascade in the peptide response If emodepside is acting to release an inhibitory (Bowman et al. 1995). Therefore it would seem that transmitter or modulator on to the muscle then the the ionic mechanism for the response to PF2 and to question arises, what is it? Previous studies have emodepside is similar.

Overall, these data suggest that emodepside may somatic muscle-cells of the parasitic nematode, Ascaris mimic the action of inhibitory neuropeptides at suum – stereoselectivity indicates similarity to a the neuromuscular junction, possibly by triggering GABA-A-type agonist recognition site. British Journal neuropeptide release from inhibitory nerve ter- of Pharmacology 98, 841–850. minals. Whether or not this contributes to the anthel- MARTIN, R. J. (1982). Electrophysiological effects of piperazine and diethylcarbamazine on Ascaris suum mintic action remains to be determined. Further somatic muscle. British Journal of Pharmacology 77, studies on the model genetic animal C. elegans are 255–265. being performed to refine this hypothesis. MARTIN, R. J., HARDER, A., LONDERSHAUSEN, M. & JESCHKE, P. (1996). Anthelmintic actions of the cyclic depsipeptide Kiran Amliwala is a BBSRC–CASE student and James Willson is a Bayer sponsored research student. PF1022A and electrophysiological effects on muscle cells of Ascaris suum. Pesticide Science 48, 343–349. PARRI, H. R., HOLDEN-DYE, L. & WALKER, R. J. (1991). Studies on the ionic selectivity of the GABA-operated chloride REFERENCES channel on the somatic muscle bag cells of the parasitic BOWMAN, J. W., WINTEROWD, C. A., FRIEDMAN, A. R., nematode Ascaris suum. Experimental Physiology 76, THOMPSON, D. P., KLEIN, R. D., DAVIS, J. P., MAULE, A. G., 597–606. BLAIR, K. V. & GEARY, T. G. (1995). Nitric oxide mediates PENNINGTON, A. J. & MARTIN, R. J. (1990). A patch-clamp the inhibitory effects of SDPNFLRFamide, a nematode study of acetylcholine-activated ion channels in Ascaris FMRFamide-related neuropeptide, in Ascaris suum. suum muscle. Journal of Experimental Biology 154, Journal of Neurophysiology 74, 1880–1887. 201–221. BROWNLEE, D. J., HOLDEN-DYE, L. & WALKER, R. J. (2000). The ROSOFF, M. L., DOBLE, K. E., PRICE, D. A. &LI, C. (1993). The range and biological activity of FMRFamide-related flp-1 propeptide is processed into multiple, highly peptides and classical neurotransmitters in nematodes. similar FMRFamide-like peptides in Caenorhabditis Advances in Parasitology 45, 109–180. elegans. Peptides 14, 331–338. CHEN, W., TERADA, M. & CHENG, J. T. (1996). SAEGER, B., SCHMITT-WREDE, H., DEHNHARDT, M., Characterization of subtypes of gamma-amino butyric BENTEN, W., KRUCKEN, J., HARDER, A., VON SAMSON- acid receptors in an Ascaris muscle preparation by HIMMELSTJERNA, G., WIEGAND, H. & WUNDERLICH, F. binding assay and binding of PF1022A, a new (2001). Latrophilin-like receptor from the parasitic anthelmintic, on the receptors. Parasitological Research nematode Haemonchus contortus as a target for the 82, 97–101. anthelmintic depsipeptide PF1022a. FASEB Journal 15, COLQUHOUN, L., HOLDEN-DYE, L. & WALKER, R. J. (1991). 1332–1334. The pharmacology of cholinoceptors on the somatic SAMSON-HIMMELSTJERNA, V. G., HARDER, A., SCHNIEDER, T., muscle-cells of the parasitic nematode Ascaris suum. KALBE, J. & MENCKE, N. (2000). In vivo activities of the new Journal of Experimental Biology 158, 509–530. anthelmintic depsipeptide PF1022A. Parasitological COWDEN, C. & STRETTON, A. W. (1993). AF2, an Ascaris Research 86, 194–199. neuropeptide – isolation, sequence, and bioactivity. SASAKI, T., TAGAKI, M., YAGUCHI, T., MIYADOH, S., OKADA, T. Peptides 14, 423–430. & KOYAMA, M. (1992). A new anthelmintic DAVLETOV, B. A., MEUNIER, F. A., ASHTON, A. C., MATSUSHITA, cyclodepsipeptide, PF1022A. Journal of Antibiotics H., HIRST, W. D., LELIANOVA, V. G., WILKIN, G. P., DOLLY, (Tokyo) 45, 692–697. J. O. & USHKARYOV, Y. A. (1998). Vesicle exocytosis SITHIGORNGUL, P., STRETTON, A. W. & COWDEN, C. (1990). stimulated by alpha-latrotoxin is mediated by latrophilin Neuropeptide diversity in Ascaris –an and requires both external and stored Ca2+. EMBO immunocytochemical study. Journal of Comparative Journal 17, 3909–3920. Neurology 294, 362–376. FRANKS, C. J., HOLDEN-DYE, L., WILLIAMS, R. G., PANG, F. Y. STRETTON, A. O. W., DAVIS, R. E., ANGSTADT, J., DONMOYER, J. & WALKER, R. J. (1994). A nematode FMRFamide-like & JOHNSON, C. D. (1985). Neural control of behaviour peptide, SDPNFLRFamide (PF1), relaxes the dorsal in Ascaris. Trends in Neurosciences 8, 294–300. muscle strip preparation of Ascaris suum. Parasitology TERADA, M. (1992). Neuropharmacological mechanism of 108, 229–236. action of PF1022A, an antinematode anthelmintic with a GEARY, T. G., PRICE, D. A., BOWMAN, J. W., WINTEROWD, C. A., new structure of cyclodepsipeptide, on Angiostrongylus MACKENZIE, C. D., GARRISON, R. D., WILLIAMS, J. F. & cantonensis and isolated frog rectus. Japanese Journal FREIEDMAN, A. R. (1992). Two FMRFamide-like peptides of Parasitology 41, 108–117. from the free-living nemaotde Panagrellus redivivus. TRIM, N., HOLDEN-DYE, L., RUDDELL, R. & WALKER, R. J. Peptides 13, 209–214. (1997). The effects of the peptides AF3 HOLDEN-DYE, L., FRANKS, C. J., WILLIAMS, R. G. & WALKER, (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) R. J. (1995). The effect of the nematode peptides on the somatic muscle of the parasitic nematodes Ascaris SDPNFLRFamide (PF1) and SADPNFLRFamide suum and Ascaridia galli. Parasitology 115, 213–222. (PF2) on synaptic transmission in the parasitic nematode WALKER, R. J., FRANKS, C. J., PEMBERTON, D., ROGERS, C. Ascaris suum. Parasitology 110, 449–455. & HOLDEN-DYE, L. (2000). Physiological and HOLDEN-DYE, L., KROGSGAARD-LARSEN, P., NIELSEN, L. pharmacological studies on nematodes. Acta Biologica & WALKER, R. J. (1989). GABA receptors on the Hungarica 51, 379–394.