Investigation of feeding behaviour in C. elegans reveals distinct pharmacological and antibacterial effects of nicotine.

Caenorhabditis elegans is an informative model to study the neural basis of feeding. A useful paradigm is one in which adult nematodes feed on a bacterial lawn which has been pre-loaded with pharmacological agents and the effect on pharyngeal pumping rate scored. A crucial aspect of this assay is the availability of good quality bacteria to stimulate pumping to maximal levels. A potential confound is the possibility that the pharmacological agent impacts bacterial viability and indirectly influences feeding rate. Here, the actions of nicotine on pharyngeal pumping of C. elegans and on the Escherichia coli bacterial food source were investigated. Nicotine caused an immediate and concentration-dependent inhibition of C. elegans pharyngeal pumping, IC50 4 mM (95% CI = 3.4 mM to 4.8 mM). At concentrations between 5 and 25 mM, nicotine also affected the growth and viability of E. coli lawns. To test whether this food depletion by nicotine caused the reduced pumping, we modified the experimental paradigm. We investigated pharyngeal pumping stimulated by 10 mM 5-HT, a food 'mimic', before testing if nicotine still inhibited this behaviour. The IC50 for nicotine in these assays was 2.9 mM (95% CI = 3.1 mM to 5.1 mM) indicating the depletion of food lawn does not underpin the potency of nicotine at inhibiting feeding. These studies show that the inhibitory effect of nicotine on C. elegans pharyngeal pumping is mediated by a direct effect rather than by its poorly reported bactericidal actions.

Actions of glutamate and ivermectin on the pharyngeal muscle of Ascaridia galli: a comparative study with Caenorhabditis elegans.

The actions of glutamate and ivermectin were examined in the pharynx of Ascaridia galli and the results compared with those on the pharynx of Caenorhabditis elegans. In both preparations glutamate elicits a depolarization and inhibition of pharyngeal pumping, but the response of the pharynx of A. galli was much less than for C. elegans. This may be either because the pharyngeal membrane potential of the former is closely linked to the equilibrium potential for chloride ions (E(Cl)) while that of C. elegans is independent of E(Cl), or that there is a lower density of glutamate receptors on the pharyngeal muscle of A. galli compared with C. elegans. The maximum depolarization to glutamate of the pharyngeal muscle was 4.5+/-0.8 mV in A. galli while it was >25 mV in C. elegans. Picrotoxin was a weak antagonist of the glutamate response in both species. Flufenamic acid, pentobarbitone and flurazepam had no significant effect on either preparation at concentrations up to 100 microM. Three glutamate receptor agonists, ibotenate, kainate and quisqualate were all more potent than glutamate on the A. galli pharyngeal muscle. In contrast, only ibotenate was more potent than glutamate in C. elegans pharynx, the other two agonists being approximately 20 times less potent. The potency of ivermectin differed markedly between the two species, being approximately three orders of magnitude less potent on the pharynx of A. galli compared with C. elegans. This study demonstrates clear differences between the properties of the pharyngeal muscle of the two species and shows that care must be taken when extrapolating data from free-living to parasitic species of nematode.

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans.

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.

Mechanisms of action of emodepside.

The research of the class of cyclic octadepsipeptides started at the beginning of the 1990s. PF1022A, the starting material of emodepside, is a natural secondary metabolite of the fungus Mycelia sterilia, which belongs to the microflora of the leaves of Camellia japonica. PF1022A consists of four N-methyl-L-leucins, two D-Iactic acids and two D-phenyllactic acids, which build up a cyclic octadepsipeptide with an alternating L-D-L-configuration. Emodepside is a semisynthetic derivative of PF1022A, which contains a morpholine attached in para position at each of both D-phenyllactic acids. Emodepside is efficacious against a variety of gastrointestinal nematodes. Emodepside binds to a presynaptic latrophilin receptor in nematodes. The following presynaptic signal transduction occurs via activation of Gqalpha protein and phospholipase-Cbeta, which leads to mobilization of diacylglycerol (DAG). DAG then activates UNC-13 and synaptobrevin, two proteins which play an important role in presynaptic vesicle-functioning. This finally leads to the release of a currently unidentified transmitter. The transmitter (or modulator) exerts its effects at the postsynaptic membrane and induces a flaccid paralysis of the pharynx and the somatic musculature in nematodes.

The range and biological activity of FMRFamide-related peptides and classical neurotransmitters in nematodes.

Nematodes include both major parasites of humans, livestock and plants in addition to free-living species such as Caenorhabditis elegans. The nematode nervous system (especially in C. elegans) is exceptionally well defined in terms of the number, location and projections of the small number of neurons in the nervous system and their integration into circuits involved in regulatory behaviours vital to their survival. This review will summarize what is known about the biological activity of neurotransmitters in nematodes: the biosynthetic pathways and genes involved, their receptors, inactivation mechanisms and secondary messenger signalling systems. It will cover the 'classical' transmitters, such as acetylcholine (ACh), GABA, glutamate, serotonin, dopamine, octopamine, noradrenaline and nitric oxide. The localization of peptides throughout the nematode nervous system is summarized, in addition to the isolation of nematode neuropeptides by both traditional biochemical techniques and more modern genetic means. The major contribution of the completion of the C. elegans genome-sequencing program is highlighted throughout. Efforts to unravel neurotransmitter action in various physiological actions such as locomotion, feeding and reproduction are detailed as well as the various inactivation mechanisms for the current complement of nematode transmitters.

The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli.

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 microM forskolin (by 22% of control; P < 0.05; n = 9) and by 500 microM isobutylmethylxanthine (IBMX, by 27% of control; P < 0.001; n = 6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721 +/- 134 pmol mg-1 protein; with 1 microM AF3, 1148 +/- 133 pmol mg-1 protein; P < 0.05, n = 5). AF3 (1 microM) also reduced the 10 microM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242 +/- 471 pmol mg-1 protein; forskolin and AF3, 1524 +/- 143 pmol mg-1 protein; P < 0.001, n = 6) and A. galli (forskolin 291 +/- 32 pmol mg-1 protein, forskolin +AF3, 185 +/- 12 pmol mg-1 protein; P < 0.005, n = 5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.

Metabolism of AF1 (KNEFIRF-NH2) in the nematode, Ascaris suum, by aminopeptidase, endopeptidase and deamidase enzymes.

We have studied the metabolism and inactivation of AF1 (KNEFIRF-NH2) by membranes prepared from the locomotory muscle of Ascaris suum. FIRF-NH2 and KNEFIRF were identified as three primary degradation products, resulting from the action of an endopeptidase, aminopeptidase and a deamidase, respectively. The endopeptidase resembled mammalian neprilysin (NEP, endopeptidase 24.11) in that the enzyme activity was inhibited by phosphoramidon and thiorphan and that it cleaved AF1 on the amino side of phenylalanine. The aminopeptidase activity was inhibited by amastatin and bestatin but not by puromycin. The deamidation of AF1 was inhibited by phenylmethylsulfonyl fluoride, p-chloromercuricphenylsulfonate and mercuric chloride, indicating that the deamidase enzyme is a serine protease with a requirement for a free thiol group for activity. AF1 (1 microM) induces an increase in tension and an increase in the frequency and amplitude of spontaneous contractions of an A. suum muscle strip. None of the aforementioned AF1 metabolites (2-20 microM) retained biological activity in this bioassay, indicating that the endopeptidase, aminopeptidase and deamidase have the potential to terminate the action of AF1 on locomotory muscle of A. suum.

ZAPA, (Z)-3-[(aminoiminomethyl)thio]-2-propenoic acid hydrochloride, a potent agonist at GABA-receptors on the Ascaris muscle cell.

This study is the first report of a compound which is equal in efficacy to gamma-aminobutyric acid (GABA) at the nematode Ascaris muscle GABA-receptor. The GABA-receptor at the Ascaris muscle cell which mediates a membrane hyperpolarization and muscle relaxation has eluded classification. The structure-activity profile of this receptor is not typical of GABAA or GABAB-receptors. Here we report that the isothiouronium compound ZAPA is as potent as GABA at this receptor. This finding has important implications for the characterization of the Ascaris GABA-receptor and the design of novel anthelmintics.

GABA receptors on the somatic muscle cells of the parasitic nematode, Ascaris suum: stereoselectivity indicates similarity to a GABAA-type agonist recognition site.

1. The gamma-aminobutyric acid (GABA) receptors on the somatic muscle cells of Ascaris, which mediate muscle cell hyperpolarization and relaxation, have been characterized by use of intracellular recording techniques. 2. These receptors are like mammalian GABAA-receptors in that the response is mediated by an increase conductance to chloride ions. The GABAA-mimetic, muscimol, has a relative potency of 0.40 +/- 0.02 (n = 3) compared to GABA. 3. The stereoselectivity of the GABA receptor on Ascaris is identical to that for the mammalian GABAA-receptor, as determined from the relative potency of three pairs of enantiomers of structural analogues of GABA. 4. The most potent agonist is (S)-(+)-dihydromuscimol which is 7.53 +/- 0.98 (n = 5) times more potent than GABA. 5. The Ascaris GABA receptor is not significantly blocked, at concentrations below 100 microM by the potent, competitive GABAA-receptor antagonist, SR95531. 6. The Ascaris GABA receptor does not recognise agents that are known to block the GABA gated chloride channel in mammalian preparations such as t-butylbicyclophosphorothionate (TBPS, 10 microM, n = 2) or the insecticide dieldrin (100 microM, n = 3). 7. GABAergic responses in Ascaris are not potentiated by pentobarbitone (100 microM, n = 3) or flurazepam (100 microM, n = 3). 8. The potencies of various GABA-mimetics in the Ascaris preparation have been compared with their potency at displacing GABAA-receptor binding in mammalian brain. Excluding the sulphonic acid derivatives of GABA, the correlation coefficient (r) between the potencies of compounds in the two systems is 0.74 (P less than 0.01). The significance of this correlation is discussed. 9. The pharmacology of the Ascaris GABA receptor is discussed in relation to other invertebrate systems and the mammalian subclassification of GABA receptors.

The effects of the peptide KPNFIRFamide (PF4) on the somatic muscle cells of the parasitic nematode Ascaris suum.

1. Commonly used anthelmintic agents act on the muscle cells of parasitic nematodes to cause paralysis of the parasite and its expulsion from the host. 2. 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, gamma-aminobutyric acid (GABA), which gates a chloride channel on Ascaris muscle. 3. Both PF4 and GABA hyperpolarized the muscle cells (EC50 values 98 nM and 59 microM, respectively; n = 6) and this was accompanied by an increase in input conductance. 4. The increase in input conductance elicited by PF4 and a supramaximal concentration of GABA were additive (10 microM PF4, 7.78 +/- 1.88 microS; 10 mM GABA, 4.68 +/- 1.39 microS; 10 mM GABA and 10 microM PF4 12.05 +/- 2.6 microS, n = 6, P < 0.02 with respect to PF4 alone; P < 0.01 with respect to GABA alone). 5. The membrane potential response to 10 microM 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; r2 = 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). 6. 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 microM PF4 was -47.5 +/- 1.5 mV (P < 0.02; n = 6). 7. The conductance increase elicited by 30 microM GABA was blocked by 10 microM ivermectin (before ivermectin 0.97 +/- 0.2 microS, after ivermectin 0.33 +/- 0.12 microS; n = 5; P < 0.05; Student's paired t test) but the conductance increase elicited by 1 microM PF4 was not (before ivermectin 0.96 +/- 0.14 microS, after ivermectin 1.07 +/- 0.19 microS; n = 0.34; Student's paired t test). 8. 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.

The characterization of [3H]sulpiride binding sites in pig striatal membranes.

Pig striatal membranes have [3H]sulpiride-binding sites similar to those identified in rat striatal membranes. The pharmacological profile indicates that this binding is to dopamine receptors. Agonist displacement of [3H]sulpiride binding in pig striatal membranes is subject to guanine nucleotide regulation. This effect is mimicked by heat treatment. N-ethyl maleamide (20 microM) and dithioerythritol (3 mM) decrease agonist affinity for the [3H]sulpiride-binding site in pig striatal membranes without significantly affecting maximal displacement.

The actions of muscle relaxants at nicotinic acetylcholine receptor isoforms.

The pharmacological diversity of the different isoforms of the nicotinic acetylcholine receptor arises from the diversity of the subunits that assemble to form the native receptors. The aim of this study was to investigate the actions of the muscle relaxants d-tubocurarine, pancuronium and vecuronium on different isoforms of nicotinic acetylcholine receptors (mouse foetal muscle, mouse adult muscle and a rat neuronal), using the Xenopus oocyte expression system. Oocytes were injected with cRNAs for alpha, beta, gamma, delta subunits (the native foetal muscle subunit combination), or with cRNAs for alpha, beta, epsilon, delta subunits (the native adult muscle subunit combination), or with cRNAs for alpha4beta2 subunits (a putative native neuronal subunit combination). Acetylcholine had a similar potency at all three subunit combinations (EC50 11.6, 17.4 and 19.1 microM, respectively). At all three receptor types, d-tubocurarine and pancuronium blocked the responses elicited by acetylcholine in a reversible manner. Furthermore, the inhibition of the acetylcholine currents for the foetal and adult nicotinic acetylcholine receptor by pancuronium and d-tubocurarine was independent of the holding voltage over the range -100 to -40 mV. In oocytes expressing the foetal muscle nicotinic acetylcholine receptors the inhibition of the current in response to 100 microM acetylcholine by 10 nM d-tubocurarine was 29 +/- 5% (mean +/- S.E.M.; n = 7), and the inhibition by 10 nM pancuronium was 39 +/- 6% (mean +/- S.E.M.; n = 8; P > 0.05 vs. d-tubocurarine). However, in the adult form of the muscle nicotinic acetylcholine receptor, 10 nM d-tubocurarine and 10 nM pancuronium were both more effective at blocking the response to 100 microM acetylcholine compared to the foetal muscle nicotinic acetylcholine receptor, with values of 55 +/- 5% (P < 0.01; n = 12) and 60 +/- 4% (P < 0.001; n = 10), respectively. Thus the developmental switch from the gamma to the epsilon subunit alters the antagonism of the nicotinic acetylcholine receptor for both pancuronium and d-tubocurarine. Vecuronium was more potent than pancuronium. One nM vecuronium reduced the response to 100 microM acetylcholine by 71 +- 6% (n = 10) for foetal and 63 +/- 5% (n = 4) for adult nicotinic acetylcholine receptors. In the alpha4beta2 neuronal nicotinic acetylcholine receptor combination, 10 nM pancuronium was a more effective antagonist of the response to 100 microM acetylcholine (69 +/- 6%, n = 6) than 10 nM d-tubocurarine (30 +/- 5%; n = 6; P < 0.05 compared to pancuronium). This is in contrast to the adult muscle nicotinic acetylcholine receptor, where pancuronium and d-tubocurarine were equieffective. The expression of the beta2 subunit with muscle alpha, epsilon and delta subunits formed a functional receptor which was blocked by pancuronium and d-tubocurarine in a similar manner to the alphabeta1epsilondelta subunit consistent with the hypothesis that the beta subunit is not a major determinant in the action of this drug at the adult muscle nicotinic acetylcholine receptor.

Actions of neurotoxins (bungarotoxins, neosurugatoxin and lophotoxins) on insect and nematode nicotinic acetylcholine receptors.

Neurotoxins of natural origin have proved to be of considerable value in the isolation and characterization of vertebrate muscle and neuronal nicotinic acetylcholine receptors (nAChRs). To date, they have been used less extensively in studies of invertebrate nAChRs. Here we examine how a variety of neurotoxins (the snake toxins alpha-bungarotoxin, alpha-BGT, and kappa-bungarotoxin, kappa-BGT, the molluscan toxin, neosurugatoxin, and the soft coral toxins, lophotoxin and bipinnatin-B) can be used to characterize nAChRs in an insect, Periplaneta americana, and in a parasitic nematode, Ascaris suum. The agonist profiles of these nAChRs are distinct, but the most striking differences are in the actions of antagonists. Whereas the insect nAChR is blocked by both alpha- and kappa-bungarotoxins, the nematode receptor is only blocked by kappa-BGT. Neosurugatoxin blocks nAChRs in both species, but the lophotoxins which block all nAChRs investigated to date are much less effective on the Ascaris muscle receptor.

Pharmacological profiles of the GABA and acetylcholine receptors from the nematode, Ascaris suum.

The pharmacological profiles of the acetylcholine and GABA receptors of Ascaris muscle have been investigated. Acetylcholine excites the muscle through activation of a nicotinic receptor which resembles the mammalian ganglionic receptor. DMPP is a potent agonist and alpha-bungarotoxin is a weak antagonist. GABA inhibits the muscle through activation of a chloride-linked receptor which in terms of agonist profile resembles the mammalian GABA-A receptor. However, bicuculline is inactive and picrotoxin is a very weak antagonist. Avermectin acts as a non-competitive antagonist at this GABA receptor with an IC-50 value in the low microM range.

The pharynx of the nematode Ascaris suum: structure and function.

The pharyngeal muscle of nematodes consists of a syncytium of radial muscle whereby feeding occurs by the process of pharyngeal pumping. It is believed that the pumping behaviour of the pharynx may be partly controlled by the enteric nervous system (ENS), a component of the nematode nervous system which is associated predominantly with the pharynx. The distribution of serotoninergic and peptidergic (especially SALMF-amide-like) immunostaining is widespread in the ENS of Ascaris, being localized within the lateral and dorsal pharyngeal nerve tracts, the pharyngeal commissures, the nerve plexuses and associated nerve cells and fibres. Immunostaining for serotonin (5-HT) was only localized within the ENS. This paper also describes a method to enable in vitro pharmacological studies on the Ascaris pharynx. The effects of "classical" neurotransmitters and native nematode peptides on pharyngeal pumping behaviour in Ascaris have been investigated. The function of the pharynx in Ascaris is discussed.

Immunocytochemical and electrophysiological investigations of central neurones in the parasitic nematode Ascaris suum.

Here we report on preliminary investigations into the morphological, neurochemical and electrophysiological characteristics of central neurones in the parasitic nematode Ascaris suum. The neurones do not display all or none action potentials and apparently have low resting membrane potentials. Neurones have been shown to respond to acetylcholine. Immunoreactivity to a diverse range of neuropeptides has been demonstrated.

Physiological and pharmacological studies on nematodes.

Classical transmitters and neuroactive peptides act as transmitters or modulators within the central and peripheral nervous systems of nematodes, for example Ascaris suum and Caenorhabditis elegans. Acetylcholine (ACh) and gamma-aminobutyric acid (GABA) are respectively the excitatory and inhibitory transmitters onto somatic body wall muscle while 5-hydroxytrypamine (5-HT) is the excitatory transmitter onto pharyngeal muscle. 5-HT also reduces ACh-induced contractions of somatic muscle and this action of 5-HT is mediated through activation of adenylate cyclase while that on pharyngeal muscle is mediated through inositol phosphate activation. Glutamate, dopamine and octopamine also have transmitter roles in nematodes. Neuroactive peptides of the RFamide family can excite somatic muscle, for example, AF-1 (KNEFIRFamide), AF-2 (KHEYLRFamide), AF-3 (AVPGVLRFamide) and AF-4 (GDVPGVLRFamide) or inhibit and relax this muscle, for example, PF-1 (SDPNFLRFamide), PF-2 (SADPNFLRFamide) and PF-4 (KPNlRFamide). In addition PF-3 (AF-8) (KSAYMRFamide) has a biphasic action on pharyngeal muscle, excitation followed by inhibition while AF-1 only inhibits this muscle. The peptide effects can be either pre- or postsynaptic or both and are likely to be mediated through second messenger systems. In addition these peptides modulate the action of classical transmitters, particularly ACh.

Neuropharmacological studies on identified central neurones of the snail, Helix aspersa.

Central neurones of the snail, Helix aspersa, possess both synaptic and somatic receptors to a wide range of classical transmitters and neuroactive peptides. The range of receptors and the type of response is reasonably constant for a specific neurone. This review will provide data concerning the pharmacology of acetylcholine, serotonin, dopamine, noradrenaline, octopamine, glutamate, GABA and purine receptors on identified neurones. Many of these neurones also respond to neuroactive peptides including molluscan peptides, e g, AMPMLRLamide, LSSFVRIamide, SGQSWRQGRPFamide, FMRFamide; coelenterate peptides, e g, pQGRFamide; echinoderm peptides, e g, GFNSALMFamide; and nematode peptides, e g, KNEFIRFamide, KHEYLRFamide, SDPNFLRFamide. Using this data, the pharmacological profiles of identified neurones will be summarised.