T.A. Day

Praziquantel: The Enigmatic Antiparasitic

Praziquantel (PZQ), a pyrazinoisoquinoline, was introduced as a novel anthelmintic in 1975. PZQ is currently the drug of choice for the treatment of a wide range of both veterinary and human trematode and cestode infections, including human schistosomiasis. Current estimates suggest that 150 million humans are infected with schistosomes, and it is expected that PZQ will play the lead role in chemotherapeutic control of those infections. Despite the time that has passed since its introduction and its obvious importance in global health care, it is not yet understood why PZQ is so selective and effective. The target molecules for PZQ have not been defined, nor are the sites of its effects within the parasites known. Here, Tim Day, James Bennett and Ralph Pax summarize some of the progress that has been made toward reaching these objectives in recent years.

Praziquantel-induced tegumental damage in vitro is diminished in schistosomes derived from praziquantel-resistant infections.

The aggressive use of praziquantel to combat schistosomiasis in Egpyt raises concern about the possible emergence of resistance. Eggs from Egyptian patients with praziquantel-resistant infections (not cured by 3 doses of praziquantel) have been used to establish infection-specific schistosome isolates in mice. The response of these worms to the drug was observed in vitro, in order to determine if the isolates obtained from these resistant infections were, in fact, less responsive to praziquantel. One of the hallmark effects of praziquantel on schistosomes in vitro is a disruption of the worms outer surface, the tegument. Here, praziquantel-induced tegumental damage is observed in 3 distinct isolates, 2 derived from resistant infections and 1 from an infection cured by a single dose. The isolates from the resistant infections were less susceptible to praziquantel-induced tegumental damage in vitro, suggesting that the worms are in some way less responsive to the drug.

Parasitic peptides! The structure and function of neuropeptides in parasitic worms.

Parasitic worms come from two very different phyla-Platyhelminthes (flatworms) and Nematoda (roundworms). Although both phyla possess nervous systems with highly developed peptidergic components, there are key differences in the structure and action of native neuropeptides in the two groups. For example, the most abundant neuropeptide known in platyhelminths is the pancreatic polypeptide-like neuropeptide F, whereas the most prevalent neuropeptides in nematodes are FMRFamide-related peptides (FaRPs), which are also present in platyhelminths. With respect to neuropeptide diversity, platyhelminth species possess only one or two distinct FaRPs, whereas nematodes have upwards of 50 unique FaRPs. FaRP bioactivity in platyhelminths appears to be restricted to myoexcitation, whereas both excitatory and inhibitory effects have been reported in nematodes. Recently interest has focused on the peptidergic signaling systems of both phyla because elucidation of these systems will do much to clarify the basic biology of the worms and because the peptidergic systems hold the promise of yielding novel targets for a new generation of antiparasitic drugs.

Platyhelminth FMRFamide-related peptides (FaRPs) contract Schistosoma mansoni (Trematoda: Digenea) muscle fibres in vitro

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10(-9) and 10(-7) M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10(-8)-10(-6) M and 10(-7)-10(-5) M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 microM FMR-D-Famide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0.5 microM EGTA was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high K+-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

Neuropeptide Signaling Systems - Potential Drug Targets for Parasite and Pest Control.

Current problems of drug resistance in parasites and pests demand the identification of new targets and their exploitation through novel drug design and development programs. Neuropeptide signaling systems in helminths (nematodes and platyhelminths = worms) and arthropods are well developed and complex, play a crucial role in many aspects of their biology, and appear to have significant potential as targets for novel drugs. The best-known neuropeptide family in invertebrates is the FMRFamide-related peptides (FaRPs). Amongst many roles, FaRPs potently influence motor function. The genome sequencing projects of Drosophila melanogaster and Caenorhabditis elegans have revealed unexpected complexity within the FaRPergic systems of arthropods and nematodes, although available evidence for platyhelminths indicates structural and functional simplicity. Regardless of these differences, FaRPs potently modulate motor function in arthropods, nematodes and platyhelminths and there appears to be at least some commonality in the FaRPergic signaling systems therein. Moreover, there is now increasing evidence of cross-phyla activity for individual FaRPs, providing clear signals of opportunities for target selection and the identification and development of broad-spectrum drugs.

A confocal microscopical study of the musculature of adult Schistosoma mansoni.

Using the filamentous actin marker, FITC-conjugated phalloidin, the major muscle systems of adult male and female schistosomes have been examined. The body wall musculature comprises an outer sheath of circular fibres, within which there is a compact layer of short, spindle-shaped longitudinal fibres and a lattice-like arrangement of inner diagonal fibres. Within the oral sucker and acetabulum 3 fibre types, circular, radial and longitudinal can be distinguished. The wall of the oesophagus is lined by a grid-like array of circular and longitudinal fibres, whereas the walls of the intestinal caeca contain only comparably broad circular fibres. Within the female reproductive system, only circular fibres are present in the oviduct, vitelline duct and uterus. In contrast, the wall of the ootype displays closely arranged circular and longitudinal muscle fibres. Antisera to previously identified myoactive compounds (serotonin [5-hydroxytryptamine, 5-HT], neuropeptide F [Moniezia expansa] and GYIRFamide [Bdelloura candida, Dugesia tigrina]) were used as neuronal markers in a preliminary study of the spatial inter-relationships of specific nerve fibres and various muscle systems. Serotoninergic fibres innervate both suckers and also constitute a subtegumental nerve net. In males they provide innervation to the dorso-ventral muscle fibres of the gynaecophoric canal, and in females they innervate the circular and longitudinal muscle fibres of the ootype. Neuropeptide F and the FMRFamide-related peptide, GYIRFamide are both localized within nerve plexuses associated with the dorso-ventral fibres of the gynaecophoric canal, and are evident in the innervation of the ventral and oral sucker.

Serotonin and its requirement for maintenance of contractility in muscle fibres isolated from Schistosoma mansoni.

Muscle fibres isolated from adult Schistosoma mansoni contracted in a dose-dependent manner when exposed to elevated K+ with a maximum response obtained with 25 mM K+. These contractions were dependent on extracellular Ca2+ since Co2+ (5 mM) or nicardipine (1 microM) blocked the high K+ contractions. Serotonin (300 nM or higher) was required for maintenance of high K+ contractions. With concentrations of serotonin less than 300 nM the response was dose dependent. 5-Methoxytryptamine or alpha-methylserotonin at 1 microM as well as 10 microM tryptamine were able to substitute for serotonin, but 1 microM 5-carboxyamidotryptamine was ineffective. The order of potency for antagonists (10 microM) was: methiothepin > metergoline > Ly-278,584 = ketanserin. This pattern of responsiveness does not fit well with any known mammalian serotonin receptor subtype. Since forskolin, an adenylate cyclase activator, is able to mimic the action of serotonin and H89, a protein kinase inhibitor, is able to block the effect of serotonin, the effect of serotonin on contractility of the muscle may be via a cAMP-dependent pathway.

Pharmacology of FMRFamide-related Peptides in Helminths

Abstract: Nervous systems of helminths are highly peptidergic. Species in the phylum Nematoda (roundworms) possess at least 50 FMRFamide‐related peptides (FaRPs), with more yet to be identified. To date, few non‐FaRP neuropeptides have been identified in these organisms, though evidence suggests that other families are present. FaRPergic systems have important functions in nematode neuromuscular control. In contrast, species in the phylum Platyhelminthes (flatworms) apparently utilize fewer FaRPs than do nematodes; those species examined possess one or two FaRPs. Other neuropeptides, such as neuropeptide F (NPF), play key roles in flatworm physiology. Although progress has been made in the characterization of FaRP pharmacology in helminths, much remains to be learned. Most studies on nematodes have been done with Ascaris suum because of its large size. However, thanks to the Caenorhabditis elegans genome project, we know most about the FaRP complement of this free‐living animal. That essentially all C. elegans FaRPs are active on at least one A. suum neuromuscular system argues for conservation of ligand‐receptor recognition features among the Nematoda. Structure‐activity studies on nematode FaRPs have revealed that structure‐activity relationship (SAR) “rules” differ considerably among the FaRPs. Second messenger studies, along with experiments on ionic dependence and anatomical requirements for activity, reveal that FaRPs act through many different mechanisms. Platyhelminth FaRPs are myoexcitatory, and no evidence exists of multiple FaRP receptors in flatworms. Interestingly, there are examples of cross‐phylum activity, with some nematode FaRPs being active on flatworm muscle. The extent to which other invertebrate FaRPs show cross‐phylum activity remains to be determined. How FaRPergic nerves contribute to the control of behavior in helminths, and are integrated with non‐neuropeptidergic systems, also remains to be elucidated.

Neuromuscular physiology and pharmacology of parasitic flatworms

The trematode and cestode flatworms include numerous parasitic forms of major medical and economic importance. A better knowledge of the neuromuscular physiology of these animals could lead to development of new control measures against these parasites. Since these animals are near the stem from which all other animals have evolved, better knowledge of these animals could also yield valuable information about the early evolution of nerve and muscle systems in the animal kingdom. This review focuses on what is known about the characteristics of the somatic muscle in these animals. The anatomy of the muscles is described along with a review of current information about their electrophysiology, including descriptions of the ion channels present. Also included is a summary of recently acquired data concerning the nature of serotonin, peptide, acetylcholine and glutamate receptors on the membranes of the muscles.

Studies on muscle cells isolated from Schistosoma mansoni : a Ca 2+ -dependent K + channel

Muscle cells from adult male Schistosoma mansoni have been isolated using a combination of papain digestions and mechanical dissociation procedures. The muscle fibres isolated in this way lacked nuclei but they did contract and relax in response to high [K+], a response which was blocked in the presence of Co2+. From this we conclude that the isolation procedure yields viable muscle fibres useful for physiological studies. Patch-clamp recordings taken from the isolated fibres show a variety of discrete ionic conductances. In inside-out patches one prominent channel was a Ca(2+)-activated K+ channel with a conductance of 195 pS and a selectivity greater than 10:1 for K+ over Na+, Cs+ or NH4+. Percentage open time was dependent on [Ca2+] at the intracellular face. With [Ca2+] at 1 microM or greater percentage open time was greater than 95%; at 0.1 microM it was less than 2%. No voltage sensitivity could be detected in the voltage range from -50 to -10 mV membrane potential. Ba2+ (10 mM), but neither tetraethylammonium nor 3,4-diaminopyridine blocked the channel from the intracellular face. This Ca(2+)-activated K+ channel in the muscle membrane of this acoelomate animal is similar in most respects to the maxi-K+ channels which have been described in a variety of cells from more highly evolved animals.