Effects Of The FMRFamide-related Neuropeptide KHEYLRF-NH2 (AF2) And Related Compounds On Muscle Tension and Second Messenge Levels In Isolated Neuromuscular Preparations from the Parasitic Nematode, Ascaris suum

Abstract

Gastrointestinal nematodes are important pathogens in both human and veterinary medicine. Incidence of infection by the parasite Ascaris lumbricoides exceeds 1 billion humans worldwide (Crompton, 1988), and closely related species, Ascaris suum, infects swine and contributes extensively to agricultural production losses. Because resistance to the few drugs available for treating these pathogens is an increasing problem, an important objective in anthelmintic discovery has become the identification of novel drug targets in parasites. The EMRFamide-related peptides (FaRPs) are not present in vertebrates, and therefore, drugs directed at FaRP systems in nematodes would likely be an effective tool against the parasites while not affecting the hosts (Thompson et al. 1996). In the present study, we used standard physiographic methods and enzyme immunoassays to measure the effects of several FaRPs, including the potent excitatory peptide AF2 (KHEYLRF-NH2), on somatic muscle function in Ascaris suum. Concentration- and time-dependent effects of these peptides on tension were correlated with second messenger levels in isolated neuromuscular preparations determined by enzyme immunoassays. Results of these studies showed that AF2 and a related excitatory FaRP, AFI (KNEFIRF-NH2) stimulate a profound increase in neuromuscular activity leading eventually to spastic paralysis of the nematode neuromusculature. Concurrent with this change in tension is profound stimulation of production of the second messenger cAMP. The effects of AF2 on these parameters occurred at 100 fold lower concentrations than AF1. The stimulation due to AF2 was unaffected by drugs that block cAMP production or its degradation in vertebrates, including Cholera toxin (CTX) and isobutylmethylxanthane (IBMX). Inhibitory FaRPs, including PF 1 and PF3, as well as the cyclodepsipeptide U-I 03426, blocked effects of AF2 on muscle tension, but did not effect the AF2-induced stimulation of cAMP, nor did they affect cGMP levels in the tissue by themselves. This indicates that it is possible to decouple muscle tension from cAMP levels in A. suum neuromuscular tissue. These results demonstrate, for the first time, that receptors for AF2 and AF 1 in A. suum are G protein-coupled and thus provide important information relating to the mechanism of action of these peptides. This information will be useful in receptor cloning and drug screening efforts toward the identification of AF2 or AF 1 mimetics, which may be effective treatments against this parasite.