Effects of PF4 on Ascaris suum somatic muscle cells: an electrophysiological study

Abstract

Nematode parasites are found in humans and other species world-wide. Infected host animals show morbidity and sometimes death, which has prompted the search for safe and effective anti-parasitic drugs (anthelmintics). The essential feature of safe anthelmintics is that they are specific for the parasite and do not damage the host. To this end, differences between the physiology of nematodes and that of their vertebrate hosts can be exploited to design safe anti-parasitic drugs. One such difference is the neuroactive peptides that have been discovered in invertebrates, including nematodes.

Known as FaRPs, from FMRFnmide Related Peptides, these neuropeptides are widespread amongst nematodes and much less common in vertebrates. This has caused interest in FaRP receptors as potential dmg targets. PF4 is a FaRP which was isolated from the free-living nematode Panagrellus redivivus and which has been shown to have activity in other nematodes, including Ascaris suum. A. suum, the large parasitic nematode found in domestic pigs, is often used as an experimental model in studying nematodes due to its large size and easy availability. The A.suum used in this study were obtained from three different sources.

The action of PF4 on the somatic muscle cells ofA suum was investigated using the two electrode current clamp technique. PF4 caused these cells to hyperpolarise by 2 to 12 mV. The FaRP also caused an increase in membrane conductance of up to 3 pS. The response to PF4 was shown to be due to the opening of chloride ion channels in the somatic muscle cell membrane. In experiments to examine the delay between application ofPF4 and the response, PF4 was found to act as rapidly as the directly gating ligand y-amino butyric acid. This observation is consistent with the argument that PF4 directly gates ion channels.

PF4 was investigated further with patch clamp experiments that were carried out on membrane vesicles produced by treating the muscle cells with collagenase. Using vesicle-attached and isolated inside-out patches, PF4 was shown to open small amplitude channels (channel conductance of 1.5 to 6.2 pS) with a relatively high probability of opening (P0pen) of 0.05 to 0.65 at [PF4] 0.003 μM. The channel The effects of PF4 on Ascaris suum muscle cells conductance and the P0pen recorded in each experiment varied with the origin of the worms, suggesting genetic diversity within A. suum.

The mean open time of the channel was found to be voltage sensitive; it varied from 522 ± 333 ms at -80 mV to 25 ± 7 ms at +120 mV. The behaviour of the channels did not alter after patch isolation, which is further evidence for them being directly gated by PF4. When the cytoplasmic side of the patches was exposed to a G-protein inhibitor (guanosine 5¹-0-(2-thiodiphosphate)), the conductance of the channels reduced and the Popen increased. This finding suggests that these ion channels can be modulated by one or more G-proteins.

The results of this project suggest that the FaRP, PF4, directly activates low conductance chloride channels by combining with a receptor on the outside of the muscle membrane. The channels are different from those described previously in nematodes or vertebrates and therefore present a potential target for anthelmintic drugs.