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 (EC(50) values 98 nM and 59 mu M, 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 mu M PF4, 7.78+/-1.88 mu S; 10 mM GABA, 4.68+/-1.39 mu S; 10 mM GABA and 10 mu M PF4 12.05+/-2.6 mu S, 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 mu M 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; r(2)=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 mu M PF4 was -47.5+/-1.5 mV (P<0.02; n=6). 7 The conductance increase elicited by 30 mu M GABA was blocked by 10 mu M ivermectin (before ivermectin 0.97+/-0.2 mu S, after ivermectin 0.33+/-0.12 mu S; n=5; P<0.05; Student's paired t test) but the conductance increase elicited by 1 mu M PF4 was not (before ivermectin 0.96+/-0.14 mu S, after ivermectin 1.07+/-0.19 mu S; 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.