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.
Holden-Dye
L
L
Department of Physiology & Pharmacology, University of Southampton.
Brownlee
D J
DJ
Walker
R J
RJ
D000818:Animals; D000969:Antinematodal Agents / Q000494:pharmacology; D017165:Ascaris suum / Q000502:physiology*; D004594:Electrophysiology; D066298:In Vitro Techniques; D007559:Ivermectin / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects; D009046:Motor Neurons / Q000187:drug effects; D009132:Muscles / Q000166:cytology* / Q000294:innervation / Q000378:metabolism; D009431:Neural Conduction / Q000187:drug effects; D009479:Neuropeptides / Q000494:pharmacology*; D009842:Oligopeptides / Q000494:pharmacology*; D013268:Stimulation, Chemical; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D000969:Antinematodal Agents; C093065:KPNFIRFamide; D009479:Neuropeptides; D009842:Oligopeptides; D005680:gamma-Aminobutyric Acid; D007559:Ivermectin