Nematode neuropeptide modulation of the vagina vera of Ascaris suum: in vitro effects of PF1, PF2, PF4, AF3 and AF4

Ascaris suum possesses a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8/PF3) have been shown to modulate the intrinsic, rhythmic activity of the vagina vera of A. suum in vitro. In the present study, the effects of the nematode FaRPs, SDPNFLRFamide (PF1), SADPNFLREamide (PF2) and KPNFIRFamide (PF4) (from Panagrellus redivivus) and AVPGVLRFamide (AF3) and GDVPGVLRFamide (AF4) (from A. suum) on the in vitro activity of the vagina vera were examined. The effects of each of the peptides were qualitatively and quantitatively distinct. All 3 FaRPs from P. redivivus were inhibitory, causing a cessation of contractions. PF2 was 3 times more potent than PF1, with a threshold of 1 nM. Although PF4 was the least potent (threshold, 10 nM), its effects at greater than or equal to 10 nM were quantitatively the greatest. Both AF3 and AF4 (1 mu M) induced complex, multiphasic responses consisting of an initial contraction and spastic paralysis followed by a return of contractile activity of increased amplitude. AF3 was 3 times more potent than AF4. The effects of these peptides had some similarities to those observed on A. suum somatic body wall muscle in vitro, with PF1, PF2 and PF4 being inhibitory and AF3 and AF4 being excitatory.

KSAYMRFamide (PF3/AF8) is present in the free-living nematode, Caenorhabditis elegans

To date, seven FMRFamide-related peptides (FaRPs) have been structurally characterized from C. elegans, of which one is structurally identical to the parasitic nematode peptide AF2 (KHEYLRFamide). The other six FaRPs have so far been identified in free-living forms only. in the present study an additional FaRP was isolated and structurally characterized from an ethanolic extract of C. elegans. The extract was screened using a C-terminally directed FaRP antiserum, and the FMRFamide-immunoreactive peptide purified to homogeneity using HPLC. Approximately 80 pmol of the peptide was subjected to Edman degradation and the unequivocal primary structure of the K-7-amide, KSAYMRFamide (PF3/AF8) was determined following a single gas-phase sequencing run. The molecular mass of the peptide was determined using a MALDI-TOF mass spectrometer and was found to be 919 (MH+), which is in agreement with the theoretical mass of C-terminally amidated PF3. A new flp-gene, designated flp-6, has recently been identified which encodes six copies of KSAYMRFamide (PF3/AF8). (C) 1998 Academic Press.

Pharmacological effects of nematode FMRFamide-related peptides (FaRPs) on muscle contractility of the trematode, Fasciola hepatica

The physiological effects of synthetic replicates of the nematode FaRPs, AF1 (KNEFIRFamide), AF2 (KHEYLRFamide), PF1 (SDPNFLRFamide), PF2 (SADPNFLRFamide), AF8/PF3 (KSAYMRFamide) and PF4 (KPNFIRFamide) were examined on muscle preparations of the liver fluke, Fasciola hepatica. Changes in contractility following the addition of the test compound were recorded using a photo-optic transducer system. Unlike the varied effects these peptides have on nematode somatic musculature, all were found to induce excitatory responses in the muscle activity of F. hepatica. While qualitative effects of the nematode peptides were similar in that they induced increases in both the amplitude and frequency of F. hepatica muscle contractions, they varied considerably in the potency of their excitatory effects. The threshold activity for each peptide was as follows: 10 mu M, PF1 and PF2; 3 mu M, AF1 and PF3; 1 mu M, AF2; and 30 nM, PF4. The results demonstrate, for the first time, the cross-phyla activity of nematode neuropeptides on the neuromuscular activity of a trematode.

Importance of the proline residue to the functional activity and metabolic stability of the nematode FMRFamide-related peptide, KPNFIRFamide (PF4)

PF4 has previously been shown to have potent inhibitory effects on myoactivity of somatic muscle strips from the nematode, Ascaris suum. This study examined the bioactivity and metabolic stability of position 2- and position 5-modified analogues of PF4. Although the analogues [Leu(5)] PF4, [Ala(2)]PF4, [Gly(2)]PF4, [Ala(2),Leu(5)]PF4, and [Gly(2),Leu(5)]PF4 all had qualitatively similar inhibitory effects on A. suum somatic muscle strips, their effects were quantitatively distinguishable and had the order of potency: PF4 = [Leu(5)] PF4 >> [Ala(2)]PF4 = [Ala(2),Leu(5)] PF4 >> [Gly(2)] PF4 = [Gly(2),Leu(5)] PF4. Leu(5) for Ile(5) substitutions in PF4 did not alter the activity of this peptide; however, Gly(2)/Ala(2) for Pro(2) substitutions reduced, but did not abolish, peptide activity. Peptide stability studies revealed that [Gly(2)]PF4(2-7) and -(3-7) and [Ala(2)]PF4(2-7), -(3-7), and -(4-7) fragments were generated following exposure to A. suum somatic muscle strips. However, the parent peptide (PF4) was not metabolized and appeared to be resistant to the sequential cleavages of native aminopeptidases. Observed analogue metabolism appeared to be due to the activity of released aminopeptidases as identical fragments were generated by incubation in medium that had been exposed to somatic muscle strips and from which the strips had been removed prior to peptide addition. It was found that the muscle stretching and bath mixing characteristics of the tension assay led to more effective release of soluble enzymes from muscle strips and thus greater peptide degradation. These studies reveal that Pro(2) in PF4 is not essential for the biological activity of this peptide; however, it does render the peptide resistant to the actions of native nematode aminopeptidases. Copyright (C) 1996 Elsevier Science Inc.

The pharmacology of nematode FMRFamide-related peptides

FMRFamide-related peptides (FaRPs) are the largest known family of invertebrate neuropeptides. Immunocytochemical screens of nematode tissues using antisera raised to these peptides have localized extensive FaRP-immunostaining to their nervous systems. Although 21 FaRPs have been isolated and sequenced from extracts of free-living and parasitic nematodes, available evidence indicates that other FaRPs await discovery. While our knowledge of the pharmacology of these native nematode neuropeptides is extremely limited, reports on their physiological activity in nematodes are ever increasing. All the nematode FaRPs examined so far have been found to have potent and varied actions on nematode neuromuscular activity. It is only through the extensive pharmacological and physiological assessment of the tissue, cell and receptor interactions of these peptidic messengers that an understanding of their activity on nematode neuromusculature will be possible. In this review, Aaron Maule and colleagues examine the current understanding of the pharmacology of nematode FaRPs.

ISOLATION AND PRELIMINARY BIOLOGICAL CHARACTERIZATION OF KPNFIRFAMIDE, A NOVEL FMRFAMIDE-RELATED PEPTIDE FROM THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS

A novel FMRFamide-related heptapeptide, Lys-Pro-Asn-Phe-Ile-Arg-Phe-NH2 (KPNFIRFamide), was isolated and characterized from acid ethanol extracts of the free-living nematode, Panagrellus redivivus. Whole-worm extracts contained greater than or equal to 9 pmol KPNFIRFamide/g wet weight. A synthetic replicate of this peptide induced a rapid relaxation of tone and inhibited spontaneous contractility in isolated innervated and denervated body-wall muscle strips of the parasitic nematode, Ascaris suum. KPNFIRFamide (0.1 nM) induced measurable relaxations in 50% of the muscle preparations examined. Concentrations greater than or equal to 0.3 nM induced relaxation in 100% of muscle preparations examined. The relaxation was short-lived at concentrations of peptide greater than or equal to 1 mu M and displayed a profile typical of receptor desensitization. These data suggest the occurrence of a closely related peptide in A. suum and add further evidence to the concept of primary structural conservation of FaRPs within the nematodes.

THE FMRFAMIDE-LIKE NEUROPEPTIDE AF2 (ASCARIS-SUUM) IS PRESENT IN THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS (NEMATODA, RHABDITIDA)

Available primary structural information suggests that the FMRFamide-related peptides (FaRPs) from parasitic and free-living nematodes are different, and that free-living forms may not represent appropriate models for the study of the neurochemistry of parasitic forms in the laboratory. However, here we report the isolation and unequivocal identification of AF2 (originally isolated from the parasite, Ascaris suum) from acidified alcoholic extracts of the free-living species, Panagrellus redivivus. While reverse-phase HPLC analysis of extracts revealed FMRFamide-immunoreactivity to be highly heterogeneous, AF2 was the predominant FMRFamide-immunoreactive peptide present (at least 26 pmol/g wet weight of worms). This peptide was also the major immunoreactant identified by an antiserum raised to the conserved C-terminal hexapeptide amide of mammalian pancreatic polypeptide (PP), which has been used previously to isolate neuropeptide F (NPF). These observations were confirmed by radioimmunoassay and chromatographic fractionation of an acidified alcoholic extract of A. suum heads. The FMRFamide-related peptides present in a nematode extract may be highly dependent on the extraction medium employed, and these data would suggest that this complement of neuropeptides may not be as different between parasitic and free-living nematodes as initial studies have suggested. Finally, all of the evidence suggests that NPF is not present in nematodes and that the PP-immunoreactant previously demonstrated immunochemically is probably AF2.

KSAYMRFAMIDE - A NOVEL FMRFAMIDE-RELATED HEPTAPEPTIDE FROM THE FREE-LIVING NEMATODE, PANAGRELLUS-REDIVIVUS, WHICH IS MYOACTIVE IN THE PARASITIC NEMATODE, ASCARIS-SUUM

In nematodes, FMRFamide-related peptides (FaRPs) have been structurally characterised from the parasite, Ascaris suum, and from two free-living species, Panagrellus redivivus and Caenorhabditis elegans. While both FaRPs isolated from P. redivivus (PF1 and PF2) have been identified in C. elegans the two heptapeptides isolated from A. suum (AF1 and AF2) have until recently been considered unique to this parasitic species. We have recently isolated AF2 from P. redivivus and, during this study, an additional novel heptapeptide amide, Lys-Ser-Ala-Tyr-Met-Arg-Phe amide (KSAYMRFamide), was structurally characterised. A synthetic replicate of this peptide induced a rapid concentration-dependent muscle tension increase in an isolated A. suum somatic muscle preparation, with a threshold of approximately 0.1 mu M. These data suggest that the complement of FaRPs in parasitic and free-living nematodes may not be as radically different as preliminary studies would suggest, and that the absence of AF1, AF2 and KSAYMRFamide on the C. elegans FMRFamide-related peptide gene (flp-1) may imply the presence of at least two different FaRP genes in nematodes. (C) 1994 Academic Press, Inc.