THE ACTION OF SEROTONIN AND THE NEMATODE NEUROPEPTIDE KSAYMRFAMIDE ON THE PHARYNGEAL MUSCLE OF THE PARASITIC NEMATODE, ASCARIS-SUUM

The pharyngeal component of the enteric nervous system of the parasitic nematode, Ascaris suum exhibits immunoreactivity for serotonin (5-hydroxytryptamine or 5-HT) and for FMRFamide-like peptides. This paper describes the application of an in vitro pharmacological approach to investigate the functional role of 5-HT and FMRFamide-like peptides. The pharyngeal pumping behaviour of Ascaris suum was monitored using a modified pressure transducer system which measures pharyngeal pressure changes and therefore pumping. The pharynx did not contract spontaneously; however, 5-HT (10-1000 mu M) stimulated pumping at a frequency of 0 . 5 Hz. FMRFamide had no apparent effect on pharyngeal pumping. The native nematode FMRFamide-related peptide (FaRP), KSAYMRFamide inhibited the pumping elicited by 5-HT. The duration of inhibition was dose-dependent (0 . 1-1000 nM) with a threshold of 0 . 1 nM. In 4 preparations, the inhibition of the pharyngeal muscle was preceded by an initial excitation and increase in the amplitude of pharyngeal pressure changes. The pharynx is involved in various nematode processes, including feeding, regulation of hydrostatic pressure and excretion. The role of 5-HT and KSAYMRFamide in the pharyngeal function of nematodes is discussed.

ULTRASTRUCTURAL-LOCALIZATION OF PANCREATIC POLYPEPTIDE AND FMRFAMIDE IMMUNOREACTIVITIES WITHIN THE CENTRAL-NERVOUS-SYSTEM OF THE NEMATODE, ASCARIS-SUUM (NEMATODA, ASCAROIDEA)

A post-embedding immunogold technique has been used to examine the subcellular distribution of immunoreactivities to vertebrate pancreatic polypeptide (PP) and to the invertebrate peptide, FMRFamide within the central nervous system (CNS) of the nematode, Ascaris suum. Gold labelling of peptide was localized exclusively over dense-cored vesicles within nerve cell bodies, nerve axons and nerve terminals of the main ganglia and nerve cords in the CNS. Double-labelling of peptides demonstrated an apparent co-localization of PP and FMRFamide immunoreactivities in the same dense-cored vesicles, although populations of dense-cored vesicles that labelled solely for FMRFamide were also evident. Antigen preabsorption studies indicated little or no cross-reactivity between the two antisera.

CHROMATOGRAPHIC AND IMMUNOLOGICAL CHARACTERIZATION OF NEUROPEPTIDE Y-LIKE AND PANCREATIC POLYPEPTIDE-LIKE PEPTIDES FROM THE NEMATODE ASCARIS-SUUM

1. Immunoreactivity (IR) towards neuropeptide Y (NPY) and pancreatic polypeptide (PP) has previously been demonstrated in nematodes by immunocytochemistry (ICC).

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.