A fish bradykinin (Arg0; Trp5; Leu8-bradykinin) from the defensive skin secretion of the European edible frog; Pelophylax kl. esculentus: structural characterization; molecular cloning of skin kininogen cDNA and pharmacological effects on mammalian smooth muscle

Extensive studies on bradykinin-related peptides (BRPs) generated from plasma kininogens in representative species of various vertebrate taxa, have confirmed that many amphibian skin BRPs reflect those present in putative vertebrate predators. For example, the (Val1, Thr6)-bradykinin, present in the defensive skin secretions of many ranids and phyllomedusines, can be generated from plasma kininogens in colubrid snakes - common predators of these frogs. Here, we report the presence of (Arg0, Trp5, Leu8)-bradykinin in the skin secretion of the European edible frog, Pelophylax kl. esculentus, and have found it to be encoded in single copy by a kininogen with an open-reading frame of 68 amino acid residues. This peptide is the archetypal bony fish bradykinin that has been generated from plasma kininogens of the bowfin (Amia calva), the long-nosed gar (Lepisosteus oseus) and the rainbow trout (Onchorhynchus mykiss). More recently, this peptide has been shown to be encoded within cloned kininogens of the Atlantic cod (Gadus morhua) spotted wolf-fish (Anarichas minor), zebrafish (Danio rerio), pufferfish (Tetraodon nigroviridis) and Northern pike (Esox lucius). The latter species is regarded as a major predator of P. kl. esculentus. Synthetic (Arg0, Trp5, Leu8)-bradykinin was previously reported as having multiphasic effects on arterial blood pressure in conscious trout and here we have demonstrated that it can antagonize the relaxation in rat arterial smooth muscle induced by canonical mammalian bradykinin. The discovery of (Arg0, Trp5, Leu8)-bradykinin in the defensive skin secretion of this amphibian completes the spectrum of vertebrate taxon-specific BRPs identified from this source.

FMRFamide-related peptides (FaRPs) in helminth parasites

Neuropeptides are ubiquitous intercellular signalling molecules in all Metazoa with nervous systems. Research over the past 10 years has confirmed through immunocytochemistry that neuropeptides are widespread and abundant in the nervous systems of helminth parasites. Biochemical isolation and characterisation studies have indentified the primary structures of numerous structurally-related peptides in helminths, the best studied being the FMRFamide-related peptides (FaRPs). While to date only four FaRPs have been identified from platyhelminths, some 60 FaRPs or FaRP-like peptides have been isolated or predicted for nematodes. Preliminary physiological studies have shown that FaRPs are strongly myoactive, but with quire different actions in the two groups of helminth parasite. The absence of FaRPs from vertebrates suggests compounds with a high affinity for FaRP receptors are likely to have selective effects against helminths and, if protected from degradation, could have therapeutic potential.

Modulation of the motility of the vagina vera of Ascaris suum in vitro by FMRFamide-related peptides

Ascaris suum contains a large number of FMRFamide-related peptides (FaRPs) of which KNEFIRFamide (AF1), KHEYLRFamide (AF2) and KSAYMRFamide (AF8, also called PF3) have been extensively studied and are known to exert actions on somatic muscle strips of the worm. In the present study, the effects of AF1, AF2 and AF8 on the activity of the vagina vera of female A. suum have been examined in vitro. The vagina vera is a muscular tube connecting the uterus and vagina uteri to the gonopore and is probably involved in regulating egg output. The tissue exhibited spontaneous, rhythmic contractions in vitro, which were modulated by each of the FaRPs tested. The effects of each of the peptides were qualitatively and quantitatively different, and in each case were reversible. AF1 (1 mu M) caused a biphasic response in the form of a transient lengthening of the preparation, followed by a shortening; contractions were initially inhibited but resumed 5 min post-addition of the peptide. Lower concentrations (less than or equal to 0.1 mu M) induced a less marked effect, with rhythmic contractions returning 5 min post-addition. AF2 and AF8 reduced contraction frequency at concentrations greater than or equal to 0.1 mu M. Both peptides also caused the tissue to shorten, although the effects of AF8 on baseline tension were inconsistent. The apparent potencies of AF1 and AF8 on contraction frequency of the vagina vera were 10-fold greater than AF2 and, unlike their actions on A. suum somatic body wall muscles, the actions of AF1 and AF2 were qualitatively different. Indeed, the effects of each of these FaRPs on the vagina vera were markedly different from those observed on the somatic muscle.

Structure-activity relationships of FMRFamide-related peptides contracting Schistosoma mansoni muscle

This study reports the potent myoactivity of flatworm FMRFamide-related peptides (FaRPs) on isolated muscle fibers of the human blood fluke, Schistosoma mansoni. The turbellarian peptides YIRFamide (EC50 4 eta M), GYIRFamide (EC50 1 eta M). and RYIRFamide (EC50 7 eta M), all induced muscle contraction more potently than the cestode FaRP GNFFRFamide (EC50 500 eta M). Using a series of synthetic analogs of the flatworm peptides YIRFamide, GYIRFamide and RYIRFamide, the structure-activity relationships of the muscle FaRP receptor were examined. With a few exceptions, each residue in YIRFamide is important in the maintenance of its myoactivity. Alanine scans resulted in peptides that were inactive (Ala(1), Ala(2), Ala(3) and Ala(4) YIRFamide; Ala(4) and Ala(5) RYIRFamide) or had much reduced potencies (Ala(1), Ala(2) and Ala(3) RYIRFamide). Substitution of the N-terminal (Tyr(1)) residue of YIRFamide with the non-aromatic residues Thr or Arg produced analogs with greatly reduced potency. Replacement of the N-terminal Tyr with aromatic amino acids resulted in myoactive peptides (FIRFamide, EC50 100 eta M; WIRFamide, EC50 0.5 eta M). The activity of YIRFamide analogs which possessed a Leu(2), Phe(2) or Met(2) residue (EC50's 10, 1 and 3 eta M, respectively) instead of Ile(2) was not significantly altered, whereas, YVRFamide had a greatly reduced (EC50 200 eta M) activity. Replacement of the Phe(4) with a Tyr(4) (YIRYamide) also greatly lowered potency. Truncated analogs were either inactive (FRFamide, YRFamide, HRFamide, RFamide, Famide) or had very low potency (IRFamide and MRFamide), with the exception of nLRFamide (EC50 20 eta M). YIRF free acid was inactive. In summary, these data show the general structural requirements of this schistosome muscle FaRP receptor to be similar, but not identical, to those of previously characterized molluscan FaRP receptors. (C) 1997 Elsevier Science Inc.

Physiological effects of platyhelminth FMRFamide-related peptides (FaRPs) on the motility of the monogenean Diclidophora merlangi

The actions of known platyhelminth FaRPs on the contractility of whole-worm preparations of the monogenean, Diclidophora merlangi have been examined in vitro for the first time. All of the peptides tested had excitatory effects on the motor activity of the worm. The order of potency for the peptides tested was: YIRFamide > GYIRFamide = RYIRFamide > GNFFRFamide = FLRFamide. However, although YIRFamide was more potent than GYIRFamide, the latter was the most efficacious on each of the motility parameters (tension, contraction amplitude and contraction frequency) examined at concentrations greater than or equal to 0.1 mu M. Serotonin, which stimulates contractility in the worm was used as a positive control. The excitatory activity of turbellarian and cestode neuropeptides on a monogenean indicates at least some structural similarities in the neuropeptide receptors of these classes of flatworm.

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.

FMRFamide-related peptides (FaRPs) in nematodes: Occurrence and neuromuscular physiology

The occurrence of classical neurotransmitter molecules and numerous peptidic messenger molecules in nematode nervous systems indicate that although structurally simple, nematode nervous systems are chemically complex. Thus far, studies on one nematode neuropeptide family, namely the FMRFamide-related peptides (FaRPs), have revealed an unexpected variety of neuropeptide structures in both free-living and parasitic species. To date 23 nematode FaRPs have been structurally characterized including 12 from Ascaris suum, 8 from Caenorhabditis elegans, 5 from Panagrellus redivivus and 1 from Haemonchus contortus. Ten FaRP-encoding genes have been identified in Caenorhabditis elegans. However, the full complement of nematode neuronal messengers has yet to be described and unidentified nematode FaRPs await detection. Preliminary characterization of the actions of nematode neuropeptides on the somatic musculature and neurones of A. suum has revealed that these peptidic messengers have potent and complex effects. Identified complexities include the biphasic effects of KNEFIRFamide/KHEYLRFamide (AF1/2; relaxation of tone followed by oscillatory contractile activity) and KPNFIRFamide (PF4; rapid relaxation of tone followed by an increase in tone), the diverse actions of KSAYMRFamide (AF8 or PF3; relaxes dorsal muscles and contracts ventral muscles) and the apparent coupling of the relaxatory effects of SDPNFLRFamide/SADPNFLRFamide (PF1/PF2) to nitric oxide release. Indeed, all of the nematode FaRPs which have been tested on somatic muscle strips of A. suum have actions which are clearly physiologically distinguishable. Although we are a very long way from understanding how the actions of these peptides are co-ordinated, not only with those of each other but also with those of the classical transmitter molecules, to control nematode behaviour, their abundance coupled with their diversity of structure and function indicates a hitherto unidentified sophistication to nematode neuromuscular intergration.

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.

Platyhelminth FMRFamide-related peptides

Platyhelminths are the most primitive metazoan phylum to possess a true central nervous system, comprising a brain and longitudinal nerve cords connected by commissures. Additional to the presence of classical neurotransmitters, the nervous systems of all major groups of flatworms examined have widespread and abundant peptidergic components, Decades of research on the major invertebrate phyla, Mollusca and Arthropoda, have revealed the primary structures and putative functions of several families of structurally related peptides, the best studied being the FMRFamide-related peptides (FaRPs). Recently, the first platyhelminth FaRP was isolated from the tapeworm, Moniezia expansa, and was found to be a hexapeptide amide, GNFFRFamide. Two additional PaRPs were isolated from species of turbellarians; these were pentapeptides, RYIRFamide (Artioposthia triangulata) and GYIRFamide (Dugesia tigrina). The primary structure of a monogenean or digenean FaRP has yet to be deduced. Preliminary physiological studies have shown that both of the turbellarian FaRPs elicit dose-dependent contractions of isolated digenean and turbellarian somatic muscle fibres. Unlike the high structural diversity of FaRPs found in molluscs, arthropods and nematodes, the complement of FaRPs in individual species of platyhelminths appears to be restricted to 1 or 2 related molecules. Much remains to be learnt about platyhelminth PaRPs, particularly from peptide isolation, molecular cloning of precursor proteins, receptor localization, and physiological studies. Copyright (C) 1996 Australian Society for Parasitology.

PLATYHELMINTH FMRFAMIDE-RELATED PEPTIDES (FARPS) CONTRACT SCHISTOSOMA-MANSONI (TREMATODA, DIGENEA) MUSCLE-FIBERS IN-VITRO

Molluscan FMRFamide and two recently discovered platyhelminth FMRFamide-related peptides (FaRPs), GNFFRFamide from the cestode Moniezia expansa and RYIRFamide from the terrestrial turbellarian Artioposthia triangulata, cause dose-dependent contractions of individual muscle fibres from Schistosoma mansoni in vitro. The most potent FaRP tested was the turbellarian peptide RYIRFamide, which produced a concentration-dependent effect between 10(-9) and 10(-7) M. FMRFamide and GNFFRFamide were less potent, inducing contractions between 10(-8)-10(-6) M and 10(-7)-10(-5) M respectively. The contractile effect of each of these peptides was blocked by the presence of 1 mu M FMR-D-Famide. FMRF free acid did not elicit contraction of the muscle fibres. The FaRP-induced contractions did not occur if the Ca2+ was omitted and 0.5 mu M EGTA. was added to the extracellular medium. The FaRP-induced contractions were not blocked by the Ca2+ channel blockers nicardipine, verapamil or diltiazem, although high Kf-induced contractions of these fibres were blocked by nicardipine. These data indicate the presence of FaRP receptors on schistosome muscle fibres and demonstrate their ability to mediate muscle contraction. The action of these endogenous flatworm peptides on schistosome muscle is the first demonstration of a direct excitatory effect of any putative neurotransmitter on the muscle of a flatworm, and establishes a role for FaRPs in neuromuscular transmission in trematodes. In addition, it provides the first evidence that the peptidergic nervous system is a rational target for chemotherapeutic attack in parasitic platyhelmiths.

PRIMARY STRUCTURES AND BIOLOGICAL-ACTIVITIES OF SUBSTANCE-P-RELATED PEPTIDES FROM THE BRAIN OF THE DOGFISH, SCYLIORHINUS-CANICULA

Two peptides with substance-P-like immunoreactivity were isolated in pure form from an extract of the brain of the elasmobranch fish, Scyliorhinus canicula (european common dogfish). One peptide was identical to scyliorhinin I, previously identified in dogfish intestine, and the second was the undecapeptide Lys-Pro-Arg-Pro-Gly-Gln-Phe-Phe-Gly-Leu-Met-CONH2 which is structurally similar to mammalian substance P Scyliorhinin II or a peptide analogous to mammalian neurokinin A were not detected in the extract. Synthetic dogfish substance P ([Lys1, Arg3, Gly5]substance P) was approximately threefold more potent than mammalian substance P (K(d) = 0.21 +/- 0.11 nM versus K(d)= 0.74 +/- 0.17 nM; mean +/- SD; n = 6) in inhibiting the binding of I-125-labelled substance P to neurokinin (NK1) receptors in rat submandibular gland membranes. The vasodilator action of tachykinins in mammals is mediated primarily through interaction with NK1 receptors. Bolus intravenous injections of [Lys1, Arg3, Gly5]substance P (100 pmol) and scyliorhinin I (100 pmol) produced appreciable (>4 kPa) decreases in arterial blood pressure in the rat whereas intravenous injections of up to 5 nmol of the peptides into conscious, unrestrained dogfish produced no change in arterial blood pressure, pulse amplitude or heart rate. Injections of greater amounts of the peptides (10-50 nmol) produced a slight increase (400-667 Pa) in blood pressure. The data indicate that mammalian-type NK1 tachykinin receptors are not involved in cardiovascular regulation in elasmobranch fish.

Ranakinestatin-PPF from the Skin Secretion of the Fukien Gold-Striped Pond Frog, Pelophylax plancyi fukienensis: A Prototype of a Novel Class of Bradykinin B2 Receptor Antagonist Peptide from Ranid Frogs

The defensive skin secretions of many amphibians are a rich source of bradykinins and bradykinin-related peptides (BRPs). Members of this peptide group are also common components of reptile and arthropod venoms due to their multiple biological functions that include induction of pain, effects on many smooth muscle types, and lowering systemic blood pressure. While most BRPs are bradykinin receptor agonists, some have curiously been found to be exquisite antagonists, such as the maximakinin gene-related peptide, kinestatin—a specific bradykinin B₂-receptor antagonist from the skin of the giant fire-bellied toad, Bombina maxima. Here, we describe the identification, structural and functional characterization of a heptadecapeptide (DYTIRTRLHQGLSRKIV), named ranakinestatin-PPF, from the skin of the Chinese ranid frog, Pelophylax plancyi fukienensis, representing a prototype of a novel class of bradykinin B₂-receptor specific antagonist. Using a preconstricted preparation of rat tail arterial smooth muscle, a single dose of 10⁻⁶ M of the peptide effectively inhibited the dose-dependent relaxation effect of bradykinin between 10⁻¹¹ M and 10⁻⁵ M and subsequently, this effect was pharmacologically-characterized using specific bradykinin B₁- (desArg-HOE140) and B₂-receptor (HOE140) antagonists; the data from which demonstrated that the antagonism of the novel peptide was mediated through B₂-receptors. Ranakinestatin—PPF—thus represents a prototype of an amphibian skin peptide family that functions as a bradykinin B₂-receptor antagonist herein demonstrated using mammalian vascular smooth muscle.

Evaluation of multiple reaction monitoring cubed for the analysis of tachykinin related peptides in rat spinal cord using a hybrid triple quadrupole–linear ion trap mass spectrometer

Targeted peptide methods generally use HPLC-MS/MRM approaches. Although dependent on the instrumental resolution, interferences may occur while performing analysis of complex biological matrices. HPLC-MS/MRM3 is a technique, which provides a significantly better selectivity, compared with HPLC-MS/MRM assay. HPLC-MS/MRM3 allows the detection and quantitation by enriching standard MRM with secondary product ions that are generated within the linear ion trap. Substance P (SP) and neurokinin A (NKA) are tachykinin peptides playing a central role in pain transmission. The objective of this study was to verify whether HPLC-HPLCMS/ MRM3 could provide significant advantages over a more traditional HPLC-MS/MRM assay for the quantification of SP and NKA in rat spinal cord. The results suggest that reconstructed MRM3 chromatograms display significant improvements with the nearly complete elimination of interfering peaks but the sensitivity (i.e. signal-to-noise ratio) was severely reduced. The precision (%CV) observed was between 3.5% - 24.1% using HPLC-MS/MRM and in the range of 4.3% - 13.1% with HPLC-MS/MRM3, for SP and NKA. The observed accuracy was within 10% of the theoretical concentrations tested. HPLC-MS/MRM3 may improve the assay sensitivity to detect difference between samples by reducing significantly the potential of interferences and therefore reduce instrumental errors.