Comparative analysis of the distribution of bradykinin-, GYIRFamide- and neuropeptide F-like immunoreactivities in the monogenean, Diclidophora merlangi

An indirect immunocytochemical technique combined with confocal scanning laser microscopy has been used to demonstrate immunoreactivities to the nonapeptide, RPPGFSPFR (bradykinin, BK) and the endogenous flatworm regulatory peptide, GYIRFamide in the nervous system of the monogenean, Diclidophora merlangi. In addition, a simultaneous double-labelling technique was employed to examine possible co-localization of GYIRFamide- and neuropeptide F (NPF) immunoreactivities, using antisera to the C-terminal nonapeptide-amide of NPF (Moniezia expansa, FAIIGRPRF.NH2). BK immunostaining was restricted to a small population of nerve cells and associated fibres within the Ventral nerve cords and to 2 pairs of nerve cells innervating the cirrus and the pharynx, respectively. No immunopositive nerve cells and fibres were identified within the brain or in association with the female reproductive apparatus. In contrast, GYIRFamide staining was abundant throughout the central and peripheral nervous systems, and appeared similar to the staining pattern revealed using an FMRFamide antiserum. GYIRFamide immunoreactivity was localized to nerve cells and fibres within the paired cerebral ganglia and the longitudinal ventral, dorsal and lateral nerve cords and their numerous interconnecting transverse commissures. The plexuses of the buccal suckers, pharynx and clamps of the haptor were strongly immunopositive for GYIRFamide, as were nerve cells innervating the ootype, the oviduct and the vitelline reservoir of the reproductive apparatus. Double-labelling experiments indicated an apparent co-localization of GYIRFamide and NPF immunoreactivities.

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.

Physiological effects of platyhelminth RFamide peptides on muscle-strip preparations of Fasciola hepatica (Trematoda: Digenea)

The effects of each of the known platyhelminth neuropeptides were determined on muscle-strip preparations from the liver fluke, Fasciola hepatica. The activity of synthetic replicates of the C-terminal nonapeptide of neuropeptide F (NPF9, Moniezia expansa), and the FMRFamide-related peptides (FaRPs), GNFFRFamide, RYIRFamide, GYIRFamide and YIRFamide, were examined. Muscle-strip activity was recorded from 1 mm segments of muscle prepared from 28 to 32-day-old worms, using a photo-optic transducer system. None of the peptides (less than or equal to 10 mu M) altered baseline tension significantly; however, each of the peptides increased the amplitude and frequency of muscle contraction. The threshold for activity of each of the peptides examined was, respectively, 1 nM (RYIRFamide), 0.3 mu M (GYIRFamide and YIRFamide), and 10 mu M (GNFFRFamide and NPF9). All of the effects were reversible and repeatable, following wash-out. Muscle-strip integrity was tested following experimentation, using arecoline (10 mu M) and high-K+ bathing medium (90 mM K+).

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.

Histamine release from human bronchoalveolar lavage mast cells by neurokinin A and bradykinin

Objective and Design: This study examined whether bradykinin and neurokinin A activate human pulmonary mast cells retrieved by bronchoalveolar lavage (BAL).

LOCALIZATION, QUANTITATION, AND CHARACTERIZATION OF NEUROPEPTIDE-F-IMMUNOREACTIVE AND FMRFAMIDE-IMMUNOREACTIVE PEPTIDES IN TURBELLARIANS AND A MONOGENEAN - A COMPARATIVE-STUDY

Over the past decade it has become clear that the nervous systems of platyhelminths are both complex and highly developed, particularly in peptidergic elements. The central position of an ancestral flatworm in the evolution of the Bilateria has placed a greater importance on the study of modern flatworms. Using antisera generated to the C-terminal region of platyhelminth neuropeptide F and the molluscan neuropeptide, FMRFamide, in immunocytochemistry at both Light and ultrastructural levels, immunoreactivities have been localised within the nervous systems of three species of triclad turbellarians, Dugesia lugubris, Dendrocoelum lacteum, and Polycelis nigra, and one species of monogenean trematode, Diclidophora merlangi. Extensive immunostaining was obtained with both antisera throughout the central and peripheral nervous systems of all species studied, but intensity and abundance was significantly greater in the turbellarians. Indirect electron-immunogold labeling demonstrated that immunoreactivity to both neuropeptides was often colocalised in neurosecretory vesicles, although discrete populations of vesicles were also observed. Radioimmunoassay of extracts of all species confirmed that neuropeptide F immunoreactivity was consistently more abundant than FMRFamide immunoreactivity, and that the levels of both in the three turbellarians were several orders of magnitude greater than those found in the monogenean. Chromatographic analyses of turbellarian extracts revealed that neuropeptide F and FMRFamide immunoreactivities were attributable to different peptides. These data imply that the neuropeptidergic systems of turbellarians are considerably more extensive than those of monogeneans, and would suggest that a regression has occurred in the latter as a consequence of the adoption of a more sedentary parasitic lifestyle. (C) 1995 Wiley-Liss, Inc.

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.

PEPTIDES RELATED TO THE DIPLOPTERA-PUNCTATA ALLATOSTATINS IN NONARTHROPOD INVERTEBRATES - AN IMMUNOCYTOCHEMICAL SURVEY

The allatostatins are a family of peptides isolated originally from the cockroach, Diploptera punctata. Related peptides have been identified in Periplaneta americana and the blowfly, Calliphora vomitoria. These peptides have been shown to be potent inhibitors of juvenile hormone synthesis in these species. A peptide inhibitor of juvenile hormone biosynthesis has also been isolated from the moth, Manduca sexta; however, this peptide has no structural homology with the D. punctata-type allatostatins. Investigations of the phylogeny of the D. punctata allatostatin peptide family have been started by examining a number of nonarthropod invertebrates for the presence of allatostatin-like molecules using immunocytochemistry with antisera directed against the conserved C-terminal region of this family. Allatostatin-like immunoreactivity (ALIR) was demonstrated in the nervous systems of Hydra oligactis (Hydrozoa), Moniezia expansa (Cestoda), Schistosoma mansoni (Trematoda), Artioposthia triangulata (Turbellaria), Ascaris suum (Nematoda), Lumbricus terrestris (Oligochaeta), Limax pseudoflavus (Gastropoda), and Eledone cirrhosa (Cephalopoda). ALIR could not be demonstrated in Ciona intestinalis (Ascidiacea). These results suggest that molecules related to the allatostatins may play an important role in nervous system function in many invertebrates as well as in insects and that they also have an ancient evolutionary lineage. (C) 1994 Wiley-Liss, Inc.

Antimicrobial/cytolytic peptides from the venom of the North African scorpion, Androctonus amoreuxi: Biochemical and functional characterization of natural peptides and a single site-substituted analog

The venoms of scorpions are complex cocktails of polypeptide toxins that fall into two structural categories: those that contain cysteinyl residues with associated disulfide bridges and those that do not. As the majority of lethal toxins acting upon ion channels fall into the first category, most research has been focused there. Here we report the identification and structural characterization of two novel 18-mer antimicrobial peptides from the venom of the North African scorpion, Androctonus amoreuxi. Named AamAP1 and AamAP2, both peptides are C-terminally amidated and differ in primary structure at just two sites: Leu?Pro at position 2 and Phe?Ile at position 17. Synthetic replicates of both peptides exhibited a broad-spectrum of antimicrobial activity against a Gram-positive bacterium (Staphylococcus aureus), a Gram-negative bacterium (Escherichia coli) and a yeast (Candida albicans), at concentrations ranging between 20µM and 150µM. In this concentration range, both peptides produced significant degrees of hemolysis. A synthetic replicate of AamAP1 containing a single substitution (His?Lys) at position 8, generated a peptide (AamAP-S1) with enhanced antimicrobial potency (3-5µM) against the three test organisms and within this concentration range, hemolytic effects were negligible. In addition, this His?Lys variant exhibited potent growth inhibitory activity (ID(50) 25-40µm) against several human cancer cell lines and endothelial cells that was absent in both natural peptides. Natural bioactive peptide libraries, such as those that occur in scorpion venoms, thus constitute a unique source of novel lead compounds with drug development potential whose biological properties can be readily manipulated by simple synthetic chemical means.