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Distribution of FMRFamide-like immunoreactivity in the brain, retina and nervus terminalis of the sockeye salmon parr, Oncorhynchus nerka.

Neurons displaying FMRFamide(Phe - Met - Arg - Phe - NH2)-like immunoreactivity have recently been implicated in neural plasticity in salmon. We now extend these findings by describing the extent of the FMRF-like immunoreactive (FMRF-IR) system in the brain, retina and olfactory system of sockeye salmon parr using the indirect peroxidase anti-peroxidase technique. FMRF-IR perikarya were found in the periventricular hypothalamus, mesencephalic laminar nucleus, nucleus nervi terminalis and retina (presumed amacrine cells), and along the olfactory nerves. FMRF-IR fibers were distributed throughout the brain with highest densities in the ventral area of the telencephalon, in the medial forebrain bundle, and at the borders between layers III/IV and IV/V in the optic tectum. High densities of immunoreactive fibers were also observed in the area around the torus semicircularis, in the medial hypothalamus, median raphe, ventromedial tegmentum, and central gray. In the retina, immunopositive fibers were localized to the inner plexiform layer, but several fiber elements were also found in the outer plexiform layer. The olfactory system displayed FMRF-IR fibers in the epithelium and along the olfactory nerves. These findings differ from those reported in other species as follows: (i) FMRF-IR cells in the retina have not previously been reported in teleosts; (ii) the presence of FMRF-IR fibers in the outer plexiform layer of the retina is a new finding for any species; (iii) the occurrence of immunopositive cells in the mesencephalic laminar nucleus has to our knowledge not been demonstrated previously.

Amino Acid Sequence↗

FMRFamide-like and allatostatin-like immunoreactivity in the lateral heart nerve of Periplaneta americana: colocalization at the electron-microscopic level.

Both allatostatin immunoreactivity (AS-IR) and FMRFamide immunoreactivity (FMRFa-IR) have been demonstrated light-microscopically in the lateral heart nerve of Periplaneta americana. The identical labeling of some fibers suggests the coexistence of the two antigens. Electron-microscopically, six granule types in the peripheral part of the lateral heart nerve can be distinguished according to their size and density (types 1-6). These granule types can be subdivided immunocytochemically by means of a new mirror-section technique. Granules of types 4 and 5 always exclusively show FMRFa-IR. In the populations of fibers containing granules of types 1 and 6, axon profiles can be found that contain granules colocalizing FMRFa-IR and AS-IR. Other axon profiles of these populations only contain immunonegative granules of the same ultrastructure. Granules of type 2 can be differentiated immunocytochemically in three forms in the same section: In some fibers, they are nonreactive; in other fibers of the same section, they show FMRFa - IR, whereas in a third fiber type, granules show AS - IR. Finally, granules of type 3 can be observed with FMRFa-IR. In other fibers, they occur with the same ultrastructure but exhibit no immunoreactivity. Two soma types occur in the lateral heart nerve. Soma type I is characterized by the production of electron-dense granules that show FMRFa-IR. Type II is in close contact with various fibers, forming different types of axosomatic synapses, hitherto unknown in Insecta.

Amino Acid Sequence↗

FMRFamide-related peptides in Hymenolepis diminuta: immunohistochemistry and radioimmunoassay.

The localization of FMRFamide-related peptide (FaRP) immunoreactivity was determined during different stages of development of the rat tapeworm Hymenolepis diminuta. In the adult worm (14 days old), FaRP immunostaining was most intense in the scolex and concentrated in the central nervous system (cerebral ganglia and transverse commissures) and around the lips of the suckers. In the strobila, medial and lateral longitudinal nerve cords (LNCs) and ladder-like connecting commissures were the only tissue stained. Immunoreactivity in the medial LNCs of the adult tapeworms extended only to and included proglottides containing developing testis and seminal receptacle but disappeared in proglottides in which primordial ovaries were first detected. Radioimmunoassay confirmed that FaRPs were concentrated in the scolex/neck region of the adult worm (3.9 +/- 1.5 pmol mg protein-1), whereas the lowest concentrations (0.2 +/- 0.19 pmol mg protein-1) were recovered from the regions of the strobila containing shelled eggs. The pattern of FaRP immunoreactivity observed in 5- and 7-day-old worms was similar to that seen in adult worms, but in 2- and 3-day-old worms the pattern of immunoreactivity observed in the cerebral ganglia, transverse commissures, and LNCs differed significantly as compared with that seen in older worms. These results indicate differential utilization and/or roles for FaRPs during development and suggest both central and sensory roles in this tapeworm.

Amino Acid Sequence↗

Inhibitory effects of nematode FMRFamide-related peptides (FaRPs) on muscle strips from Ascaris suum.

A large number of FMRFamide-related peptides (FaRPs) are found in nematodes, and some of these are known to influence tension and contractility of neuromuscular strips isolated from Ascaris suum body wall. Relaxation of these strips has been noted with five nematode FaRPs. The inhibitory actions of SDPNFLRFamide (PF1) and SADPNFLRFamide (PF2) appear to be mediated by nitric oxide, as previously demonstrated with inhibitors of nitric oxide synthase (NOS). This present study showed that the effects of PF1 were also depended on external Ca++ and were reduced by the Ca(++)-channel blocker verapamil, observations consistent with the finding that nematode NOS is Ca(++)-dependent. KSAYMRFamide (PF3), KNIRFamide (PF4) and KNAFIRFamide (an alanine substituted analog of KNEFIRFamide, AF1, termed A3AF1) also relaxed A. suum muscle strips, but these responses were not affected by NOS inhibitors. PF3 inhibited the activity of strips prepared from the dorsal side of the worm, but contracted ventral strips. Both effects were dependent on the presence of ventral/dorsal nerve cords (unlike PF1/PF2) and were attenuated in medium which contained high K+ or low Ca++. PF4-induced muscle relaxation and hyperpolarization were independent of nerve cords, but were reversed in Cl-free medium, unlike PF1 or PF3. The PF4 effect physiologically desensitized muscle strips to subsequent treatment with PF4 and/or GABA. However, PF4 and GABA were not synergistic in this preparation. The effects of GABA, but not PF4, were reduced in muscle strips treated with the GABA antagonist, NCS 281-93. Following PF4 (or GABA) relaxation, subsequent treatment with higher doses of PF4 caused muscle strip contraction. A3AF1 was found to relax muscle strips and hyperpolarize muscle cells independently of the ventral and dorsal nerve cords, K+, Ca++, and Cl-, and mimicked the inhibitory phase associated with the exposure of these strips to AF1. On the basis of anatomical and ionic dependence, these data have delineated at least four distinct inhibitory activities attributable to nematode FaRPs. Clearly, a remarkably complex set of inhibitory mechanisms operate in the nematode neuromuscular system.

Animals↗

Isolation and structural characterization of Drosophila TDVDHVFLRFamide and FMRFamide-containing neural peptides.

An extract of adult Drosophila melanogaster was separated by gel exclusion, ion exchange, and reversed-phase chromatography. Four peptides, each with an -ArgPheNH2 C-terminal sequence, were identified by radioimmunoassay. The primary sequences were determined by Edman degradation and confirmed by mass spectrometry and sequence-specific radioimmunoassay. Three of the peptides are encoded by Drosophila proFMRFamide: AspProLysGlnAspPheMetArgPheNH2 (DPKQDFMRFamide), ThrProAlaGluAspPheMetArgPheNH2 (TPAEDFMRFamide), and SerAspAsnPheMetArgPheNH2 (SDNFMRFamide). A novel Drosophila peptide ThrAspValAspHisValPheLeuArgPheNH2 (TDVDHVFLRFamide) was also isolated. TDVDHVFLRFamide is structurally related to peptides isolated from chicken, cockroach, locust, and snail; the cockroach, fruitfly, and locust peptides differ only by the N-terminal amino acid residue. Two Drosophila neural genes, dsk and FMRFamide, are known to encode -ArgPheNH2-containing peptides; however, neither encodes TDVDHVFLRFamide, indicating that Drosophila contains another precursor encoding -ArgPheNH2 peptides.

Amino Acid Sequence↗

FMRFamide-like immunocytochemistry in the brain and subesophageal ganglion of Triatoma infestans (Insecta: Heteroptera). Coexpression with beta-pigment-dispersing hormone and small cardioactive peptide B.

The distribution of FMRFamide (FMRFa)-like immunoreactivity (LI) was studied in the brain and subesophageal ganglion of Triatoma infestans, the insect vector of Chagas' disease. The neuropeptide displayed a widespread distribution with immunostained somata in the optic lobe, in the anterior, lateral, and posterior soma rinds of the protocerebrum, and around the antennal sensory and mechanosensory and motor neuropils of the deutocerebrum. FMRFa-immunoreactive profiles of the subesophageal ganglion were seen in the mandibular, maxillary, and labial neuromeres. Immunostained neurites were detected in the medulla and lobula of the optic lobe, the lateral protocerebral neuropil, the median bundle, the calyces and the stalk of the mushroom bodies, and the central body. In the deutocerebrum, the sensory glomeruli showed a higher density of immunoreactive processes than the mechanosensory and motor neuropil, whereas the neuropils of each neuromere of the subesophageal ganglion displayed a moderate density of immunoreactive neurites. Colocalization of FMRFa-LI and crustacean pigment-dispersing hormone-LI was found in perikarya of the proximal optic lobe, the lobula, the sensory deutocerebrum, and the labial neuromere of the subesophageal ganglion. The distribution pattern of small cardioactive peptide B (SCP(B))-LI was also widespread, with immunolabeled somata surrounding every neuropil region of the brain and subesophageal ganglion, except for the optic lobe. FMRFa- and SCP(B)-LIs showed extensive colocalization in the brain of this triatomine species. The presence of immunolabeled perikarya displaying either FMRFa- or SCP(B)-LI confirmed that each antisera identified different peptide molecules. The distribution of FMRFa immunostaining in T. infestans raises the possibility that FMRFa plays a role in the regulation of circadian rhythmicity. The finding of immunolabeling in neurosecretory somata of the protocerebrum suggests that this neuropeptide may also act as a neurohormone.

Animals↗

Comparative analysis of FMRFamide-like immunoreactivity in caiman (Caiman crocodilus) and turtle (Trachemys scripta elegans) brains.

The distribution of FMRFamide (FMRFa)-like peptides in caiman (Caiman crocodilus) and turtle (Trachemys scripta elegans) brains was studied by immunohistochemistry. In both species, distinct groups of FMRFa-like immunoreactive (ir) perikarya were present in the medial septal nucleus, accumbens nucleus, nucleus of the diagonal band of Broca, suprachiasmatic area, lateral hypothalamic area, and periventricular hypothalamic nucleus. A few FMRFa-ir neurons in the hypothalamic area were located in the neuroepithelial cell lining of the third ventricle. FMRFa-ir fibers were scattered in all major areas of the brain, from the olfactory bulbs to the rhombencephalon. They formed dense aggregates in the medial septal area, basal telencephalon, median eminence, and infundibulum, and adjacent to the fourth ventricle. The most obvious difference between the FMRFa-ir systems in caimans and turtles concerned the number of nuclei that contained neurons with this immunoreactivity. Eight such clusters were present in the caiman brain, whereas thirteen clusters were found in the turtle brain. The turtle also displayed scattered FMRFa-ir somata in the anterior olfactory nucleus, striatum, lateral septal nucleus, medial and lateral cortex, medial forebrain bundle, lateral preoptic area, and lateral geniculate nucleus. In the caiman brain, a few FMRFa-ir neurons were noted in the ventrolateral area of the pallial commissure and an even smaller number of ir neurons was found dispersed in the optic tracts. Neither formed nuclear aggregates. The results are compared with those described for other vertebrates.

Alligators and Crocodiles↗

Evidence for peptidergic innervation of the endocrine optic gland in Sepia by neurons showing FMRFamide-like immunoreactivity.

The innervation of the endocrine optic gland of Sepia, which controls sexual maturation, was studied by immunocytochemistry. Anti-FMRFamide (Phe-Met-Arg-Phe-NH2) serum revealed immunoreactive neurons in the olfactory and basal-dorsal lobe of the supra-esophageal brain mass. The axons of these neurons form a network from which fibers run to the optic gland. The fibers form many varicosities on the glandular cells, indicating synaptic innervation. Apparently, the two brain lobes containing the immunopositive cells function as a unit where visual and olfactory cues are integrated to regulate the endocrine activity of the optic gland.

Animals↗

Dual inhibitory action of FMRFamide on neurosecretory cells controlling egg laying behavior in the pond snail.

We describe here the electrophysiological characterization of a dual inhibitory action of FMRFamide (FMRFa, Phe-Met-Arg-Phe-NH2) on the caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis: (i) a transient hyperpolarizing response (H-response) and (ii) a suppression of the excitability of the cells, which lasted as long as the peptide was present. Both effects of FMRFa occurred in silent, excitable cells as well as discharging cells. The effects were reversible and dose-dependent in the range of 10(-9) to 10(-5) M. The H-response was not blocked by any of the antagonists to classical neurotransmitters that were tested. The reversal potential of the H-response was dependent on the [K+]o, which suggests that K+ is the major charge carrier in this response. 4-Aminopyridine (4-AP) blocked the H-response but did not affect the suppression of the excitability by FMRFa. This indicates that the effects of the peptide on these cells are independent. Experiments on the mechanism of the inhibition of the excitability indicated that FMRFa blocks the cAMP-dependent activation of the pacemaking mechanism of the CDCs. In experiments with isolated cells it was demonstrated that the actions of FMRFa are mediated directly through receptors on CDCs (H-response: ED50 = 10(-8) M). Finally, anti-FMRFa-positive varicosities and axons close to the somata, the axons and the neurohaemal endings of the CDCs were demonstrated immunocytochemically. The duality of the action of FMRFa on the neural activity of CDCs indicates its role of high priority in the regulation of egg laying behavior.

Animals↗

Oxytocin-like peptide: a novel epitope colocalized with the FMRFamide-like peptide in the supernumerary neurons of the sex segmental ganglia of leeches--morphological and biochemical characterization; putative anti-diuretic function.

A large number of oxytocin (OT)-like neurons were detected in the sex segmental ganglia (SG5, SG6) of three species of leeches belonging to different orders: Theromyzon tessulatum, Hirudo medicinalis and Erpobdella octoculata. In this latter species, an epitope close to the vertebrate OT by its C-terminal part (MSH release inhibiting factor: MIF), localized in granules of a size diameter of ca 120 nm and colocalized with FMRFamide(FMRFa)-like material was demonstrated. With reverse phase-high performance liquid chromatography, evidence was given that the two epitopes (OT and FMRFa) colocalized in the same neurons were biochemically different. A titration of OT per SG indicated that the OT-like amount was considerably higher in sex SG than in non-sex SG (ca. 5 pmol vs. ca. 0.5 pmol). Moreover, at the level of sex SG, this amount was ca. 3-fold higher in immature leeches than in mature specimens. Injections of extracts of SG of E. octoculata and of fragments of OT (Tocinoic acid or MIF) to T. tessulatum, indicated that MIF (the epitope found in the sex SG) and sex SG have the same anti-diuretic effect on the leeches injected. These results pointed to an anti-diuretic role of the leech OT-like substance.

Animals↗

The pharmacology of FMRFamide-related neuropeptides in nematodes: new opportunities for rational anthelmintic discovery?

The chemotherapeutic control of helminth parasites is compromised by the limited number of classes of anthelmintic drugs. Discovery of novel anthelmintics is impeded by the lack of novel screening technologies that overcome the difficulties inherent in screens based on whole organism toxicity. The development and implementation of mechanism-based screens for new anthelmintics offers great promise for the revitalization of antiparasitic drug discovery. However, mechanism-based screens must be based on a thorough understanding of the proteins or processes that offer the best chance for selective chemotherapeutic intervention. Basic research on the characterization of nematode FMRFamide-related peptides (FaRPs) has revealed that these peptides are ubiquitously distributed in helminths. Chemical identification of a number of nematode FaRPs has been achieved, and these peptides have potent and profound effects on the nematode neuromuscular system. Physiological processes mediated by nematode FaRPs (and other helminth neuropeptides) offer potential targets for the discovery of novel anthelmintics.

Amino Acid Sequence↗

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 FaRPs 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 FaRPs, particularly from peptide isolation, molecular cloning of precursor proteins, receptor localization, and physiological studies.

Amino Acid Sequence↗

FMRFamide- and pancreatic polypeptide-like immunoreactivity of endocrine cells in the midgut of a mosquito.

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.

Aedes↗

Modulation of human lymphocyte proliferation by FLFQPQRFamide, a FMRFamide-like peptide with anti-opiate properties.

The octapeptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F8Fa), originally detected in mammalian brain by antisera raised against the invertebrate peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a neuropeptide able to antagonize the actions of both endogenous and exogenous opiates. Since it is well accepted that lymphocytes are targets for opiates, we have tested the effect of F8Fa on T cell proliferation from normal human peripheral blood lymphocytes. Our study shows that F8Fa exerts a concentration-dependent diphasic modulation of human T lymphocyte proliferation. Thus, despite a great variability between individuals, 10(-13) M F8Fa was found to enhance the proliferation of T cells induced by phytohemagglutinin or anti-CD2 monoclonal antibodies, while 10(-7) M F8Fa inhibited T cell proliferation, without affecting cell viability. When F8Fa was tested on monocyte-depleted cell preparations, only the inhibitory effect was observed. These results indicate that F8Fa may stimulate T cells via monocytes, but may also directly inhibit T lymphocyte proliferation. Given the presence of F8Fa-like peptide in human plasma, we suggest that F8Fa may act as a neurohormone in the control of the immune system.

Carbohydrate Sequence↗

A-18-famide and F-8-famide, endogenous mammalian equivalents of the molluscan neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2), inhibit colonic bead expulsion time in mice.

Morphine and the two endogenous mammalian FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides known as morphine-modulating neuropeptides, F-8-Famide (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and A-18-Famide (Ala-Gly-Glu-Gly-Leu-Ser-Ser-Pro-Phe-Trp-Ser-Leu-Ala-Ala-Pro-Gln-Arg-Phe -NH2), were administered intracerebroventricularly (ICV) to mice and the effect of each on colonic bead expulsion time was measured. Each of the three compounds delayed expulsion of a 3 mm glass bead placed in the distal colon. A-18-Famide was more potent than F-8-Famide [ED 50 = 2.3 micrograms (1.2 nmole) and 13.9 micrograms (13.0 nmole), respectively]. A-18-Famide: 1) did not block morphine-induced delay of bead expulsion time, and 2) was blocked by simultaneous administration (ICV) of 1.0 microgram of the competitive opiate antagonist naloxone. These data demonstrate apparent opioid modulatory or agonist-like, rather than antagonist-like, properties of A-18-Famide and F-8-Famide.

Animals↗

The identification and structure-activity relations of a cardioactive FMRFamide-related peptide from the blue crab Callinectes sapidus.

The pericardial organs and thoracic ganglia of the blue crab Callinectes sapidus were resected and extracted. The extracts were fractionated by HPLC and the fractions analyzed by a radioimmunoassay (RIA) to FMRFamide. Multiple peaks of immunoreactivity were present and one of these, upon fast atom bombardment mass spectrometry (FAB-ms) and microsequencing, yielded the sequence GYNRSFLRFamide. The amount of this peptide in each crab is between 7 and 13 pmol. Several incomplete sequences were also characterized, suggesting a precursor with multiple copies of peptides related to GYNRSFLRFamide might occur. The peptide caused a dose-dependent increase in heart rate; threshold was 10 to 30 nM, and the EC50 was 323 +/- 62 nM. A structure-activity study of GYNRSFLRFamide on the crab heart suggests that, for full potency, a peptide should be at least a heptapeptide with the sequence XXZFLRFamide, where X is any amino acid and Z is either asparagine or serine.

Amino Acid Sequence↗

Two FMRFamide-like peptides from the free-living nematode Panagrellus redivivus.

Peptides of the FXRFamide family, where X = M, I or L, are broadly distributed among invertebrates. Two such peptides were purified and sequenced from the free-living nematode, Panagrellus redivivus. Immunohistochemical techniques localized FMRFamide-like material in several regions of these organisms, including the nerve cords and, most prominently, in paired groups of cells located caudally to the base of the pharynx. RIA determinations gave an estimate of 2.8 nmol immunoreactive peptide/g of an acetone extract of P. redivivus. Four sequential HPLC purification steps, followed by sequencing by automated Edman degradation and FAB-MS, led to the identification of Ser-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SDPNFLRFamide) and Ser-Ala-Asp-Pro-Asn-Phe-Leu-Arg-Phe-amide (SADPNFLRFamide) as members of the FXRFamide family in this nematode.

Amino Acid Sequence↗

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 > or = 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 > or = 0.3 nM induced relaxation in 100% of muscle preparations examined. The relaxation was short-lived at concentrations of peptide > or = 1 microM 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.

Amino Acid Sequence↗