FMRFamide-related peptides and serotonin regulate Drosophila melanogaster heart rate: mechanisms and structure requirements

Drosophila melanogaster FMRFamide-related peptides (FaRPs) include SDNFMRFamide, PDNFMRFamide, and TDVDHVFLRFamide (dromyosuppressin, DMS); each peptide contains a C-terminal FMRFamide but a different N-terminal extension. FaRPs and serotonin (5-HT) each affect the frequency of D. melanogaster heart contractions in vivo. We examined the cellular expression of FaRPs and 5-HT, and the activities of FMRFamide, SDNFMRFamide, PDNFMRFamide, or DMS and 5-HT on heart rate. FaRPs and 5-HT were not co-localized; FaRP-and 5-HT-immunoreactive fibers extended from different brain cells and innervated the anterior D. melanogaster dorsal vessel. However, no neuron expressed both a FaRP and 5-HT. The effect of FMRFamide and 5-HT was not different from the effect of 5-HT alone on heart rate. The effect of PDNFMRFamide and 5-HT showed an additive effect on heart rate. SDNFMRFamide and 5-HT or DMS and 5-HT resulted in non-additive effects on heart rate. Our data provide evidence for the complexity of FaRP and 5-HT interactions to regulate frequency of heart contractions in vivo. Our results also confirm the biological importance of FaRP N-terminal amino acid extensions.     

Immunoaffinity-based mass spectrometric characterization of the FMRFamide-related peptide family in the pericardial organ of Cancer borealis

The tetrapeptide, FMRFamide, was first discovered in 1977 in the molluscan nervous system and was found to affect the contractile force of molluscan cardiac muscle and other muscles [1]. Since then, numerous FMRFamide-related peptides (FaRPs) have been reported in both invertebrate and vertebrate species [2], [3], [4], [5], [6], [7], [8] and [9]. We have previously reported the detection and identification of numerous FaRPs in Cancer borealis pericardial organs (POs), one of the major neurosecretory structures in the crustaceans [2] and [3]. Here, we have developed two immunoaffinity-based methods, immunoprecipitation (IP) and immuno-dot blot screening assay, for the enrichment of FaRPs in C. borealis POs. A combined mass spectrometry (MS)-based approach involving both matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI-FTMS) and nanoscale liquid chromatography coupled to electrospray ionization quadrupole time-of-flight tandem mass spectrometry (nanoLC-ESI-QTOF MS/MS) is used for a more comprehensive characterization of the FaRP family by utilizing high mass accuracy measurement and efficient peptide sequencing. Overall, 17 FMRFamide-related peptides were identified using these two complementary immuno-based approaches. Among them, three novel peptides were reported for the first time in this study.     

Drosophila melanogaster FMRFamide-containing peptides: redundant or diverse functions?

FMRFamide-related peptides (FaRPs) are expressed throughout the animal kingdom and regulate a multitude of physiological activities. FaRPs have an RFamide C-terminal consensus structure that is important for interaction with the receptor. The ease of genetic manipulation and availability of genomic sequences makes Drosophila melanogaster an important experimental organism. Multiple classes of FaRPs encoded by different genes have been identified within this species. Here, we review FMRFamide-containing peptides encoded by the D. melanogaster FMRFamide gene in order to review the data on the expression, regulation, and activity of these peptides as well as acknowledge further endeavors required to elucidate FaRP signaling.     

FaRP cell distribution in the developing CNS suggests the involvement of FaRPs in all parts of the chromatophore control pathway in Sepia officinalis (Cephalopoda)

The FMRFamide-related peptide (FaRP) family includes a wide range of neuropeptides that have a role in many biological functions. In cephalopods, these peptides intervene in the peculiar body patterning system used for communication and camouflage. This system is particularly well developed in the cuttlefish and is functional immediately after hatching (stage 30). In this study, we investigate when and how the neural structures involved in the control of body patterning emerge and combine during Sepia embryogenesis, by studying the expression or the production of FaRPs. We detected FaRP expression and production in the nervous system of embryos from the beginning of organogenesis (stage 16). The wider FaRP expression was observed concomitantly with brain differentiation (around stage 22). Until hatching, FaRP-positive cells were located in specific areas of the central and peripheral nervous system (CNS and PNS). Most of these areas were implicated in the control of body patterns, suggesting that FaRPs are involved in all parts of the neural body pattern control system, from the ‘receptive areas’ via the CNS to the chromatophore effectors.     

Authentic FMRFamide is present in the polychaete Nereis virens

The tetrapeptide FMRFamide is but one member of a large family of invertebrate neuropeptides which includes another tetrapeptide, FLRFamide, and several longer peptides terminating in one or the other of these tetrapeptide sequences. These peptides have been isolated from both molluscs and arthropods, but so far not one has been isolated from an annelid. Since the annelid worms are believed to share a common ancestor with molluscs and arthropods, they should contain FMRFamide-like peptides. We found two immunoreactive peaks in Nereis virens, but microsequencing and fast atom bombardment mass spectrometry revealed that they represent only one native peptide, FMRFamide. (The other peak is its methionyl sulfoxide derivative.) Each worm contained only 100 to 600 fmols of peptide, which is at least 10-100 times less than the levels in molluscs. Our identification of a tetrapeptide, and only a tetrapeptide, in this worm suggests that the tetrapeptides are the more ancient members of the family, and were probably present in the common ancestors of the annelids, arthropods, and molluscs.     

FMRFamide-related peptides: anti-opiate transmitters acting in apoptosis

Members of the FMRFamide-related peptide (FaRP) family are neurotransmitters, hormone-like substances and tumor suppressor peptides. In mammals, FaRPs are considered as anti-opiate peptides due to their ability to inhibit opioid signaling. Some FaRPs are asserted to attenuate opiate tolerance. A recently developed chimeric FaRP (Met-enkephalin-FMRFa) mimics the analgesic effects of opiates without the development of opiate-dependence, displaying a future therapeutical potential in pain reduction. In this review we support the notion, that opiates and representative members of the FaRP family show overlapping effects on apoptosis. Binding of FaRPs to opioid receptors or to their own receptors (G-protein linked membrane receptors and acid-sensing ion channels) evokes or suppresses cell death, in a cell- and receptor-type manner. With the dramatically increasing incidence of opiate abuse and addiction, understanding of opioid-induced cell death, and in this context FaRPs will deserve growing attention.     

FMRFamide and related peptides in the phylum mollusca

FMRFamide is one of the well-known peptides studied within the phylum Mollusca. It was first isolated from the clam Macrocallista nimbosa during the end of the 1960s. Since then, a number of reports related to FMRFamide have been published from different experimental approaches, revealing that it and its related peptides (FaRPs) are implicated in a variety of physiological processes. As this year is the 30th anniversary since its discovery, this review focuses on diverse findings related to both FMRFamide and FaRPs in the phylum Mollusca.     

RFamide Peptides in the Leech, Hirudo medicinalis

RFamide peptides have been localized to a number of neuronsof the CNS of the leech, Hirudo medicinalis, using immunocytochemicaltechniques. The majority of this immunoreactivity appears tobe due to the peptide FMRFamide. Most of the identified RFamideimmunoreactive cells are cholinergic motor neurons, though someare interneurons. Superfused FMRFamide is active on the targetsof these identified neurons; in a few well studied cases, ithas been possible to show that FMRFamide mimics a specific physiologicalaction of an identified neuron on its target. In the leech as in other phyla where they occur, RFamide peptidesare widely distributed in neurons, and are neuromodulators withdiverse physiological effects.     

Role of FMRFamide in the reproduction of Octopus vulgaris: molecular analysis and effect on visual input

As a part of continuous research on the neurobiology of the cephalopods in general, and the neuroendocrine control of reproduction in Octopus vulgaris in particular, the presence, the molecular analysis and the effect of FMRFamide on the screening-pigment migration in the visual system have been analysed. FMRFamide immunoreactive fibres are present in the outer plexiform layer of the retina as well as in the plexiform zone of the deep retina. These fibres presumably come from optic and olfactory lobes. We isolated an incomplete Octopus FMRFamide cDNA which encodes an amino terminal truncated precursor containing several FMRFamide-related peptides (FaRPs) showing a high degree of identity with the FaRPs encoded in the precursor of Sepia officinalis, except for the presence of an Rpamide related peptide, present only in cnidarians. Finally, stimulation of isolated retina demonstrated that the effect of this tetrapeptide, coupled with dopamine, is the induction of an extreme adaptation of the retina to the light condition. This situation de facto inhibits sexual maturation. Our results on the effect of FMRFamide on the retina confirm the suggested hypothesis that this peptide plays an inhibitory role on the activity of optic gland.     

Functional identification of an FMRFamide-related peptide gene on diapause induction of the migratory locust,Locusta migratoria L

FMRFamide-related peptides (FaRPs) are a type of neuropeptide, which participate in a variety of physiological processes in insects. Previous study showed that myosuppressin, being a member of FaRPs, initiated pupal diapause in Mamestra brassicae. We presumed that FaRPs genes might play a critical role in photoperiodic diapause induction of L. migratoria. To verify our hypothesis, flrf, a precursor gene of FaRP from L. migratoria, was initially cloned under long and short photoperiods that encoded by flrf gene identified from central nervous system (CNS). Phylogenetic analysis showed that the protein encoded by L. migratoria flrf gene, clustered together with Nilaparvata lugens (Hemiptera: Delphacidae) with 100% bootstrap support, was basically an FMRFamide precursor homologue. We noticed the availability of -RFamide peptides (GSERNFLRFa, DRNFIRFa) under short photoperiod only, which suggested their functions related to photoperiodic diapause induction. RNAi and quantitative real-time PCR (qRT-PCR) results further confirmed that the flrf gene promoted locust's diapause.     

Presence and distribution of FMRFamide-like immunoreactivity in the sea cucumber Holothuria scabra (Jaeger, 1833)

Since the discovery of FMRFamide, a cardioactive neuropeptide in the mollusk Macrocallista nimbosa (Lightfoot, 1786) by Price and Greenberg (Science 197:670–671, 1977), there has been a steady increase in our understanding of the distribution and diversity of FMRFamide and FMRFamide-related peptides (FaRPs) across phyla. The sea cucumber Holothuria scabra (Jaeger, 1833) (Echinodermata: Holothuroidea) is a high-premium tropical economic species that is overexploited globally and may be in imminent danger of extinction in some areas. Conservation of this species and its amenability to culture is however hampered by the limited knowledge about its biology, including the neurohormonal system. Using indirect immunofluorescence technique with antisera raised against the tetrapeptide FMRFamide as well as reverse-phase HPLC, we have demonstrated the widespread distribution of FMRFamide-like immunoreactivity in the tissues of H. scabra, including the coelomic fluid, suggesting that FaRPs might have neurohormonal activities in this species.     

Regulation of Drosophila FMRFamide neuropeptide gene expression

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell‐ or tissue‐specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide‐related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C‐terminal structure but different N‐terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide‐immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 347–358, 1999     

Evolution of a Molluscan Cardioregulatory Neuropeptide

SYNOPSIS. The cardioexcitatory neuropeptide FMRFamide was firstidentified from a clam, but has now been demonstrated in severalother molluscs. It is probably present throughout the molluscanphylum though co-existing with related peptides in some species.For example, I report here the finding of the peptide phenylalanyl-leucyl-arginyl-phenylalanineamide (FLRFamide) in the mesogastropod Pomacea paludosa whereit accounts for 10–20% of the total FMRFamide-like activity.This peptide may be a minor component of the FMRFamide-likeactivity in other species as well. The pulmonate snails haveseveral, closely-related, heptapeptide analogs of FLRFamidethat are unique to them, such as pyroglutamyl-aspartyl-prolyl-phenylalanyl-leucyl-arginyl-phenylalanineamide (pQDPFLRFamide) which was isolated from Helix aspersa.Two additional pulmonate heptapeptides that have been isolatedprobably differ from pQDPFLRFamide only in their N-terminalamino acid residues. The heptapeptides account for most of theFMRFamidelike activity in the species in which they occur. Though the tetrapeptides FMRFamide and FLRFamide have virtuallyidentical activities on various molluscan tissues, the heptapeptideshave activity that is distinct from the tetrapeptides on somepulmonate muscles. 1 have attempted to explain the evolutionof this diversity of peptide structure and function found inthe modern pulmonates by postulating a gene duplication in thegastropod line leading to them.     

FMRFamide elicits chromatophore expansion and retraction depending on its type and development in the squid, Sepioteuthis lessoniana

To examine chromatophore control by FMRFamide-related peptide (FaRP), we investigated the pharmacological effect of FMRFamide on the chromatophores and the FMRFamide-immunoreactivity of nerves surrounding the muscles in the coastal squid, Sepioteuthis lessoniana. Applications of FMRFamide elicited expansion of black chromatophores and retraction of yellow chromatophores in the adult squid. FMRFamide-immunoreactive terminals were distributed along black chromatophore muscles but were not observed around the yellow ones. This means that FMRFamide functions differently for each of the two types of chromatophores in the adult squid. Moreover, the pharmacological effect of FMRFamide on the black chromatophores differed between adults and hatchlings; application of FMRFamide retracted black chromatophores in hatchlings but not in adults. These results indicate that certain squid species have an FaRP system for controlling the chromatophores in their skin and that the system changes during development.     

Regulation of Drosophila FMRFamide neuropeptide gene expression.

Physiologically important peptides are often encoded in precursors that contain several gene products; thus, regulation of expression of polypeptide proteins is crucial to transduction pathways. Differential processing of precursors by cell- or tissue-specific proteolytic enzymes can yield messengers with diverse distributions and dissimilar activities. FMRFamide-related peptides (FaRPs) are present throughout the animal kingdom and affect both neural and gastrointestinal functions. Organisms have several genes encoding numerous FaRPs with a common C-terminal structure but different N-terminal amino acid extensions. We have isolated SDNFMRFamide, DPKQDFMRFamide, and TPAEDFMRFamide contained in the Drosophila FMRFamide gene. To investigate the regulation of expression of FMRFamide peptides, we generated antisera to distinguish among the three neuropeptides. We have previously reported the distribution of SDNFMRFamide and DPKQDFMRFamide. In this article, we describe TPAEDFMRFamide expression. TPAEDFMRFamide antisera stain cells in embryonic, larval, pupal, and adult thoracic and abdominal ganglia. In addition, TPAEDFMRFamide-immunoreactive material is present in a lateral protocerebrum cell in adult. Thus, TPAEDFMRFamide antisera staining of neural tissue is different from SDNFMRFamide or DPKQDFMRFamide. In addition, TPAEDFMRFamide antisera stain larval, pupal, and adult gut, while SDNFMRFamide and DPKQDFMRFamide do not. TPAEDFMRFamide immunoreactivity is present in cells stained by FMRFamide antisera. Taken together, these data support the conclusion that TPAEDFMRFamide is differentially processed from the FMRFamide polypeptide protein precursor and may act in both neural and gastrointestinal tissue.     

Conorfamide-Sr2, a gamma-carboxyglutamate-containing FMRFamide-related peptide from the venom of Conus spurius with activity in mice and mollusks

A novel peptide, conorfamide-Sr2 (CNF-Sr2), was purified from the venom extract of Conus spurius, collected in the Caribbean Sea off the Yucatan Peninsula. Its primary structure was determined by automated Edman degradation and amino acid analysis, and confirmed by electrospray ionization mass spectrometry. Conorfamide-Sr2 contains 12 amino acids and no Cys residues, and it is only the second FMRFamide-related peptide isolated from a venom. Its primary structure GPM gammaDPLgammaIIRI-nh2, (gamma, gamma-carboxyglutamate; -nh2, amidated C-terminus; calculated monoisotopic mass, 1468.72Da; experimental monoisotopic mass, 1468.70Da) shows two features that are unusual among FMRFamide-related peptides (FaRPs, also known as RFamide peptides), namely the novel presence of gamma-carboxyglutamate, and a rather uncommon C-terminal residue, Ile. CNF-Sr2 exhibits paralytic activity in the limpet Patella opea and causes hyperactivity in the freshwater snail Pomacea paludosa and in the mouse. The sequence similarities of CNF-Sr2 with FaRPs from marine and freshwater mollusks and mice might explain its biological effects in these organisms. It also resembles FaRPs from polychaetes (the prey of C. spurius), which suggests a natural biological role. Based on these similarities, CNF-Sr2 might interact with receptors of these three distinct types of FaRPs, G-protein-coupled receptors, Na+ channels activated by FMRFamide (FaNaCs), and acid-sensing ion channels (ASICs). The biological activities of CNF-Sr2 in mollusks and mice make it a potential tool to study molecular targets in these and other organisms.     

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.     

The action of FMRFamide (Phe-Met-Arg-Phe-NH2) and related peptides on mammals

First purified 11 years ago from clam ganglia, FMRFamide (Phe-Met-Arg-Phe-NH2) was quickly demonstrated to be cardioactive in several molluscan species. Subsequent discovery that FMRFamide, or FMRFamide-related peptides (FaRPs), were present in mammalian central nervous system and gastrointestinal tract prompted investigations into the effect of FMRFamide on mammals. FMRFamide has now been shown to be cardioexcitatory in mammals, to inhibit morphine-induced antinociception, and to block morphine-, defeat-, and deprivation-induced feeding. It also inhibits colonic propulsive motility, induces behavioral effects when administered intrathecally, and has been reported to have amnesic effects in rodents. A proposal has arisen that a FMRFamide-like substance is an endogenous opioid antagonist and has stimulated a search for such a substance. However, FMRFamide has only weak affinity for opioid receptors and not all the actions of FMRFamide appear to be explained by actions at opioid receptors. Alternative mechanisms have been proposed which suggest that FMRFamide acts as a neuromodulator.     

Examination of the role of FMRFamide-related peptides in the circadian clock of the cockroach Leucophaea maderae

The accessory medulla, the circadian clock of the cockroach Leucophaea maderae, is abundant in neuropeptides. Among these neuropeptides are the FMRFamide-related peptides (FaRPs), which generally share the C-terminal RFamide. As a first step toward understanding the functional role of FaRPs in the circadian clock of the cockroach, immunocytochemistry with antisera against various FaRPs, MALDI-TOF mass spectrometry, and injections of two FaRPs combined with running-wheel assays were performed. Prominent FMRFamide-like immunoreactivity was found in maximally four soma clusters associated with the accessory medulla and in most neuropils of the protocerebrum. By MALDI-TOF mass spectrometry, various extended FMRFamides of the cockroach L. maderae were partially identified in thoracic perisympathetic organs, structures known to accumulate extended FMRFamides in insects. By mass match, several of these peptides were also detected in the accessory medulla. Injections of FMRFamide and Pea-FMRFa-7 (DRSDNFIRF-NH₂) into the vicinity of the accessory medulla caused time-dependent phase-shifts of locomotor activity rhythms at circadian times 8, 18, and 4. Thus, our data suggest a role for the different FaRPs in the control of circadian locomotor activity rhythms in L. maderae.