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[D-Met2]-FMRFamide (DMFa): production of naloxone-sensitive antinociception in mouse tail-flick test.

Considerable data support the hypothesis that mammalian FMRFamide (Phe-Met-Arg-Phe-NH2) or mammalian FMRFamide-related peptides (FaRPs) function as endogenous antiopiates (for review see Raffa, 1988). We report here that central administration (i.c.v.) of a FaRP with D-amino acid substitution in the second position, i.e. [D-Met2]-FMRFamide (DMFa), produces dose-related, naloxone-reversible antinociception in the mouse tail-flick test. Hence, this modification appears to confer agonist-like activity.

Analgesics↗

Cardioactive neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) and novel related peptides are encoded in multiple copies by a single gene in the snail Lymnaea stagnalis.

The neuropeptide Phe-Met-Arg-Phe-NH2 (FMRFamide) is a potent cardioactive neuropeptide in Lymnaea stagnalis. Isolation and sequencing of 2 cDNAs and a genomic clone shows that a single gene encodes a precursor protein which contains 9 copies of the FMRFamide peptide, 2 copies of the related peptide Phe-Leu-Arg-Phe-NH2 (FLRFamide), and single copies of the putative pentapeptides Gln-Phe-Tyr-Arg-lle-NH2 (posttranslationally modified to pQFYRlamide) and Glu-Phe-Leu-Arg-lle-NH2 (EFLRlamide). The gene is transcribed in the CNS and gives rise to a single RNA of 1.7 kb in size. The organization of the Lymnaea gene is significant with respect to the evolution of FMRFamide and related peptides in other organisms.

Animals↗

FMRFamide-like immunoreactivity in the brain and pituitary of the teleost. Eigenmannia lineata (Gymnotiformes).

The distribution of cells immunoreactive for the molluscan tetrapeptide FMRFamide in the brain and the pituitary of Eigenmannia was investigated immunohistochemically by the use of the peroxidase-antiperoxidase (PAP) technique and unlabelled antibodies. FMRFi neurons were located in the ganglion of the nervus terminalis at the rostroventral side of the bulbus olfactorius. FMRFi perikarya were also found in a dorsomedial diencephalic nucleus, in the nucleus ventromedialis, in some liquor-contacting neurons of the nucleus lateralis tuberis and of the nucleus recessus lateralis and posterior. The perikarya of the midbrain pre-pacemaker nucleus were only weakly immunoreactive for FMRFamide while large FMRFi neurons (T-cells) occurred in lamina VI of the torus semicircularis, in the brain stem, in dorsal and medial layers of the lobus lineae lateralis posterior (LLLp) and in the medullary electric organ pacemaker nucleus (pm). FMRFi fibers and nerve endings were found in the bulbus olfactorius, in medial areas of the telencephalon, and rather densely in the rostral diencephalon. Ventrocaudally to most of the hypothalamic nuclei the occurrence of immunoreactive fibres increased; many coursed to the pituitary through the pituitary stalk. FMRFi fibres also appeared in the deep layers of the tectum opticum, in the torus semicircularis, in the medial and lateral medulla and below the pacemaker nucleus. Wherever FMRFamide-immunoreactivity occurred fibres and nerve endings could be found in close contact with blood vessels.

Animals↗

Inward rectification in response to FMRFamide in Aplysia neuron L2: summation with transient K current.

The response of Aplysia abdominal ganglion neuron L2 to the molluscan neuroactive peptide Phe-Met-Arg-Phe-NH2 (FMRFamide) was studied in voltage-clamp experiments. In all of the experiments, focal application of the peptide to the soma activated an inward rectifier current and reduced the apparent amplitude of the transient K current, IA. In a few cells, Na and K currents were activated in addition to these effects. Voltage-jump experiments were performed to study the ionic dependence, kinetics, and voltage dependence of the inward rectifier. Inward rectification increased exponentially during hyperpolarizing pulses and recovered exponentially on return to the resting potential. The reversal potential was variable, but was near -40 mV at the beginning of experiments. Inward rectification was insensitive to changes in external Na, Ca, or K concentration, but lowering the external Cl concentration had complicated effects on current amplitude. When KCl microelectrodes were used, perfusion with low-Cl external saline increased the amplitude of the peptide-dependent inward rectifier and shifted its reversal potential to a more positive voltage. With KAc microelectrodes, perfusion with low-Cl saline reduced the amplitude of the current. Inward rectification increased when a KAc microelectrode was withdrawn and replaced with a low-resistance KCl electrode, even when there was no measurable change in reversal potential. These results suggest that the FMRFamide-dependent inward rectifier is a Cl current that, like the current described by Chesnoy-Marchais (1982, 1983), is modulated by intracellular Cl. FMRFamide reduced the apparent amplitude of IA without affecting the voltage dependence of IA activation or inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Functional redundancy of FMRFamide-related peptides at the Drosophila larval neuromuscular junction.

The Drosophila FMRFamide gene encodes multiple FMRFamide-related peptides. These peptides are expressed by neurosecretory cells and may be released into the blood to act as neurohormones. We analyzed the effects of eight of these peptides on nerve-stimulated contraction (twitch tension) of Drosophila larval body-wall muscles. Seven of the peptides strongly enhanced twitch tension, and one of the peptides was inactive. Their targets were distributed widely throughout the somatic musculature. The effects of one peptide, DPKQDFMRFamide, were unchanged after the onset of metamorphosis. The seven active peptides showed similar dose-response curves. Each had a threshold concentration near 1 nM, and the EC50 for each peptide was approximately 40 nM. At concentrations <0.1 microM, the responses to each of the seven excitatory peptides followed a time course that matched the fluctuations of the peptide concentration in the bath. At higher concentrations, twitch tension remained elevated for 5-10 min or more after wash-out of the peptide. When the peptides were presented as mixtures predicted by their stoichiometric ratios in the dFMRFamide propeptide, the effects were additive, and there were no detectable higher-order interactions among them. One peptide was tested and found to enhance synaptic transmission. At 0.1 microM, DPKQDFMRFamide increased the amplitude of the excitatory junctional current to 151% of baseline within 3 min. Together, these results indicate that the products of the Drosophila FMRFamide gene function as neurohormones to modulate the strength of contraction at the larval neuromuscular junction. In this role these seven peptides appear to be functionally redundant.

Amino Acid Sequence↗

The FMRFamide-like neuropeptide AF2 is present in the parasitic nematode Haemonchus contortus.

Peptides belonging to the FMRFamide family are widely distributed amongst invertebrates. We report here on the isolation of the FMRFamide neuropeptide AF2 (Lys-His-Glu-Tyr-Leu-Arg-Phe-NH2) from the parasitic nematode Haemonchus contortus. Immunocytochemical techniques showed that FMRFamide-like material was distributed in several regions of these organisms including nerve cords and cell bodies of the central nervous system. AF2 was isolated using a method that employed 6 steps of reverse-phase HPLC. The concentration of AF2 in this organism was approximately 30 pmol/g of nematode.

Amino Acid Sequence↗

Alternative RNA splicing generates diversity of neuropeptide expression in the brain of the snail Lymnaea: in situ analysis of mutually exclusive transcripts of the FMRFamide gene.

In the CNS of the snail Lymnaea stagnalis, Phe-Met-Arg-Phe-amide (FMRFamide)-like and additional novel neuropeptides are encoded by a common, multi-exon gene. This complex locus, comprising at least five exons, is subject to post-transcriptional regulation at the level of alternative RNA splicing. Our aim was first to analyse the pattern by which exons of this neuropeptide locus combine during splicing of the primary RNA transcript, and second to investigate the functional significance of splicing by mapping the expression and neuronal localization in the CNS of the alternative mRNA transcripts, in the context of defined neuronal networks and single identified neurons. The approach was a combination of comparative in situ hybridization and immunocytochemistry, using a battery of exon-specific oligonucleotides and anti-peptide antisera. The analysis illustrated that exons III, IV and V were always coexpressed and colocalized whereas the expression of exon II was always differential and mutually exclusive. Both sets of exons were, however, coexpressed with exon I: the total number of exon I-expressing neurons was equal to the combined number of neurons expressing exon III/IV/V and neurons expressing exon II. In addition, it was revealed that the extreme 5' of exon II, encoding a potential hydrophobic leader signal, was not expressed in the CNS of Lymnaea but was apparently spliced out during RNA processing. Both mRNA transcripts of the FMRFamide locus, type 1 (exons I/II) and type 2 (exons I/III/IV/V), were translated in the CNS and the resulting protein precursors were also expressed in a mutually exclusive fashion, as were their respective transcripts. The expression of alternative transcripts within identified networks or neuronal clusters was heterogeneous, as exemplified by the cardiorespiratory network. On the basis of this work and a previous cDNA analysis, we put forward a revised model of differential splicing and expression of the FMRFamide gene in the CNS of Lymnaea.

Amino Acid Sequence↗

FMRFamide immunoreactivity in the nervous system of the medusa Polyorchis penicillatus.

Three different antisera to the molluscan neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and two different antisera to the fragment RFamide were used to stain sections or whole mounts of the hydrozoan medusa Polyorchis penicillatus. All antisera stained the same neuronal structures. Strong immunoreactivity was found in neurons of the ectodermal nerve nets of the manubrium and tentacles, in neurons of the sensory epithelium, and in neurons at the periphery of the sphincter muscle. Strong immunoreactivity was also present in processes and perikarya of the whole outer nerve ring, in the ocellar nerves, and in nerve cells lying at the periphery of the ocellus. The inner nerve ring contained a moderate number of immunoreactive processes and perikarya, which were distinct from the swimming motor neurons. In contrast to the situation in the hydrozoan polyp Hydra attenuata, no immunoreactivity was found with several antisera to oxytocin/vasopressin and bombesin/gastrin-releasing peptide. The morphology and location of most FMRFamide-immunoreactive neurons in Polyorchis coincides with two identified neuronal systems, which have been recently discovered from neurophysiological studies.

Animals↗

Serotonin immunoreactivity in the optic lobes of the sphinx moth Manduca sexta and colocalization with FMRFamide and SCPB immunoreactivity.

In the optic lobes (OLs) of the sphinx moth Manduca sexta, 300-350 neurons per hemisphere are immunoreactive with an antiserotonin antiserum. Two groups of weakly serotonin-immunoreactive cells (OL1) appear to be amacrine cells of the medulla, whereas more intensely immunoreactive cells (OL2) are probably centrifugal neurons that innervate the lobula, medulla, and lamina, as well as the superior protocerebrum. At least one other OL2 cell is a local optic-lobe interneuron with arborizations in the dorsal medulla and lobula. The serotonin-immunoreactive cells are also immunoreactive with an antiserum against Drosophila melanogaster DOPA decarboxylase. All OL2 cells, but not the OL1 cells, are furthermore immunoreactive with an anti-FMRFamide antiserum and an anti-SCPB antiserum. This suggests that neuropeptides related or identical to FMRFamide and SCPB are localized and may serve as cotransmitters with serotonin in OL2 optic-lobe interneurons.

Animals↗

Substance P-, FMRFamide-, and gastrin/cholecystokinin-like immunoreactive neurons in the thoraco-abdominal ganglia of the flies Drosophila and Calliphora.

Immunocytochemical analysis of the thoraco-abdominal ganglia of the flies Drosophila melanogaster and Calliphora vomitoria revealed neurons displaying substance P- (SPLI), FMRFamide-(FLI), and cholecystokinin-like (CCKLI) immunoreactivity. It could be demonstrated that a number of neurons contain peptides reacting with antisera against all the three types of substances, others were either FLI or CCKLI alone. No neurons displayed only SPLI. Instead, the total number (about 30) of SPLI neurons constitute a subpopulation of the FLI/CCKLI neurons. Many of the identifiable immunoreactive neurons seem to be homologous in the two fly species. One set of six large neurons, termed ventral thoracic neurosecretory neurons (VTNCs), are among those that are SPLI, FLI, and CCKLI in both Drosophila and Calliphora. With the present immunocytochemical technique, the detailed morphology of the VTNCs could be resolved. These neurosecretory neurons supply the entire dorsal neural sheath of the thoraco-abdominal ganglia with terminals, thus forming an extensive neurohaemal area. The VTNCs also have processes connecting the thoracic neuromeres to the cephalic suboesophageal ganglion, as well as extensive arborizations in the thoracic ganglia, suggesting an important role in integrating and/or regulating large portions of the central nervous system, in addition to their neurosecretory function. Most of the other SPLI, FLI, and CCKLI neurons in the thoraco-abdominal ganglia seem to be interneurons. However, there are four FLI neurons that appear to be efferents innervating the hindgut and a few abdominal FLI and CCKLI neurons may be additional neurosecretory cells. From the present study it appears as if neuropeptides related to substance P, FMRFamide and CCK have roles as neurotransmitters/neuromodulators and circulating neurohormones in Drosophila and Calliphora.

Animals↗

Localization and differential expression of FMRFamide-like immunoreactivity in the nematode Ascaris suum.

By immunocytochemical and immunohistochemical methods, FMRFamide-like immunoreactivity (FLI) was localized to many neurons and processes in the Ascaris nervous system, including the head, tail, and lateral lines. Some of these cells were identified; they included sensory neurons, interneurons, and motor neurons. FLI was also present in the pharyngeal neurons and in their varicosities near the surface of the pharynx. By HPLC analysis of extracts, only a subset of the FMRFamide-like peptides (FLPs) expressed in Ascaris heads, and heads from which the pharynx had been removed, were expressed in the pharynx. Furthermore, FLPs appeared to be differentially expressed in female heads and tails and male heads and tails. Acetone and acid methanol differentially extracted subforms of FLI from Ascaris heads and from C. elegans.

Animals↗

Neural network controlling feeding in Lymnaea stagnalis: immunocytochemical localization of myomodulin, small cardioactive peptide, buccalin, and FMRFamide-related peptides.

This paper investigates the distribution of four classes of neuropeptides, myomodulin, small cardioactive peptide (SCP), buccalin, and FMRFamide, in central neurons forming the network that underlies feeding behavior in the snail Lymnaea stagnalis. Intracellular dye-marking and immunocytochemical analysis, using antisera to the different classes of peptides, were applied to identified neurons of all three levels of the hierarchy of the circuitry: modulatory interneurons (cerebral giant cells, CGC; slow oscillator, SO), central pattern generator (CPG) interneurons (N1, N2, N3), motoneurons (B1-B10), and their peripheral target organs. Myomodulin immunoreactivity was detected in the CGC interneurons, in the SO, and in ventral N2-type CPG interneurons. Several large buccal motoneurons, the paired B1, B2, B3, B7, and neurons located in the dorsal posterior area (putative B4 cluster types) were also myomodulin immunoreactive. Target organs of buccal motoneurons, the buccal mass, salivary glands, and oesophagus contained myomodulin-immunopositive fibers. SCP appeared in N2-type interneurons and was found colocalized with myomodulin in the B1 and B2 motoneurons. SCP-containing neurons in the B4 cluster area were also detected. The buccal mass and salivary glands exhibited SCP-immunoreactive fibers. Buccalin immunoreactivity was scarce in the buccal ganglia and was identified only in N1-type interneurons and three pairs of dorsal posterior neurons. In the periphery, immunoreactive fibers were localized in the oesophagus only. None of the buccal neuronal types examined revealed immunoreactivity to SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY"), a peptide encoded in the FMRFamide precursor protein of Lymnaea. SEEPLY immunoreactivity was confined to a pair of novel ventral neurons with projections to the laterobuccal nerve innervating the buccal mass. Immunoreactive fibers were also traced in this organ.

Amino Acid Sequence↗

GNFFRFamide: a novel FMRFamide-immunoreactive peptide isolated from the sheep tapeworm, Moniezia expansa.

The widespread distribution of FMRFamide-immunoreactivity in platyhelminth nervous systems has been demonstrated immunocytochemically. Here we report the isolation and primary structure of the first platyhelminth FMRFamide-related peptide (FaRP) from the tapeworm, Moniezia expansa. The peptide was found to have a molecular mass of 785 Da and a primary structure of Gly-Asn-Phe-Phe-Arg- Phe-NH2 (GNFFRFamide). Acid alcohol extracts of this platyhelminth contained only this single FaRP which had a phenylalanyl (F) residue in the variable position 3 from the C-terminal occupied by either methionyl (M), leucyl (L) or isoleucyl (I) residues in most other FaRPs.

Amino Acid Sequence↗

KSAYMRFamide: a novel FMRFamide-related heptapeptide from the free-living nematode, Panagrellus redivivus, which is myoactive in the parasitic nematode, Ascaris suum.

In nematodes, FMRFamide-related peptides (FaRPs) have been structurally characterised from the parasite, Ascaris suum, and from two free-living species, Panagrellus redivivus and Caenorhabditis elegans. While both FaRPs isolated from P. redivivus (PF1 and PF2) have been identified in C. elegans, the two heptapeptides isolated from A. suum (AF1 and AF2) have until recently been considered unique to this parasitic species. We have recently isolated AF2 from P. redivivus and, during this study, an additional novel heptapeptide amide, Lys-Ser-Ala-Tyr-Met-Arg-Phe amide (KSAYMRFamide), was structurally characterised. A synthetic replicate of this peptide induced a rapid concentration-dependent muscle tension increase in an isolated. A. suum somatic muscle preparation, with a threshold of approximately 0.1 microM. These data suggest that the complement of FaRPs in parasitic and free-living nematodes may not be as radically different as preliminary studies would suggest, and that the absence of AF1, AF2 and KSAYMRFamide on the C.elegans FMRFamide-related peptide gene (flp-1) may imply the presence of at least two different FaRP genes in nematodes.

Amino Acid Sequence↗

Expression of mRNA encoding FMRFamide-related peptides (FaRPs) in the nervous system of Helix aspersa.

The FMRFamide-related peptides (FaRPs) of Helix fall into two groups with often different pharmacological effects: the tetrapeptides FMRFamide and FLRFamide (tetraFaRPs) and the heptapeptides, which have the general structure XDP(F or Y)LRFamide (heptaFaRPs). Previously, we have shown that each group of FaRPs is encoded within a separate type of cDNA clone, a situation which corresponds to two distinct mRNA species existing in the CNS of Helix. Here, we report on the expression patterns of the two FaRP mRNAs both through embryo-genesis and in the fully differentiated regions of the adult nervous system. The levels and locations of FaRP mRNAs were studied by molecular and in situ hybridization using antisense riboprobes. The onset of expression of FaRP mRNAs occurs in Helix embryos about half-way between egg laying and hatching. First detection of the FaRPs themselves occurs about 2 days later. In embryos, as in the adult CNS, the heptaFaRP mRNA is at least five times more abundant than the tetraFaRP mRNA. In adults, the tetraFaRP mRNA is located primarily in the cerebral ganglia, most obviously in the C3 neuron, but also in a crescent-shaped cluster of small neurons lying anterior to C3. Occasional neurons expressing the tetraFaRP mRNA are detected in the parietal ganglia, but these have not yet been mapped. In contrast, the heptaFaRP mRNA is expressed almost exclusively in the parietal ganglia: in large clusters of about 100 neurons lying near to the anterior surface. The most interesting aspect of FaRP mRNAs is that their expression is not only exclusive to a relatively small number of specified neurons, but that expression appears to be mutually exclusive, that is, a particular neuron expresses only the mRNA for tetra-FaRPs or heptaFaRPs, never both. These results are discussed in relation to what we now know about the structure of the individual mRNA molecules.

Age Factors↗

Localization of substance P-like, leucine-enkephalin-like, methionine-enkephalin-like, and FMRFamide-like immunoreactivity in the eyestalk of the fiddler crab, Uca pugilator.

The occurrence and distribution of substance P (SP)-like, methionine-(Met)- and leucine-(Leu)-enkephalin-like, and FMRFamide-like immunoreactivities were determined in the neuroendocrine complex of the eyestalk of the fiddler crab, Uca pugilator, by immunocytochemistry. SP-like immunoreactivity was found in the optic peduncle, sinus gland, medulla externa, medulla interna, lamina ganglionaris, and retinular cells. Met-enkephalin-like and Leu-enkephalin-like immunoreactivity was observed in most of the retinular cells, optic peduncle, sinus gland, medulla terminalis, and lamina ganglionaris. However, Met-enkephalin-like, but no Leu-enkephalin-like, immunoreactivity was seen in the medulla terminalis X-organ. FMRFamide-like immunoreactivity could be seen in all parts of the eyestalk except in the sinus gland, lamina ganglionaris, and retinular cells. FMRF-amide-like activity was especially strong in the three chiasmatic regions connecting the optic ganglia. The possibility that these four peptides may function as neuroregulators in the fiddler crab is discussed.

Animals↗

Localization of pigment-dispersing hormone (PDH) immunoreactivity in the central nervous system of Carcinus maenas and Orconectes limosus (Crustacea), with reference to FMRFamide immunoreactivity in O. limosus.

By use of antisera raised against synthetic pigment-dispersing hormone (PDH) of Uca pugilator and FMRFamide, the distribution of immunoreactive structures in the central nervous system (CNS) of Carcinus maenas and Orconectes limosus was studied by light microscopy. In both species, a total of 10-12 PDH-positive perikarya occur amongst the anterior medial, dorsal lateral and angular somata of the cerebral ganglion (CG). In C. maenas, one PDH-perikaryon was found in each commissural ganglion (COG) and several more in the thoracic ganglion. In O. limosus, only four immunopositive perikarya could be demonstrated in the ventral nerve cord, i.e., two somata in the anterior and two in the posterior region of the suboesophageal ganglion (SOG). PDH-immunoreactive tracts and fiber plexuses were present in all central ganglia of both species, and individual axons were observed in the connectives. FMRFamide-immunoreactivity was studied in O. limosus only. Neurons of different morphological types were found throughout the entire CNS, including numerous perikarya in the anterior medial, anterior olfactory, dorsal lateral and posterior cell groups of the CG. Four perikarya were found in the COG, six large and numerous smaller ones in the SOG, and up to eight cells in each of the thoracic and abdominal ganglia. In each ganglion, the perikarya form fiber plexuses. Axons from neurons belonging to the CG could be traced into the ventral nerve cord; nerve fibers arising from perikarya in the SOG appeared to project to the posterior ganglia. In none of the structures examined colocalization of PDH- and FMRF-amide-immunoreactivity was observed.

Animals↗

Mapping of neurons in the central nervous system of the guinea pig by use of antisera specific to the molluscan neuropeptide FMRFamide.

Immunoreactive neurons were mapped in the central nervous system of colchicine-treated and untreated guinea pigs with the use of two antisera to the molluscan neuropeptide FMRFamide. These antisera were especially selected for their incapability to react with peptides of the pancreatic polypeptide family. Only one group of perikarya was stained by both antisera; this group was mainly located in the nucleus dorsomedialis hypothalami and extended to the nucleus paraventricularis and nucleus periventricularis hypothalami. The perikarya were found to project fibers to all regions of the hypothalamus, to the septum, nucleus proprius striae terminalis, nucleus paraventricularis thalami, nucleus centralis thalami, nucleus reuniens, medial, central and basal amygdala, area praetectalis, area tegmentalis ventralis of Tsai, substantia grisea centralis mesencephali, formatio reticularis mesencephali, nucleus centralis superior, locus coeruleus, nuclei parabrachiales, nucleus raphe magnus, A 5-region, vagus-solitarius complex, ventral medulla, nucleus spinalis nervi trigemini, and substantia gelatinosa of the spinal cord. In many brain regions FMRFamide-immunoreactive processes were found in close contact with blood vessels.

Animals↗